@article {pmid31711197,
year = {2019},
author = {Herrera-Carrillo, E and Gao, Z and Berkhout, B},
title = {CRISPR therapy towards an HIV cure.},
journal = {Briefings in functional genomics},
volume = {},
number = {},
pages = {},
doi = {10.1093/bfgp/elz021},
pmid = {31711197},
issn = {2041-2657},
abstract = {Tools based on RNA interference (RNAi) and the recently developed clustered regularly short palindromic repeats (CRISPR) system enable the selective modification of gene expression, which also makes them attractive therapeutic reagents for combating HIV infection and other infectious diseases. Several parallels can be drawn between the RNAi and CRISPR-Cas9 platforms. An ideal RNAi or CRISPR-Cas9 therapeutic strategy for treating infectious or genetic diseases should exhibit potency, high specificity and safety. However, therapeutic applications of RNAi and CRISPR-Cas9 have been challenged by several major limitations, some of which can be overcome by optimal design of the therapy or the design of improved reagents. In this review, we will discuss some advantages and limitations of anti-HIV strategies based on RNAi and CRISPR-Cas9 with a focus on the efficiency, specificity, off-target effects and delivery methods.},
}

@article {pmid31709036,
year = {2019},
author = {Tang, L and Yang, F and He, X and Xie, H and Liu, X and Fu, J and Xi, H and Lu, X and Liu, C and Song, Z and Qu, J and Zhao, J and Gu, F},
title = {Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells.},
journal = {Cell regeneration (London, England)},
volume = {8},
number = {2},
pages = {44-50},
doi = {10.1016/j.cr.2019.08.002},
pmid = {31709036},
issn = {2045-9769},
abstract = {Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) of bacterial adaptive immunity have been adopted as a powerful and versatile tool for manipulation of the genome. This paradigm has been widely applied in biological research and treatments of animal or cellular disease models. A critical feature of CRISPR-Cas is the protospacer adjacent motif (PAM), which dictates the DNA target recognition mechanism of Cas proteins. While, direct identifying functional PAM sequences in human cells remains a challenge. Here, we developed a positive screen system termed PAM-DOSE (PAM Definition by Observable Sequence Excision) to delineate the functional PAMs in human cells. Specifically, the PAM libraries for CRISPR-Cas (SpCas9, SpCas9-NG, FnCas12a, AsCas12a, LbCas12a and MbCas12a) were generated and the corresponding CRISPR-Cas mediated cleaved fragments with functional PAM in human cells were harvested for DNA sequencing, which could be tracked and visualized with either florescence microscopy or flow cytometry analysis. With this system, we identified the functional PAMs of CRISPR-Cas members. We also found that spacer sequence affects the PAM preference of Cas proteins. This method will facilitate identification of functional PAMs for Cas-mediated human genome editing applications.},
}

@article {pmid31708910,
year = {2019},
author = {Li, Y and Peng, N},
title = {Endogenous CRISPR-Cas System-Based Genome Editing and Antimicrobials: Review and Prospects.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {2471},
doi = {10.3389/fmicb.2019.02471},
pmid = {31708910},
issn = {1664-302X},
abstract = {CRISPR-Cas systems adapt "memories" via spacers from viruses and plasmids to develop adaptive immunity against mobile genetic elements. Mature CRISPR RNAs guide CRISPR-associated nucleases to site-specifically cleave target DNA or RNA, providing an efficient genome engineering tool for organisms of all three kingdoms. Cas9, Cas12, and Cas13 are single proteins with multiple domains that are the most widely used CRISPR nucleases of the Class 2 system. However, these CRISPR endonucleases are large in size, leading to difficulty for manipulation and toxicity for cells. Most archaeal genomes and half of the bacterial genomes encode different types of CRISPR-Cas systems. Therefore, developing endogenous CRISPR-Cas systems-based genome editing will simplify manipulations and increase editing efficiency in prokaryotic cells. Here, we review the current applications and discuss the prospects of using endogenous CRISPR nucleases for genome engineering and CRISPR-based antimicrobials.},
}

@article {pmid31640958,
year = {2019},
author = {Guo, R and Tian, Y and Jin, X and Chen, H and Wang, G and Huang, X and Li, B and Li, Z and Yang, J},
title = {[Generation of a novel HBeAg transgenic mice using CRISPR/Cas9 technique].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {39},
number = {9},
pages = {1017-1022},
doi = {10.12122/j.issn.1673-4254.2019.09.03},
pmid = {31640958},
issn = {1673-4254},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Genetic Vectors ; Hepatitis B e Antigens/*genetics ; Hepatitis B virus ; Mice ; Mice, Inbred C57BL ; *Mice, Transgenic ; },
abstract = {OBJECTIVE: To generate a new strain of HBeAg transgenic mice using CRISPR/Cas9 technique.

METHODS: Hepatitis B virus (HBV) HBeAg gene was cloned and inserted in the pliver-HBeAg expression frame at the site of Rosa26 gene using CRISPR/Cas9 and homologous recombination techniques to construct the pliver-HBeAg expression vector containing HBeAg gene. The linear DNA fragment containing HBeAg gene was obtained by enzyme digestion. Cas9 mRNA, gRNA and the donor vector were microinjected into fertilized eggs of C57BL/6J mice, which were then transplanted into the uterus of C57BL/6J female surrogate mice to obtain F0 generation mice. The F0 generation mice were identified by long fragment PCR to obtain F0 transgenic mice with HBeAg gene. The positive F0 generation mice were bred with wild-type C57BL/6J mice to produce the F1 mice, which were identified by PCR and sequencing. The positive F1 transgenic mice carrying HBeAg gene were backcrossed until the homozygous offspring transgenic mice were obtained. The genotypes of the offspring mice were identified. The expressions of HBeAg and HBeAb in the heterozygous and homozygous HBeAg transgenic mice were detected by automatic chemiluminescence immunoassay, immune colloidal gold technique and immunohistochemistry method.

RESULTS: A total of 56 F0 mice were obtained, and 2 of them carried homologous recombined HBeAg gene. Six positive F1 mice were obtained, from which 22 homozygous and 29 heterozygous F2 generation HBeAg transgenic mice were obtained. High concentration of HBeAg protein was detected in the peripheral blood of all the positive HBeAg transgenic mice without HBeAb expression. HBeAg expression was detected in the hepatocytes of HBeAg transgenic mice.

CONCLUSIONS: We obtained a new strain of HBeAg transgenic mice with stable expression of HBeAg in the hepatocytes and immune tolerance to HBeAg using CRISPR/Cas9 technique, which provide a new animal model for studying HBV.},
}

Animals
*CRISPR-Cas Systems
Female
Genetic Vectors
Hepatitis B e Antigens/*genetics
Hepatitis B virus
Mice
Mice, Inbred C57BL
*Mice, Transgenic

@article {pmid31075206,
year = {2019},
author = {Gou, Y and Liu, W and Wang, JJ and Tan, L and Hong, B and Guo, L and Liu, H and Pan, Y and Zhao, Y},
title = {CRISPR-Cas9 knockout of qseB induced asynchrony between motility and biofilm formation in Escherichia coli.},
journal = {Canadian journal of microbiology},
volume = {65},
number = {9},
pages = {691-702},
doi = {10.1139/cjm-2019-0100},
pmid = {31075206},
issn = {1480-3275},
mesh = {Biofilms/*growth & development ; CRISPR-Cas Systems ; Escherichia coli/cytology/*genetics/metabolism/physiology ; Escherichia coli Proteins/genetics/*metabolism ; Fimbriae, Bacterial/genetics ; Gene Expression Regulation, Bacterial/*genetics ; Microscopy, Electron, Scanning ; Sequence Deletion ; Virulence ; },
abstract = {Generally, cell motility and biofilm formation are tightly regulated. The QseBC two-component system (TCS) serves as a bridge for bacterial signal transmission, in which the protein QseB acts as a response regulator bacterial motility, biofilm formation, and virulence. The mechanisms that govern the interaction between QseBC and their functions have been studied in general, but the regulatory role of QseB on bacterial motility and biofilm formation is unknown. In this study, the CRISPR-Cas9 system was used to construct the Escherichia coli MG1655ΔqseB strain (strain ΔqseB), and the effects of the qseB gene on changes in motility and biofilm formation in the wild type (WT) were determined. The motility assay results showed that the ΔqseB strain had higher (p < 0.05) motility than the WT strain. However, there was no difference in the formation of biofilm between the ΔqseB and WT strains. Real-time quantitative PCR illustrated that deletion of qseB in the WT strain downregulated expression of the type I pili gene fimA. Therefore, we might conclude that the ΔqseB induced the downregulation of fimA, which led to asynchrony between motility and biofilm formation in E. coli, providing new insight into the functional importance of QseB in regulating cell motility and biofilm formation.},
}

Biofilms/*growth & development
CRISPR-Cas Systems
Escherichia coli/cytology/*genetics/metabolism/physiology
Escherichia coli Proteins/genetics/*metabolism
Fimbriae, Bacterial/genetics
Gene Expression Regulation, Bacterial/*genetics
Microscopy, Electron, Scanning
Sequence Deletion
Virulence

@article {pmid31039344,
year = {2019},
author = {Khosravi, MA and Abbasalipour, M and Concordet, JP and Berg, JV and Zeinali, S and Arashkia, A and Azadmanesh, K and Buch, T and Karimipoor, M},
title = {Targeted deletion of BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta thalassemia disease.},
journal = {European journal of pharmacology},
volume = {854},
number = {},
pages = {398-405},
doi = {10.1016/j.ejphar.2019.04.042},
pmid = {31039344},
issn = {1879-0712},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Carrier Proteins/*genetics ; Fetal Hemoglobin/*metabolism ; *Gene Deletion ; Gene Editing ; Genetic Therapy/*methods ; Humans ; K562 Cells ; Nuclear Proteins/*deficiency/*genetics ; beta-Thalassemia/genetics/metabolism/*therapy ; gamma-Globins/genetics ; },
abstract = {Hemoglobinopathies, such as β-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of β-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous β-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of β-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating β-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.},
}

Base Sequence
CRISPR-Cas Systems/*genetics
Carrier Proteins/*genetics
Fetal Hemoglobin/*metabolism
*Gene Deletion
Gene Editing
Genetic Therapy/*methods
Humans
K562 Cells
Nuclear Proteins/*deficiency/*genetics
beta-Thalassemia/genetics/metabolism/*therapy
gamma-Globins/genetics

@article {pmid30214055,
year = {2018},
author = {Kusano, H and Ohnuma, M and Mutsuro-Aoki, H and Asahi, T and Ichinosawa, D and Onodera, H and Asano, K and Noda, T and Horie, T and Fukumoto, K and Kihira, M and Teramura, H and Yazaki, K and Umemoto, N and Muranaka, T and Shimada, H},
title = {Establishment of a modified CRISPR/Cas9 system with increased mutagenesis frequency using the translational enhancer dMac3 and multiple guide RNAs in potato.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {13753},
pmid = {30214055},
issn = {2045-2322},
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Vectors/genetics ; Mutagenesis/genetics ; Plants, Genetically Modified/*genetics/growth & development ; RNA, Guide/*genetics ; Regulatory Sequences, Nucleic Acid/genetics ; Solanum tuberosum/*genetics/growth & development ; Starch Synthase/*genetics ; },
abstract = {CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5'-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF. To enrich the amount of Cas9, we applied the dMac3 translational enhancer to the Cas9 expression system with multiple gRNA genes. CRISPR/Cas9 systems targeting the potato granule-bound starch synthase I (GBSSI) gene examined the frequency of mutant alleles in transgenic potato plants. The efficiency of the targeted mutagenesis strongly increased when the dMac3-installed Cas9 was used. In this case, the ratio of transformants containing four mutant alleles reached approximately 25% when estimated by CAPS analysis. The mutants that exhibited targeted mutagenesis in the GBSSI gene showed characteristics of low amylose starch in their tubers. This result suggests that our system may facilitate genome-editing events in polyploid plants.},
}

Alleles
CRISPR-Cas Systems/genetics
Gene Editing
Genetic Vectors/genetics
Mutagenesis/genetics
Plants, Genetically Modified/*genetics/growth & development
RNA, Guide/*genetics
Regulatory Sequences, Nucleic Acid/genetics
Solanum tuberosum/*genetics/growth & development
Starch Synthase/*genetics

@article {pmid31406383,
year = {2019},
author = {Campa, CC and Weisbach, NR and Santinha, AJ and Incarnato, D and Platt, RJ},
title = {Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts.},
journal = {Nature methods},
volume = {16},
number = {9},
pages = {887-893},
doi = {10.1038/s41592-019-0508-6},
pmid = {31406383},
issn = {1548-7105},
support = {26606//Brain and Behavior Research Foundation (Brain & Behavior Research Foundation)/International ; },
mesh = {Acidaminococcus/enzymology ; *CRISPR-Cas Systems ; Endonucleases/genetics/*metabolism ; *Gene Editing ; *Gene Regulatory Networks ; *Genetic Engineering ; *Genome, Human ; HEK293 Cells ; Humans ; Plasmids/genetics ; RNA, Guide/*genetics ; Transcriptional Activation ; },
abstract = {The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.},
}

Acidaminococcus/enzymology
*CRISPR-Cas Systems
Endonucleases/genetics/*metabolism
*Gene Editing
*Gene Regulatory Networks
*Genetic Engineering
*Genome, Human
HEK293 Cells
Humans
Plasmids/genetics
RNA, Guide/*genetics
Transcriptional Activation

@article {pmid31308554,
year = {2019},
author = {Choi, GCG and Zhou, P and Yuen, CTL and Chan, BKC and Xu, F and Bao, S and Chu, HY and Thean, D and Tan, K and Wong, KH and Zheng, Z and Wong, ASL},
title = {Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.},
journal = {Nature methods},
volume = {16},
number = {8},
pages = {722-730},
doi = {10.1038/s41592-019-0473-0},
pmid = {31308554},
issn = {1548-7105},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; *Mutagenesis ; *Mutation ; Protein Engineering ; RNA, Guide/*genetics ; *Software ; Streptococcus pyogenes/enzymology ; Substrate Specificity ; },
abstract = {The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.},
}

CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/*genetics
*Gene Editing
Humans
*Mutagenesis
*Mutation
Protein Engineering
RNA, Guide/*genetics
*Software
Streptococcus pyogenes/enzymology
Substrate Specificity

@article {pmid31039782,
year = {2019},
author = {Lagziel, S and Lee, WD and Shlomi, T},
title = {Inferring cancer dependencies on metabolic genes from large-scale genetic screens.},
journal = {BMC biology},
volume = {17},
number = {1},
pages = {37},
doi = {10.1186/s12915-019-0654-4},
pmid = {31039782},
issn = {1741-7007},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor/*metabolism ; Genes, Essential ; *Genetic Testing ; Humans ; Neoplasms/*genetics ; RNA Interference ; },
abstract = {BACKGROUND: Cancer cells reprogram their metabolism to survive and propagate. Thus, targeting metabolic rewiring in tumors is a promising therapeutic strategy. Genome-wide RNAi and CRISPR screens are powerful tools for identifying genes essential for cancer cell proliferation and survival. Integrating loss-of-function genetic screens with genomics and transcriptomics datasets reveals molecular mechanisms that underlie cancer cell dependence on specific genes; though explaining cell line-specific essentiality of metabolic genes was recently shown to be especially challenging.

RESULTS: We find that variability in tissue culture medium between cell lines in a genetic screen is a major confounding factor affecting cell line-specific essentiality of metabolic genes-while, quite surprisingly, not being previously accounted for. Additionally, we find that altered expression level of a metabolic gene in a certain cell line is less indicative of its essentiality than for other genes. However, cell line-specific essentiality of metabolic genes is significantly correlated with changes in the expression of neighboring enzymes in the metabolic network. Utilizing a machine learning method that accounts for tissue culture media and functional association between neighboring enzymes, we generated predictive models for cancer cell line-specific dependence on 162 metabolic genes (representing a ~ 2.2-fold increase compared to previous studies). The generated predictive models reveal numerous novel associations between molecular features and cell line-specific dependency on metabolic genes. Specifically, we demonstrate how cancer cell dependence on one-carbon metabolic enzymes is explained based on cancer lineage, oncogenic mutations, and RNA expression of neighboring enzymes.

CONCLUSIONS: Considering culture media as well as accounting for molecular features of functionally related metabolic enzymes in a metabolic network significantly improves our understanding of cancer cell line-specific dependence on metabolic genes. We expect our approach and predictive models of metabolic gene essentiality to be a useful tool for investigating metabolic abnormalities in cancer.},
}

CRISPR-Cas Systems
Cell Line, Tumor/*metabolism
Genes, Essential
*Genetic Testing
Humans
Neoplasms/*genetics
RNA Interference

@article {pmid30740978,
year = {2019},
author = {Savage, DF},
title = {Cas14: Big Advances from Small CRISPR Proteins.},
journal = {Biochemistry},
volume = {58},
number = {8},
pages = {1024-1025},
doi = {10.1021/acs.biochem.9b00035},
pmid = {30740978},
issn = {1520-4995},
support = {DP2 EB018658/EB/NIBIB NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/*metabolism ; *Gene Editing ; Humans ; *RNA Interference ; RNA, Guide/genetics/*metabolism ; },
}

*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA/genetics/*metabolism
*Gene Editing
Humans
*RNA Interference
RNA, Guide/genetics/*metabolism

@article {pmid30591527,
year = {2019},
author = {Marconi, C and Di Buduo, CA and LeVine, K and Barozzi, S and Faleschini, M and Bozzi, V and Palombo, F and McKinstry, S and Lassandro, G and Giordano, P and Noris, P and Balduini, CL and Savoia, A and Balduini, A and Pippucci, T and Seri, M and Katsanis, N and Pecci, A},
title = {Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia.},
journal = {Blood},
volume = {133},
number = {12},
pages = {1346-1357},
doi = {10.1182/blood-2018-07-859496},
pmid = {30591527},
issn = {1528-0020},
mesh = {Adolescent ; Adult ; Animals ; Blood Platelets/metabolism/*pathology ; CRISPR-Cas Systems ; Child ; Female ; Follow-Up Studies ; *Genetic Predisposition to Disease ; Hematopoiesis ; Humans ; Male ; Megakaryocytes/metabolism/*pathology ; Middle Aged ; *Mutation ; Pedigree ; Prognosis ; Receptor-Like Protein Tyrosine Phosphatases, Class 3/antagonists & inhibitors/genetics ; Thrombocytopenia/etiology/genetics/*pathology ; Zebrafish ; },
abstract = {Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.},
}

Adolescent
Adult
Animals
Blood Platelets/metabolism/*pathology
CRISPR-Cas Systems
Child
Female
Follow-Up Studies
*Genetic Predisposition to Disease
Hematopoiesis
Humans
Male
Megakaryocytes/metabolism/*pathology
Middle Aged
*Mutation
Pedigree
Prognosis
Receptor-Like Protein Tyrosine Phosphatases, Class 3/antagonists & inhibitors/genetics
Thrombocytopenia/etiology/genetics/*pathology
Zebrafish

@article {pmid30027940,
year = {2018},
author = {Hudacsek, V and Gyõrffy, B},
title = {[Genome engineering using the CRISPR-Cas9 system and applications in cancer research].},
journal = {Magyar onkologia},
volume = {62},
number = {2},
pages = {119-127},
pmid = {30027940},
issn = {0025-0244},
mesh = {Biomedical Research/*methods ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Mutation ; Neoplasms/*genetics/*pathology ; Reproducibility of Results ; },
abstract = {Today, we have to investigate the effects of selected mutations and molecular alterations - this is made possible by the new genome editing technologies. In these, either a section of the DNA is deleted or moved, or a targeted mutation is introduced into the cell. Here, we describe in detail the CRISPR-Cas9 genome editing method. The technology is based on a feature used by bacteria to combat recurrent viral infections. At present, commencing a set of modifications, the method is functional and can be applied in eukaryotic cells as well. The method has three major steps: first, the system has to be carefully designed and constructed, then the construct has to be introduced into the target cells by transfection, and finally, the achieved effect has to be functionally validated. The reliability of the method enables multiple applications in oncology, including the detailed and efficient investigation of oncogenes, tumor suppressor genes, chromosomal translocations and other molecular changes. At the moment, available CRISPR-Cas9 protocols enable both in vitro and in vivo application. All these already made CRISPR-Cas9 one of the basic methods required for future-proof oncology research.},
}

Biomedical Research/*methods
*CRISPR-Cas Systems
*Gene Editing
Humans
*Mutation
Neoplasms/*genetics/*pathology
Reproducibility of Results

@article {pmid31696232,
year = {2019},
author = {Davies, B},
title = {The technical risks of human gene editing.},
journal = {Human reproduction (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/humrep/dez162},
pmid = {31696232},
issn = {1460-2350},
abstract = {A recent report from Dr He Jiankui concerning the birth of twin girls harbouring mutations engineered by CRISPR/Cas nucleases has been met with international condemnation. Beside the serious ethical concerns, there are known technical risks associated with CRISPR/Cas gene editing which further raise questions about how these events could have been allowed to occur. Numerous studies have reported unexpected genomic mutation and mosaicism following the use of CRISPR/Cas nucleases, and it is currently unclear how prevalent these disadvantageous events are and how robust and sensitive the strategies to detect these unwanted events may be. Although Dr Jiankui's study appears to have involved certain checks to ascertain these risks, the decision to implant the manipulated embryos, given these unknowns, must nonetheless be considered reckless. Here I review the technical concerns surrounding genome editing and consider the available data from Dr Jiankui in this context. Although the data remains unpublished, preventing a thorough assessment of what was performed, it seems clear that the rationale behind the undertaking was seriously flawed; the procedures involved substantial technical risks which, when added to the serious ethical concerns, fully justify the widespread criticism that the events have received.},
}

@article {pmid31695072,
year = {2019},
author = {Scott, T and Urak, R and Soemardy, C and Morris, KV},
title = {Improved Cas9 activity by specific modifications of the tracrRNA.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {16104},
doi = {10.1038/s41598-019-52616-5},
pmid = {31695072},
issn = {2045-2322},
support = {AI111139-01//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; 113407-01//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; },
abstract = {CRISPR/Cas is a transformative gene editing tool, that offers a simple and effective way to target a catalytic Cas9, the most widely used is derived from Streptococcus pyogenes (SpCas9), with a complementary small guide RNA (sgRNA) to inactivate endogenous genes resulting from insertions and deletions (indels). CRISPR/Cas9 has been rapidly applied to basic research as well as expanded for potential clinical applications. Utilization of spCas9 as an ribonuclearprotein complex (RNP) is considered the most safe and effective method to apply Cas9 technology, and the efficacy of this system is critically dependent on the ability of Cas9 to generate high levels of indels. We find here that novel sequence changes to the tracrRNA significantly improves Cas9 activity when delivered as an RNP. We demonstrate that a dual-guide RNA (dgRNA) with a modified tracrRNA can improve reporter knockdown and indel formation at several targets within the long terminal repeat (LTR) of HIV. Furthermore, the sequence-modified tracrRNAs improved Cas9-mediated reduction of CCR5 surface receptor expression in cell lines, which correlated with higher levels of indel formation. It was demonstrated that a Cas9 RNP with a sequence modified tracrRNA enhanced indel formation at the CCR5 target site in primary CD4+ T-cells. Finally, we show improved activity at two additional targets within the HBB locus and the BCL11A GATA site. Overall, the data presented here suggests that novel facile tracrRNA sequence changes could potentially be integrated with current dgRNA technology, and open up the possibility for the development of sequence modified tracrRNAs to improve Cas9 RNP activity.},
}

@article {pmid31693906,
year = {2019},
author = {Xu, Z and Li, M and Li, Y and Cao, H and Miao, L and Xu, Z and Higuchi, Y and Yamasaki, S and Nishino, K and Woo, PCY and Xiang, H and Yan, A},
title = {Native CRISPR-Cas-Mediated Genome Editing Enables Dissecting and Sensitizing Clinical Multidrug-Resistant P. aeruginosa.},
journal = {Cell reports},
volume = {29},
number = {6},
pages = {1707-1717.e3},
doi = {10.1016/j.celrep.2019.10.006},
pmid = {31693906},
issn = {2211-1247},
abstract = {Despite being fundamentally important and having direct therapeutic implications, the functional genomics of the clinical isolates of multidrug-resistant (MDR) pathogens is often impeded by the lack of genome-editing tools. Here, we report the establishment of a highly efficient, in situ genome-editing technique applicable in clinical and environmental isolates of the prototypic MDR pathogen P. aeruginosa by harnessing the endogenous type I-F CRISPR-Cas systems. Using this approach, we generate various reverse mutations in an epidemic MDR genotype, PA154197, and identify underlying resistance mechanisms that involve the extensive synergy among three different resistance determinants. Screening a series of "ancestor" mutant lines uncovers the remarkable sensitivity of the MDR line PA154197 to a class of small, cationic peptidomimetics, which sensitize PA154197 cells to antibiotics by perturbing outer-membrane permeability. These studies provide a framework for molecular genetics and anti-resistance drug discovery for clinically isolated MDR pathogens.},
}

@article {pmid31693467,
year = {2019},
author = {Valetdinova, KR and Ovechkina, VS and Zakian, SM},
title = {Methods for Correction of the Single-Nucleotide Substitution c.840C>T in Exon 7 of the SMN2 Gene.},
journal = {Biochemistry. Biokhimiia},
volume = {84},
number = {9},
pages = {1074-1084},
doi = {10.1134/S0006297919090104},
pmid = {31693467},
issn = {1608-3040},
abstract = {The CRISPR/Cas technology has a great potential in the treatment of many hereditary diseases. One of the prospective models for the CRISPR/Cas-mediated therapy is spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene that encodes the SMN protein required for the survival of motor neurons. SMA patients' genomes contain either single or several copies of SMN2 gene, which is a paralog of SMN1. Exon 7 of SMN2 has the single-nucleotide substitution c.840C>T leading to the defective splicing and decrease in the amounts of the full-length SMN. The objective of this study was to create and test gene-editing systems for correction of the single-nucleotide substitution c.840C>T in exon 7 of the SMN2 gene in fibroblasts, induced pluripotent stem cells, and motor neuron progenitors derived from a SMA patient. For this purpose, we used plasmid vectors expressing CRISPR/Cas9 and CRISPR/Cpf1, plasmid donor, and 90-nt single-stranded oligonucleotide templates that were delivered to the target cells by electroporation. Although sgRNA_T2 and sgRNA_T3 guiding RNAs were more efficient than sgRNA_T1 in fibroblasts (p < 0.05), no significant differences in the editing efficiency of sgRNA_T1, sgRNA_T2, and sgRNA_T3 was observed in patient-specific induced pluripotent stem cells and motor neuron progenitors. The highest editing efficiency in induced pluripotent stem cells and motor neuron progenitors was demonstrated by the sgRNA_T1 and 90-nt single-stranded oligonucleotide donors.},
}

@article {pmid31691023,
year = {2019},
author = {Barman, A and Deb, B and Chakraborty, S},
title = {A glance at genome editing with CRISPR-Cas9 technology.},
journal = {Current genetics},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00294-019-01040-3},
pmid = {31691023},
issn = {1432-0983},
abstract = {In recent years, CRISPR-Cas9 technology is widely acknowledged for having major applications in the field of biotechnology for editing genome of any organism to treat a variety of complex diseases and for other purposes. The acronym 'CRISPR-Cas' stands for clustered regularly interspaced short palindromic repeats-CRISPR-associated genes. This genetic organization exists in prokaryotic organisms and aids in the development of adaptive immunity since a protein called Cas9 nuclease cleaves specific target nucleic acid sequences from foreign invaders and destroys them. This mode of action has gained interest of the researchers to understand the insights of CRISPR-Cas9 technology. Here, we review that CRISPR-Cas organization is restricted to two classes and possesses different protein effectors. We also review the architecture of CRISPR loci, mechanism involved in genome editing by CRISPR-Cas9 technology and pathways of repairing double-strand breaks (DSBs) generated during the process of genome editing. This review also presents the strategies to increase the Cas9 specificity and reduce off-target activity to achieve accurate genome editing. Further, this review provides information on CRISPR tools used for genome editing, databases that are required for storing data on loci, strategies for delivering CRISPR-Cas9 to cells under study and applications of CRISPR-Cas9 to various fields. Safety measures are implemented on this technology to avoid misuse or ethical issues. We also discuss about the future aspects and potential applications of CRISPR-Cas9 technology required mainly for the treatment of dreadful diseases, crop improvement as well as genetic improvement in human.},
}

@article {pmid31689989,
year = {2019},
author = {Vats, S and Kumawat, S and Kumar, V and Patil, GB and Joshi, T and Sonah, H and Sharma, TR and Deshmukh, R},
title = {Genome Editing in Plants: Exploration of Technological Advancements and Challenges.},
journal = {Cells},
volume = {8},
number = {11},
pages = {},
doi = {10.3390/cells8111386},
pmid = {31689989},
issn = {2073-4409},
support = {Ramalingaswami Fellowship//Department of Biotechnology , Ministry of Science and Technology/ ; },
abstract = {Genome-editing, a recent technological advancement in the field of life sciences, is one of the great examples of techniques used to explore the understanding of the biological phenomenon. Besides having different site-directed nucleases for genome editing over a decade ago, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing approach has become a choice of technique due to its simplicity, ease of access, cost, and flexibility. In the present review, several CRISPR/Cas based approaches have been discussed, considering recent advances and challenges to implicate those in the crop improvement programs. Successful examples where CRISPR/Cas approach has been used to improve the biotic and abiotic stress tolerance, and traits related to yield and plant architecture have been discussed. The review highlights the challenges to implement the genome editing in polyploid crop plants like wheat, canola, and sugarcane. Challenges for plants difficult to transform and germline-specific gene expression have been discussed. We have also discussed the notable progress with multi-target editing approaches based on polycistronic tRNA processing, Csy4 endoribonuclease, intron processing, and Drosha ribonuclease. Potential to edit multiple targets simultaneously makes it possible to take up more challenging tasks required to engineer desired crop plants. Similarly, advances like precision gene editing, promoter bashing, and methylome-editing will also be discussed. The present review also provides a catalog of available computational tools and servers facilitating designing of guide-RNA targets, construct designs, and data analysis. The information provided here will be useful for the efficient exploration of technological advances in genome editing field for the crop improvement programs.},
}

@article {pmid31685954,
year = {2019},
author = {Zlotorynski, E},
title = {CRISPR-Cas in its prime.},
journal = {Nature reviews. Molecular cell biology},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41580-019-0194-3},
pmid = {31685954},
issn = {1471-0080},
}

@article {pmid31685795,
year = {2019},
author = {Moon, SB and Kim, DY and Ko, JH and Kim, YS},
title = {Recent advances in the CRISPR genome editing tool set.},
journal = {Experimental & molecular medicine},
volume = {51},
number = {11},
pages = {130},
doi = {10.1038/s12276-019-0339-7},
pmid = {31685795},
issn = {2092-6413},
support = {NRF-2016M3A9B6903343//National Research Foundation of Korea (NRF)/ ; NRF-2016M3A9B6903343//National Research Foundation of Korea (NRF)/ ; NRF-2016M3A9B6903343//National Research Foundation of Korea (NRF)/ ; NRF-2016M3A9B6903343//National Research Foundation of Korea (NRF)/ ; CAP-15-03-KRIBB//National Research Council of Science and Technology (National Research Council of Science & Technology)/ ; CAP-15-03-KRIBB//National Research Council of Science and Technology (National Research Council of Science & Technology)/ ; CAP-15-03-KRIBB//National Research Council of Science and Technology (National Research Council of Science & Technology)/ ; CAP-15-03-KRIBB//National Research Council of Science and Technology (National Research Council of Science & Technology)/ ; },
abstract = {Genome editing took a dramatic turn with the development of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) system. The CRISPR-Cas system is functionally divided into classes 1 and 2 according to the composition of the effector genes. Class 2 consists of a single effector nuclease, and routine practice of genome editing has been achieved by the development of the Class 2 CRISPR-Cas system, which includes the type II, V, and VI CRISPR-Cas systems. Types II and V can be used for DNA editing, while type VI is employed for RNA editing. CRISPR techniques induce both qualitative and quantitative alterations in gene expression via the double-stranded breakage (DSB) repair pathway, base editing, transposase-dependent DNA integration, and gene regulation using the CRISPR-dCas or type VI CRISPR system. Despite significant technical improvements, technical challenges should be further addressed, including insufficient indel and HDR efficiency, off-target activity, the large size of Cas, PAM restrictions, and immune responses. If sophisticatedly refined, CRISPR technology will harness the process of DNA rewriting, which has potential applications in therapeutics, diagnostics, and biotechnology.},
}

@article {pmid31683605,
year = {2019},
author = {Kurilovich, E and Shiriaeva, A and Metlitskaya, A and Morozova, N and Ivancic-Bace, I and Severinov, K and Savitskaya, E},
title = {Genome Maintenance Proteins Modulate Autoimmunity Mediated Primed Adaptation by the Escherichia coli Type I-E CRISPR-Cas System.},
journal = {Genes},
volume = {10},
number = {11},
pages = {},
doi = {10.3390/genes10110872},
pmid = {31683605},
issn = {2073-4425},
support = {19-74-20130 (E.S.)//Russian Science Foundation grant/ ; IP-2016-06-8861 (I.I.B.)//Croatian Science Foundation grant/ ; },
abstract = {Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders' genomes into CRISPR arrays. The acquisition of fragments of host genomes leads to autoimmunity and may drive chromosomal rearrangements, negative cell selection, and influence bacterial evolution. In this study, we investigated the role of proteins involved in genome stability maintenance in spacer acquisition by the Escherichia coli type I-E CRISPR-Cas system targeting its own genome. We show here, that the deletion of recJ decreases adaptation efficiency and affects accuracy of spacers incorporation into CRISPR array. Primed adaptation efficiency is also dramatically inhibited in double mutants lacking recB and sbcD but not in single mutants suggesting independent involvement and redundancy of RecBCD and SbcCD pathways in spacer acquisition. While the presence of at least one of two complexes is crucial for efficient primed adaptation, RecBCD and SbcCD affect the pattern of acquired spacers. Overall, our data suggest distinct roles of the RecBCD and SbcCD complexes and of RecJ in spacer precursor selection and insertion into CRISPR array and highlight the functional interplay between CRISPR-Cas systems and host genome maintenance mechanisms.},
}

@article {pmid31624056,
year = {2019},
author = {Zhang, K and Liu, W and Liu, XF and Chen, YS and Liu, XH and He, ZY},
title = {[Generation of cell strains containing point mutations in HPRT1 by CRISPR/Cas9].},
journal = {Yi chuan = Hereditas},
volume = {41},
number = {10},
pages = {939-949},
doi = {10.16288/j.yczz.19-108},
pmid = {31624056},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems ; HEK293 Cells ; HeLa Cells ; Humans ; Hypoxanthine Phosphoribosyltransferase/*genetics ; Lesch-Nyhan Syndrome/genetics ; *Point Mutation ; },
abstract = {Mutations in Hypoxanthine-guanine Phosphoribosyltransferase1 (HPRT1) gene can lead to metabolic disorder of hypoxanthine and guanine metabolism, and other severe symptoms such as hypophrenia, gout, and kidney stones, called the Lesch-Nyhan disease (LND). Although the mutations are widely distributed throughout the HPRT1 gene, there are some isolated hot spots. In this study, we aim to introduce two previously reported hot spots, c.508 C>T and c.151 C>T, which could lead to premature translational termination in HPRT1 gene. Through CRISPR/Cas9 mediated homology-directed repair (HDR) by using single-stranded oligo-deoxyribonucleotides (ssODN) as donor template, we obtained cell clones containing these two mutations in HEK293T or HeLa cells. Targeted mutation of c.508 C>T and c.151 C>T reached to 16.3% and 10%, respectively. We further detect HPRT1 protein levels with Western blot and enzyme activity with 6-TG in 5 different cell clones. HPRT1 protein and its enzymatic activity both was hardly detected in homozygous mutant cells, while reduced HPRT1 protein expression and enzymatic activity was detected in heterozygous mutant cells. Our study will be beneficial to those who working on generation of cell or animal models of HRPT1 mutations, and provides a basis for further investigations on the genetic mechanism of Lesch-Nyhan disease.},
}

*CRISPR-Cas Systems
HEK293 Cells
HeLa Cells
Humans
Hypoxanthine Phosphoribosyltransferase/*genetics
Lesch-Nyhan Syndrome/genetics
*Point Mutation

@article {pmid31501558,
year = {2019},
author = {Culp, EJ and Yim, G and Waglechner, N and Wang, W and Pawlowski, AC and Wright, GD},
title = {Hidden antibiotics in actinomycetes can be identified by inactivation of gene clusters for common antibiotics.},
journal = {Nature biotechnology},
volume = {37},
number = {10},
pages = {1149-1154},
doi = {10.1038/s41587-019-0241-9},
pmid = {31501558},
issn = {1546-1696},
support = {MT-14981//CIHR/Canada ; },
mesh = {Anti-Bacterial Agents/*biosynthesis ; CRISPR-Cas Systems ; DNA, Bacterial/genetics ; Escherichia coli ; Gene Expression Regulation, Bacterial ; Multigene Family ; Mutation ; Streptomyces/genetics/*metabolism ; },
abstract = {Actinobacteria, which are one of the largest bacterial phyla and comprise between 13 and 30% of the soil microbiota, are the main source of antibiotic classes in clinical use1. During screens for antimicrobials, as many as 50% of actinomycete strains are discarded because they produce a known antibiotic (Supplementary Fig. 1) (ref. 2). Despite each strain likely having the capacity to produce many compounds, strains are abandoned because the already characterized antibiotic could interfere with screening for, or purification of, newly discovered compounds3. We applied CRISPR-Cas9 genome engineering to knockout genes encoding two of the most frequently rediscovered antibiotics, streptothricin or streptomycin, in 11 actinomycete strains. We report that this simple approach led to production of different antibiotics that were otherwise masked. We were able to rapidly discover rare and previously unknown variants of antibiotics including thiolactomycin, amicetin, phenanthroviridin and 5-chloro-3-formylindole. This strategy could be applied to existing strain collections to realize their biosynthetic potential.},
}

Anti-Bacterial Agents/*biosynthesis
CRISPR-Cas Systems
DNA, Bacterial/genetics
Escherichia coli
Gene Expression Regulation, Bacterial
Multigene Family
Mutation
Streptomyces/genetics/*metabolism

@article {pmid31477922,
year = {2019},
author = {Grünewald, J and Zhou, R and Iyer, S and Lareau, CA and Garcia, SP and Aryee, MJ and Joung, JK},
title = {CRISPR DNA base editors with reduced RNA off-target and self-editing activities.},
journal = {Nature biotechnology},
volume = {37},
number = {9},
pages = {1041-1048},
doi = {10.1038/s41587-019-0236-6},
pmid = {31477922},
issn = {1546-1696},
support = {R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {APOBEC Deaminases/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; Cloning, Molecular ; Flow Cytometry ; Gene Editing/*methods ; Gene Expression Regulation, Enzymologic ; Gene Targeting ; HEK293 Cells ; Humans ; Petromyzon ; Protein Conformation ; RNA ; RNA, Guide/genetics ; Streptococcus pyogenes ; Transcriptome ; },
abstract = {Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1-4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE6, human activation-induced cytidine deaminase-based CBE7, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID4 induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.},
}

APOBEC Deaminases/genetics/metabolism
Animals
*CRISPR-Cas Systems
Cloning, Molecular
Flow Cytometry
Gene Editing/*methods
Gene Expression Regulation, Enzymologic
Gene Targeting
HEK293 Cells
Humans
Petromyzon
Protein Conformation
RNA
RNA, Guide/genetics
Streptococcus pyogenes
Transcriptome

@article {pmid31371821,
year = {2019},
author = {Kwon, JB and Gersbach, CA},
title = {Jumping at the chance for precise DNA integration.},
journal = {Nature biotechnology},
volume = {37},
number = {9},
pages = {1004-1006},
doi = {10.1038/s41587-019-0210-3},
pmid = {31371821},
issn = {1546-1696},
mesh = {*CRISPR-Cas Systems ; DNA Damage ; *Gene Targeting ; *Genome ; *Transposases ; },
}

*CRISPR-Cas Systems
DNA Damage
*Gene Targeting
*Genome
*Transposases

@article {pmid31359007,
year = {2019},
author = {Leenay, RT and Aghazadeh, A and Hiatt, J and Tse, D and Roth, TL and Apathy, R and Shifrut, E and Hultquist, JF and Krogan, N and Wu, Z and Cirolia, G and Canaj, H and Leonetti, MD and Marson, A and May, AP and Zou, J},
title = {Large dataset enables prediction of repair after CRISPR-Cas9 editing in primary T cells.},
journal = {Nature biotechnology},
volume = {37},
number = {9},
pages = {1034-1037},
doi = {10.1038/s41587-019-0203-2},
pmid = {31359007},
issn = {1546-1696},
support = {P50 AI150476/AI/NIAID NIH HHS/United States ; U19 AI135990/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; Gene Expression Regulation ; Genome ; Genomics ; Humans ; Induced Pluripotent Stem Cells/physiology ; RNA, Guide/*genetics ; T-Lymphocytes/*physiology ; },
abstract = {Understanding of repair outcomes after Cas9-induced DNA cleavage is still limited, especially in primary human cells. We sequence repair outcomes at 1,656 on-target genomic sites in primary human T cells and use these data to train a machine learning model, which we have called CRISPR Repair Outcome (SPROUT). SPROUT accurately predicts the length, probability and sequence of nucleotide insertions and deletions, and will facilitate design of SpCas9 guide RNAs in therapeutically important primary human cells.},
}

*CRISPR-Cas Systems
Cell Line
Gene Editing/*methods
Gene Expression Regulation
Genome
Genomics
Humans
Induced Pluripotent Stem Cells/physiology
RNA, Guide/*genetics
T-Lymphocytes/*physiology

@article {pmid31332326,
year = {2019},
author = {Thuronyi, BW and Koblan, LW and Levy, JM and Yeh, WH and Zheng, C and Newby, GA and Wilson, C and Bhaumik, M and Shubina-Oleinik, O and Holt, JR and Liu, DR},
title = {Continuous evolution of base editors with expanded target compatibility and improved activity.},
journal = {Nature biotechnology},
volume = {37},
number = {9},
pages = {1070-1079},
doi = {10.1038/s41587-019-0193-0},
pmid = {31332326},
issn = {1546-1696},
support = {R35 GM118062/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; },
mesh = {Adenosine Deaminase/genetics/*metabolism ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Cell Line ; *Directed Molecular Evolution ; *Gene Editing ; Gene Expression Regulation, Enzymologic ; Gene Targeting ; Humans ; INDEL Mutation ; Mice ; },
abstract = {Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.},
}

Adenosine Deaminase/genetics/*metabolism
Animals
Base Sequence
CRISPR-Cas Systems
Cell Line
*Directed Molecular Evolution
*Gene Editing
Gene Expression Regulation, Enzymologic
Gene Targeting
Humans
INDEL Mutation
Mice

@article {pmid31225941,
year = {2019},
author = {de Waal, E and Tran, T and Abbondanza, D and Dey, A and Peterson, C},
title = {An undergraduate laboratory module that uses the CRISPR/Cas9 system to generate frameshift mutations in yeast.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {47},
number = {5},
pages = {573-580},
doi = {10.1002/bmb.21280},
pmid = {31225941},
issn = {1539-3429},
mesh = {CRISPR-Cas Systems/*genetics ; Frameshift Mutation/*genetics ; Humans ; *Laboratories ; Learning ; Saccharomyces cerevisiae/*genetics ; Students ; Universities ; },
abstract = {The CRISPR/Cas9 system is a powerful tool for gene editing and it has become increasingly important for biology students to understand this emerging technique. Most CRISPR laboratory teaching modules use complex metazoan systems or mammalian cell culture which can be expensive. Here, we present a lab module that engages students in learning the fundamentals of CRISPR/Cas9 methodology using the simple and inexpensive model system, Saccharomyces cerevisiae. Students use CRISPR/Cas9 and nonhomologous end joining to generate frameshift insertion and deletion mutations in the CAN1 gene, which are easily selected for using media plates that have canavanine. DNA sequencing is also performed to determine what type of mutation occurred in gene-edited cells. This easy to implement set of experiments has been run as both a 5-week and a shorter 3-week lab module. Learning assessments demonstrate increased understanding in CRISPR-related concepts as well as increased confidence using molecular techniques. Thus, this CRISPR/Cas9 lab module can be added to an existing Genetics, Microbiology, or Molecular Biology lab course to help undergraduate students learn current gene editing techniques with limited effort and cost. © 2019 International Union of Biochemistry and Molecular Biology, 47(5):573-580, 2019.},
}

CRISPR-Cas Systems/*genetics
Frameshift Mutation/*genetics
Humans
*Laboratories
Learning
Saccharomyces cerevisiae/*genetics
Students
Universities

@article {pmid31209374,
year = {2019},
author = {Simon, AJ and d'Oelsnitz, S and Ellington, AD},
title = {Synthetic evolution.},
journal = {Nature biotechnology},
volume = {37},
number = {7},
pages = {730-743},
doi = {10.1038/s41587-019-0157-4},
pmid = {31209374},
issn = {1546-1696},
mesh = {Animals ; CRISPR-Cas Systems ; DNA/chemical synthesis/*genetics ; *Directed Molecular Evolution ; Genetic Engineering ; Genome ; Mutation ; },
abstract = {The combination of modern biotechnologies such as DNA synthesis, λ red recombineering, CRISPR-based editing and next-generation high-throughput sequencing increasingly enables precise manipulation of genes and genomes. Beyond rational design, these technologies also enable the targeted, and potentially continuous, introduction of multiple mutations. While this might seem to be merely a return to natural selection, the ability to target evolution greatly reduces fitness burdens and focuses mutation and selection on those genes and traits that best contribute to a desired phenotype, ultimately throwing evolution into fast forward.},
}

Animals
CRISPR-Cas Systems
DNA/chemical synthesis/*genetics
*Directed Molecular Evolution
Genetic Engineering
Genome
Mutation

@article {pmid31197230,
year = {2019},
author = {Rybicki, EP},
title = {CRISPR-Cas9 strikes out in cassava.},
journal = {Nature biotechnology},
volume = {37},
number = {7},
pages = {727-728},
doi = {10.1038/s41587-019-0169-0},
pmid = {31197230},
issn = {1546-1696},
mesh = {*CRISPR-Cas Systems ; *Geminiviridae ; Gene Editing ; Genetic Engineering/methods ; Genetic Predisposition to Disease ; Host-Pathogen Interactions/genetics ; Manihot/genetics ; Plant Diseases/*genetics/*virology ; Plants, Genetically Modified/*virology ; },
}

*CRISPR-Cas Systems
*Geminiviridae
Gene Editing
Genetic Engineering/methods
Genetic Predisposition to Disease
Host-Pathogen Interactions/genetics
Manihot/genetics
Plant Diseases/*genetics/*virology
Plants, Genetically Modified/*virology

@article {pmid31159719,
year = {2019},
author = {Li, T and Fang, Z and Peng, H and Zhou, J and Liu, P and Wang, Y and Zhu, W and Li, L and Zhang, Q and Chen, L and Li, L and Liu, Z and Zhang, W and Zhai, W and Lu, L and Gao, L},
title = {Application of high-throughput amplicon sequencing-based SSR genotyping in genetic background screening.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {444},
doi = {10.1186/s12864-019-5800-4},
pmid = {31159719},
issn = {1471-2164},
support = {14043//Jianghan University/ ; 1002-0606001//Wuhan City Partner/ ; 31300999//National Natural Science Foundation of China/ ; 31501028//National Natural Science Foundation of China/ ; 31601995//National Natural Science Foundation of China/ ; 31800306//National Natural Science Foundation of China/ ; 2016ZX08001-002//Ministry of Agriculture of the People's Republic of China/ ; 2017CFB229//Natural Science Foundation of Hubei Province/ ; },
mesh = {CRISPR-Cas Systems ; Gene Editing ; Gene Transfer Techniques ; Genetic Engineering ; Genetic Testing/*methods ; High-Throughput Nucleotide Sequencing/*methods ; *Microsatellite Repeats ; Oryza/*genetics ; Plant Proteins/antagonists & inhibitors/*genetics ; Plants, Genetically Modified/*genetics ; Polymorphism, Genetic ; Protein-Serine-Threonine Kinases/antagonists & inhibitors/*genetics ; },
abstract = {BACKGROUND: Host genetic backgrounds affect gene functions. The genetic backgrounds of genetically engineered organisms must be identified to confirm their genetic backgrounds identity with those of recipients. Marker-assisted backcrossing (MAB), transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) editing are three commonly used genetic engineering techniques. However, methods for genetic background screening between genetically engineered organisms and corresponding recipients suffer from low efficiency, low accuracy or high cost.

RESULTS: Here, we improved our previously reported AmpSeq-SSR method, an amplicon sequencing-based simple sequence repeat (SSR) genotyping method, by selecting SSR loci with high polymorphism among varieties. Ultimately, a set of 396 SSRs was generated and applied to evaluate the genetic backgrounds identity between rice lines developed through MAB, transgenesis, and CRISPR/Cas9 editing and the respective recipient rice. We discovered that the percentage of different SSRs between the MAB-developed rice line and its recipient was as high as 23.5%. In contrast, only 0.8% of SSRs were different between the CRISPR/Cas9-system-mediated rice line and its recipient, while no SSRs showed different genotypes between the transgenic rice line and its recipient. Furthermore, most differential SSRs induced by MAB technology were located in non-coding regions (62.9%), followed by untranslated regions (21.0%) and coding regions (16.1%). Trinucleotide repeats were the most prevalent type of altered SSR. Most importantly, all altered SSRs located in coding regions were trinucleotide repeats.

CONCLUSIONS: This method is not only useful for the background evaluation of genetic resources but also expands our understanding of the unintended effects of different genetic engineering techniques. While the work we present focused on rice, this method can be readily extended to other organisms.},
}

CRISPR-Cas Systems
Gene Editing
Gene Transfer Techniques
Genetic Engineering
Genetic Testing/*methods
High-Throughput Nucleotide Sequencing/*methods
*Microsatellite Repeats
Oryza/*genetics
Plant Proteins/antagonists & inhibitors/*genetics
Plants, Genetically Modified/*genetics
Polymorphism, Genetic
Protein-Serine-Threonine Kinases/antagonists & inhibitors/*genetics

@article {pmid31109137,
year = {2019},
author = {Ji, J and Zhang, C and Sun, Z and Wang, L and Duanmu, D and Fan, Q},
title = {Genome Editing in Cowpea Vigna unguiculata Using CRISPR-Cas9.},
journal = {International journal of molecular sciences},
volume = {20},
number = {10},
pages = {},
doi = {10.3390/ijms20102471},
pmid = {31109137},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Plant ; Mutagenesis ; Nitrogen Fixation ; RNA, Guide/genetics ; Vigna/*genetics/metabolism ; },
abstract = {Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume.},
}

CRISPR-Cas Systems
*Gene Editing/methods
Genome, Plant
Mutagenesis
Nitrogen Fixation
RNA, Guide/genetics
Vigna/*genetics/metabolism

@article {pmid30811422,
year = {2019},
author = {Raitskin, O and Schudoma, C and West, A and Patron, NJ},
title = {Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering.},
journal = {PloS one},
volume = {14},
number = {2},
pages = {e0211598},
doi = {10.1371/journal.pone.0211598},
pmid = {30811422},
issn = {1932-6203},
support = {BB/L014130/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/CCG1720/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/T/000PR9816/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/genetics ; Gene Editing/methods ; Genetic Engineering/methods ; Genome, Plant/*genetics ; Plants/*genetics ; },
abstract = {Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called 'off-targets' by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content of the target correlates with efficiency and that Cas9 from Staphylococcus aureus (SaCas9) is comparatively most efficient at inducing mutations. We also demonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Endonucleases/genetics
Gene Editing/methods
Genetic Engineering/methods
Genome, Plant/*genetics
Plants/*genetics

@article {pmid30753226,
year = {2019},
author = {Kim, EK and Kim, S and Maeng, YS},
title = {Generation of TGFBI knockout ABCG2+/ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing.},
journal = {PloS one},
volume = {14},
number = {2},
pages = {e0211864},
doi = {10.1371/journal.pone.0211864},
pmid = {30753226},
issn = {1932-6203},
mesh = {ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics ; ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics ; CRISPR-Cas Systems/*genetics ; Corneal Dystrophies, Hereditary/*genetics/pathology/therapy ; Epithelial Cells/metabolism/pathology ; Extracellular Matrix Proteins/*genetics ; Extremities/growth & development/pathology ; Gene Editing ; Gene Expression Regulation/genetics ; Gene Knockout Techniques ; *Genetic Therapy ; Humans ; Primary Cell Culture ; RNA, Guide/genetics ; Sequence Deletion/genetics ; Single-Cell Analysis ; Stem Cells/metabolism ; Transforming Growth Factor beta/*genetics ; },
abstract = {Corneal dystrophy is an autosomal dominant disorder caused by mutations of the transforming growth factor β-induced (TGFBI) gene on chromosome 5q31.8. This disease is therefore ideally suited for gene therapy using genome-editing technology. Here, we isolated human limbal epithelial stem cells (ABCG2+/ABCB5+ double-positive LESCs) and established a TGFBI knockout using RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. An LESC clone generated with a single-guide RNA (sgRNA) targeting exon 4 of the TGFBI gene was sequenced in order to identify potential genomic insertions and deletions near the Cas9/sgRNA-target sites. A detailed analysis of the differences between wild type LESCs and the single LESC clone modified by the TGFBI-targeting sgRNA revealed two distinct mutations, an 8 bp deletion and a 14 bp deletion flanked by a single point mutation. These mutations each lead to a frameshift missense mutation and generate premature stop codons downstream in exon 4. To validate the TGFBI knockout LESC clone, we used single cell culture to isolate four individual sub-clones, each of which was found to possess both mutations present in the parent clone, indicating that the population is homogenous. Furthermore, we confirmed that TGFBI protein expression is abolished in the TGFBI knockout LESC clone using western blot analysis. Collectively, our results suggest that genome editing of TGFBI in LESCs by CRISPR/Cas9 may be useful strategy to treat corneal dystrophy.},
}

ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics
ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics
CRISPR-Cas Systems/*genetics
Corneal Dystrophies, Hereditary/*genetics/pathology/therapy
Epithelial Cells/metabolism/pathology
Extracellular Matrix Proteins/*genetics
Extremities/growth & development/pathology
Gene Editing
Gene Expression Regulation/genetics
Gene Knockout Techniques
*Genetic Therapy
Humans
Primary Cell Culture
RNA, Guide/genetics
Sequence Deletion/genetics
Single-Cell Analysis
Stem Cells/metabolism
Transforming Growth Factor beta/*genetics

@article {pmid30692104,
year = {2019},
author = {Knoppers, BM and Kleiderman, E},
title = {"CRISPR babies": What does this mean for science and Canada?.},
journal = {CMAJ : Canadian Medical Association journal = journal de l'Association medicale canadienne},
volume = {191},
number = {4},
pages = {E91-E92},
doi = {10.1503/cmaj.181657},
pmid = {30692104},
issn = {1488-2329},
mesh = {Bioethical Issues ; *CRISPR-Cas Systems ; Canada ; Embryo Research/*ethics ; Female ; Gene Editing/*ethics/*legislation & jurisprudence ; Humans ; Infant, Newborn ; Pregnancy ; Reproductive Techniques, Assisted/*ethics/*legislation & jurisprudence ; },
}

Bioethical Issues
*CRISPR-Cas Systems
Canada
Embryo Research/*ethics
Female
Gene Editing/*ethics/*legislation & jurisprudence
Humans
Infant, Newborn
Pregnancy
Reproductive Techniques, Assisted/*ethics/*legislation & jurisprudence

@article {pmid30653821,
year = {2019},
author = {Wen, X and Iwata, K and Ikuta, K and Zhang, X and Zhu, K and Ibi, M and Matsumoto, M and Asaoka, N and Liu, J and Katsuyama, M and Yabe-Nishimura, C},
title = {NOX1/NADPH oxidase regulates the expression of multidrug resistance-associated protein 1 and maintains intracellular glutathione levels.},
journal = {The FEBS journal},
volume = {286},
number = {4},
pages = {678-687},
doi = {10.1111/febs.14753},
pmid = {30653821},
issn = {1742-4658},
mesh = {Acrolein/pharmacology ; Animals ; Butanones/pharmacology ; CRISPR-Cas Systems ; Cell Survival ; Cells, Cultured ; Gene Expression Regulation/*drug effects ; Glutathione/*metabolism ; Multidrug Resistance-Associated Proteins/antagonists & inhibitors/genetics/*metabolism ; Myocytes, Cardiac/cytology/drug effects/*metabolism ; NADPH Oxidase 1/antagonists & inhibitors/genetics/*metabolism ; Pyrazoles/pharmacology ; Pyrimidines/pharmacology ; Rats ; Reactive Oxygen Species/metabolism ; },
abstract = {The involvement of superoxide-generating NADPH oxidase (NOX) in the cytotoxic effects of cigarette smoke extracts has been documented. However, the underlying molecular mechanisms and NOX isoform involved have not been fully clarified. Among the different NADPH oxidase isoforms identified so far, NOX1 and NOX4 were found to be expressed in rat H9c2 cardiomyocytes. When H9c2 cells were exposed to acrolein or methyl vinyl ketone (MVK), major toxic components of cigarette smoke extracts, a dose-dependent decline in cell viability was observed. Unexpectedly, disruption of Nox1 as well as Nox4 significantly exacerbated cytotoxicity induced by acrolein or MVK. Compared with Nox4-disrupted cells, Nox1-disrupted cells were more vulnerable to acrolein and MVK at lower concentrations. Disruption of Nox1 markedly attenuated the levels of total and reduced glutathione (GSH) in H9c2 clones. Reduction in the cystine level in the culture medium to deplete intracellular GSH significantly exacerbated acrolein or MVK-induced cytotoxicity. Nox1 disruption neither attenuated the level of glutamate-cystine antiporter protein nor the activity of glutamate-cysteine ligase, both rate-limiting factors for GSH synthesis. On the other hand, increased expression of multidrug resistance-associated protein 1 (MRP1), which mediates glutathione efflux, was demonstrated in Nox1-disrupted cells. The augmented toxicity of acrolein and MVK in these cells was partially but significantly blunted in the presence of an MRP1 inhibitor, reversan. Taken together, these results show that NOX1/NADPH oxidase regulates the expression of MRP1 to maintain intracellular GSH levels in cardiomyocytes and protect against cytotoxic components of cigarette smoke extracts. A novel crosstalk between NOX1 and MRP1 was demonstrated in this study.},
}

Acrolein/pharmacology
Animals
Butanones/pharmacology
CRISPR-Cas Systems
Cell Survival
Cells, Cultured
Gene Expression Regulation/*drug effects
Glutathione/*metabolism
Multidrug Resistance-Associated Proteins/antagonists & inhibitors/genetics/*metabolism
Myocytes, Cardiac/cytology/drug effects/*metabolism
NADPH Oxidase 1/antagonists & inhibitors/genetics/*metabolism
Pyrazoles/pharmacology
Pyrimidines/pharmacology
Rats
Reactive Oxygen Species/metabolism

@article {pmid30595124,
year = {2019},
author = {Barton, M and Meyer, MR},
title = {Permissive Role of GPER for Arterial Hypertension.},
journal = {Hypertension (Dallas, Tex. : 1979)},
volume = {73},
number = {2},
pages = {e9-e10},
doi = {10.1161/HYPERTENSIONAHA.118.12159},
pmid = {30595124},
issn = {1524-4563},
mesh = {Animals ; CRISPR-Cas Systems ; *Dysbiosis ; Hypertension/*genetics ; Rats ; Receptors, G-Protein-Coupled ; },
}

Animals
CRISPR-Cas Systems
*Dysbiosis
Hypertension/*genetics
Rats
Receptors, G-Protein-Coupled

@article {pmid30595121,
year = {2019},
author = {Waghulde, H and Galla, S and Chakraborty, S and Joe, B},
title = {Response to Permissive Role of GPER for Arterial Hypertension.},
journal = {Hypertension (Dallas, Tex. : 1979)},
volume = {73},
number = {2},
pages = {e11},
doi = {10.1161/HYPERTENSIONAHA.118.12366},
pmid = {30595121},
issn = {1524-4563},
support = {R01 HL143082/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Dysbiosis ; Hypertension/*genetics ; Rats ; Receptors, G-Protein-Coupled ; },
}

Animals
CRISPR-Cas Systems
*Dysbiosis
Hypertension/*genetics
Rats
Receptors, G-Protein-Coupled

@article {pmid29898989,
year = {2018},
author = {Liu, Q and Thoms, JAI and Nunez, AC and Huang, Y and Knezevic, K and Packham, D and Poulos, RC and Williams, R and Beck, D and Hawkins, NJ and Ward, RL and Wong, JWH and Hesson, LB and Sloane, MA and Pimanda, JE},
title = {Disruption of a -35 kb Enhancer Impairs CTCF Binding and MLH1 Expression in Colorectal Cells.},
journal = {Clinical cancer research : an official journal of the American Association for Cancer Research},
volume = {24},
number = {18},
pages = {4602-4611},
doi = {10.1158/1078-0432.CCR-17-3678},
pmid = {29898989},
issn = {1078-0432},
mesh = {Adult ; Aged ; Aged, 80 and over ; CCCTC-Binding Factor/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Colorectal Neoplasms/*genetics/pathology ; Colorectal Neoplasms, Hereditary Nonpolyposis/*genetics/pathology ; DNA Methylation/genetics ; DNA Mismatch Repair/genetics ; Enhancer Elements, Genetic/genetics ; Female ; Gene Expression Regulation, Neoplastic ; Germ-Line Mutation/genetics ; Humans ; Male ; Middle Aged ; MutL Protein Homolog 1/*genetics ; Promoter Regions, Genetic/genetics ; },
abstract = {Purpose:MLH1 is a major tumor suppressor gene involved in the pathogenesis of Lynch syndrome and various sporadic cancers. Despite their potential pathogenic importance, genomic regions capable of regulating MLH1 expression over long distances have yet to be identified.Experimental Design: Here, we use chromosome conformation capture (3C) to screen a 650-kb region flanking the MLH1 locus to identify interactions between the MLH1 promoter and distal regions in MLH1-expressing and nonexpressing cells. Putative enhancers were functionally validated using luciferase reporter assays, chromatin immunoprecipitation, and CRISPR-Cas9-mediated deletion of endogenous regions. To evaluate whether germline variants in the enhancer might contribute to impaired MLH1 expression in patients with suspected Lynch syndrome, we also screened germline DNA from a cohort of 74 patients with no known coding mutations or epimutations at the MLH1 promoter.Results: A 1.8-kb DNA fragment, 35 kb upstream of the MLH1 transcription start site enhances MLH1 gene expression in colorectal cells. The enhancer was bound by CTCF and CRISPR-Cas9-mediated deletion of a core binding region impairs endogenous MLH1 expression. A total of 5.4% of suspected Lynch syndrome patients have a rare single-nucleotide variant (G > A; rs143969848; 2.5% in gnomAD European, non-Finnish) within a highly conserved CTCF-binding motif, which disrupts enhancer activity in SW620 colorectal carcinoma cells.Conclusions: A CTCF-bound region within the MLH1-35 enhancer regulates MLH1 expression in colorectal cells and is worthy of scrutiny in future genetic screening strategies for suspected Lynch syndrome associated with loss of MLH1 expression. Clin Cancer Res; 24(18); 4602-11. ©2018 AACR.},
}

Adult
Aged
Aged, 80 and over
CCCTC-Binding Factor/*genetics
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Colorectal Neoplasms/*genetics/pathology
Colorectal Neoplasms, Hereditary Nonpolyposis/*genetics/pathology
DNA Methylation/genetics
DNA Mismatch Repair/genetics
Enhancer Elements, Genetic/genetics
Female
Gene Expression Regulation, Neoplastic
Germ-Line Mutation/genetics
Humans
Male
Middle Aged
MutL Protein Homolog 1/*genetics
Promoter Regions, Genetic/genetics

@article {pmid29898403,
year = {2018},
author = {Zhao, Y and Zhang, Z and Gao, J and Wang, P and Hu, T and Wang, Z and Hou, YJ and Wan, Y and Liu, W and Xie, S and Lu, T and Xue, L and Liu, Y and Macho, AP and Tao, WA and Bressan, RA and Zhu, JK},
title = {Arabidopsis Duodecuple Mutant of PYL ABA Receptors Reveals PYL Repression of ABA-Independent SnRK2 Activity.},
journal = {Cell reports},
volume = {23},
number = {11},
pages = {3340-3351.e5},
pmid = {29898403},
issn = {2211-1247},
support = {R01 GM059138/GM/NIGMS NIH HHS/United States ; },
mesh = {Abscisic Acid/*pharmacology ; Arabidopsis/drug effects/growth & development/*metabolism ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Gene Expression Regulation, Plant/*drug effects ; Mutagenesis ; Osmotic Pressure/drug effects ; Plant Growth Regulators/*pharmacology ; Protein-Serine-Threonine Kinases/*metabolism ; },
abstract = {Abscisic acid (ABA) is an important phytohormone controlling responses to abiotic stresses and is sensed by proteins from the PYR/PYL/RCAR family. To explore the genetic contribution of PYLs toward ABA-dependent and ABA-independent processes, we generated and characterized high-order Arabidopsis mutants with mutations in the PYL family. We obtained a pyl quattuordecuple mutant and found that it was severely impaired in growth and failed to produce seeds. Thus, we carried out a detailed characterization of a pyl duodecuple mutant, pyr1pyl1/2/3/4/5/7/8/9/10/11/12. The duodecuple mutant was extremely insensitive to ABA effects on seed germination, seedling growth, stomatal closure, leaf senescence, and gene expression. The activation of SnRK2 protein kinases by ABA was blocked in the duodecuple mutant, but, unexpectedly, osmotic stress activation of SnRK2s was enhanced. Our results demonstrate an important role of basal ABA signaling in growth, senescence, and abscission and reveal that PYLs antagonize ABA-independent activation of SnRK2s by osmotic stress.},
}

Abscisic Acid/*pharmacology
Arabidopsis/drug effects/growth & development/*metabolism
Arabidopsis Proteins/genetics/*metabolism
CRISPR-Cas Systems/genetics
Gene Editing
Gene Expression Regulation, Plant/*drug effects
Mutagenesis
Osmotic Pressure/drug effects
Plant Growth Regulators/*pharmacology
Protein-Serine-Threonine Kinases/*metabolism

@article {pmid29898387,
year = {2018},
author = {Cunningham, TJ and Lancman, JJ and Berenguer, M and Dong, PDS and Duester, G},
title = {Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development.},
journal = {Cell reports},
volume = {23},
number = {11},
pages = {3146-3151},
pmid = {29898387},
issn = {2211-1247},
support = {R01 AR067731/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/metabolism ; CRISPR-Cas Systems/genetics ; Enhancer Elements, Genetic/*genetics ; Forelimb/*growth & development/metabolism ; *Gene Editing ; Introns ; Mice ; T-Box Domain Proteins/*genetics ; Zebrafish/*genetics/metabolism ; },
abstract = {A standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 forelimb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals. We used CRISPR/Cas9 gene editing to knockout the endogenous elements and unexpectedly found that deletion of the intron 2 and downstream elements, either singly or together in double knockouts, resulted in no effect on forelimb development. Our findings show that reporter transgenes may not identify endogenous enhancers and that in vivo genetic loss-of-function studies are required, such as CRISPR/Cas9, which is similar in effort to production of animals carrying reporter transgenes.},
}

Animals
Animals, Genetically Modified/metabolism
CRISPR-Cas Systems/genetics
Enhancer Elements, Genetic/*genetics
Forelimb/*growth & development/metabolism
*Gene Editing
Introns
Mice
T-Box Domain Proteins/*genetics
Zebrafish/*genetics/metabolism

@article {pmid29847792,
year = {2018},
author = {Kuo, CY and Long, JD and Campo-Fernandez, B and de Oliveira, S and Cooper, AR and Romero, Z and Hoban, MD and Joglekar, AV and Lill, GR and Kaufman, ML and Fitz-Gibbon, S and Wang, X and Hollis, RP and Kohn, DB},
title = {Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome.},
journal = {Cell reports},
volume = {23},
number = {9},
pages = {2606-2616},
pmid = {29847792},
issn = {2211-1247},
support = {K23 CA222659/CA/NCI NIH HHS/United States ; U54 AI082973/AI/NIAID NIH HHS/United States ; T32 GM007185/GM/NIGMS NIH HHS/United States ; K12 HD034610/HD/NICHD NIH HHS/United States ; T32 AI060567/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Antigens, CD34/metabolism ; Base Sequence ; CD40 Ligand/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Colony-Forming Units Assay ; DNA Repair ; DNA, Complementary/genetics ; *Gene Editing ; Genetic Diseases, X-Linked/*genetics ; Hematopoietic Stem Cells/*metabolism ; Humans ; Hyper-IgM Immunodeficiency Syndrome/*genetics ; Mice ; T-Lymphocytes/metabolism ; Transcription Activator-Like Effector Nucleases/metabolism ; },
abstract = {X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.},
}

Animals
Antigens, CD34/metabolism
Base Sequence
CD40 Ligand/metabolism
CRISPR-Associated Protein 9/metabolism
CRISPR-Cas Systems/genetics
Cell Differentiation
Cell Line
Colony-Forming Units Assay
DNA Repair
DNA, Complementary/genetics
*Gene Editing
Genetic Diseases, X-Linked/*genetics
Hematopoietic Stem Cells/*metabolism
Humans
Hyper-IgM Immunodeficiency Syndrome/*genetics
Mice
T-Lymphocytes/metabolism
Transcription Activator-Like Effector Nucleases/metabolism

@article {pmid31679813,
year = {2019},
author = {Ophinni, Y and Palatini, U and Hayashi, Y and Parrish, NF},
title = {piRNA-Guided CRISPR-like Immunity in Eukaryotes.},
journal = {Trends in immunology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.it.2019.09.003},
pmid = {31679813},
issn = {1471-4981},
abstract = {Eukaryotic genomes contain virus-derived sequences called endogenous virus elements (EVEs). The majority of EVEs are related to retroviruses, which integrate into the host genome in order to replicate. Some retroviral EVEs encode a function; for example, some produce proteins that block infection by related viruses. EVEs derived from nonretroviral viruses - also recently found in many eukaryotic genomes - are more enigmatic. Here, we summarize the evidence that EVEs can act as templates to generate Piwi-interacting RNAs (piRNAs), whose canonical function is sequence-specific silencing of transposable elements (TEs) to maintain genomic integrity. We argue that EVEs may thus enable heritable, sequence-specific antiviral immune memory in eukaryotes - analogous to CRISPR-Cas immunity in prokaryotes.},
}

@article {pmid31363223,
year = {2019},
author = {Rusk, N},
title = {Human CRISPR.},
journal = {Nature methods},
volume = {16},
number = {8},
pages = {677},
doi = {10.1038/s41592-019-0526-4},
pmid = {31363223},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *RNA ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Humans
*RNA

@article {pmid31101683,
year = {2019},
author = {Schertzer, MD and Thulson, E and Braceros, KCA and Lee, DM and Hinkle, ER and Murphy, RM and Kim, SO and Vitucci, ECM and Calabrese, JM},
title = {A piggyBac-based toolkit for inducible genome editing in mammalian cells.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {8},
pages = {1047-1058},
doi = {10.1261/rna.068932.118},
pmid = {31101683},
issn = {1469-9001},
support = {T32 ES007126/ES/NIEHS NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; R25 GM055336/GM/NIGMS NIH HHS/United States ; T32 GM119999/GM/NIGMS NIH HHS/United States ; T32 GM007092/GM/NIGMS NIH HHS/United States ; R01 GM121806/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Expression Regulation ; Genetic Engineering ; Humans ; Plasmids/*genetics ; Transposases/genetics/*metabolism ; },
abstract = {We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is cotransfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. We show that CRISPR-Bac can be used to knock down proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.},
}

Animals
CRISPR-Cas Systems
Gene Editing/*methods
Gene Expression Regulation
Genetic Engineering
Humans
Plasmids/*genetics
Transposases/genetics/*metabolism

@article {pmid31088860,
year = {2019},
author = {Lorenzini, PA and Chew, RSE and Tan, CW and Yong, JY and Zhang, F and Zheng, J and Roca, X},
title = {Human PRPF40B regulates hundreds of alternative splicing targets and represses a hypoxia expression signature.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {8},
pages = {905-920},
doi = {10.1261/rna.069534.118},
pmid = {31088860},
issn = {1469-9001},
mesh = {*Alternative Splicing ; CRISPR-Cas Systems ; Carrier Proteins/*genetics ; Cell Hypoxia ; Down-Regulation ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; *Gene Regulatory Networks ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics ; K562 Cells ; Kruppel-Like Transcription Factors/genetics ; Leukemia, Myeloid, Acute/*genetics ; Mutation ; Myelodysplastic Syndromes/*genetics ; Sequence Analysis, RNA/methods ; },
abstract = {Altered splicing contributes to the pathogenesis of human blood disorders including myelodysplastic syndromes (MDS) and leukemias. Here we characterize the transcriptomic regulation of PRPF40B, which is a splicing factor mutated in a small fraction of MDS patients. We generated a full PRPF40B knockout (KO) in the K562 cell line by CRISPR/Cas9 technology and rescued its levels by transient overexpression of wild-type (WT), P383L or P540S MDS alleles. Using RNA sequencing, we identified hundreds of differentially expressed genes and alternative splicing (AS) events in the KO that are rescued by WT PRPF40B, with a majority also rescued by MDS alleles, pointing to mild effects of these mutations. Among the PRPF40B-regulated AS events, we found a net increase in exon inclusion in the KO, suggesting that this splicing factor primarily acts as a repressor. PRPF40B-regulated splicing events are likely cotranscriptional, affecting exons with A-rich downstream intronic motifs and weak splice sites especially for 5' splice sites, consistent with its PRP40 yeast ortholog being part of the U1 small nuclear ribonucleoprotein. Loss of PRPF40B in K562 induces a KLF1 transcriptional signature, with genes involved in iron metabolism and mainly hypoxia, including related pathways like cholesterol biosynthesis and Akt/MAPK signaling. A cancer database analysis revealed that PRPF40B is lowly expressed in acute myeloid leukemia, whereas its paralog PRPF40A expression is high as opposed to solid tumors. Furthermore, these factors negatively or positively correlated with hypoxia regulator HIF1A, respectively. Our data suggest a PRPF40B role in repressing hypoxia in myeloid cells, and that its low expression might contribute to leukemogenesis.},
}

*Alternative Splicing
CRISPR-Cas Systems
Carrier Proteins/*genetics
Cell Hypoxia
Down-Regulation
Gene Expression Profiling/*methods
Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
*Gene Regulatory Networks
Humans
Hypoxia-Inducible Factor 1, alpha Subunit/genetics
K562 Cells
Kruppel-Like Transcription Factors/genetics
Leukemia, Myeloid, Acute/*genetics
Mutation
Myelodysplastic Syndromes/*genetics
Sequence Analysis, RNA/methods

@article {pmid31043511,
year = {2019},
author = {Lavalou, P and Eckert, H and Damy, L and Constanty, F and Majello, S and Bitetti, A and Graindorge, A and Shkumatava, A},
title = {Strategies for genetic inactivation of long noncoding RNAs in zebrafish.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {8},
pages = {897-904},
doi = {10.1261/rna.069484.118},
pmid = {31043511},
issn = {1469-9001},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Expression Regulation ; Gene Knock-In Techniques ; *Gene Silencing ; Organ Specificity ; RNA, Long Noncoding/*genetics ; Sequence Deletion ; Transcription Initiation Site ; Zebrafish/*genetics ; },
abstract = {The number of annotated long noncoding RNAs (lncRNAs) continues to grow; however, their functional characterization in model organisms has been hampered by the lack of reliable genetic inactivation strategies. While partial or full deletions of lncRNA loci disrupt lncRNA expression, they do not permit the formal association of a phenotype with the encoded transcript. Here, we examined several alternative strategies for generating lncRNA null alleles in zebrafish and found that they often resulted in unpredicted changes to lncRNA expression. Removal of the transcription start sites (TSSs) of lncRNA genes resulted in hypomorphic mutants, due to the usage of either constitutive or tissue-specific alternative TSSs. Deletions of short, highly conserved lncRNA regions can also lead to overexpression of truncated transcripts. In contrast, knock-in of a polyadenylation signal enabled complete inactivation of malat1, the most abundant vertebrate lncRNA. In summary, lncRNA null alleles require extensive in vivo validation, and we propose insertion of transcription termination sequences as the most reliable approach to generate lncRNA-deficient zebrafish.},
}

Animals
CRISPR-Cas Systems
Gene Expression Regulation
Gene Knock-In Techniques
*Gene Silencing
Organ Specificity
RNA, Long Noncoding/*genetics
Sequence Deletion
Transcription Initiation Site
Zebrafish/*genetics

@article {pmid30792150,
year = {2019},
author = {Bäck, S and Necarsulmer, J and Whitaker, LR and Coke, LM and Koivula, P and Heathward, EJ and Fortuno, LV and Zhang, Y and Yeh, CG and Baldwin, HA and Spencer, MD and Mejias-Aponte, CA and Pickel, J and Hoffman, AF and Spivak, CE and Lupica, CR and Underhill, SM and Amara, SG and Domanskyi, A and Anttila, JE and Airavaara, M and Hope, BT and Hamra, FK and Richie, CT and Harvey, BK},
title = {Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.},
journal = {Neuron},
volume = {102},
number = {1},
pages = {105-119.e8},
doi = {10.1016/j.neuron.2019.01.035},
pmid = {30792150},
issn = {1097-4199},
mesh = {Adult Germline Stem Cells/*metabolism ; Animals ; Brain/*metabolism ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Deoxyribonuclease I/genetics ; Dependovirus ; Disease Models, Animal ; Dopamine Plasma Membrane Transport Proteins/genetics ; Dopaminergic Neurons/*metabolism ; Gene Editing/*methods ; Gene Knock-In Techniques/methods ; Gene Knockout Techniques ; Gene Targeting/*methods ; Genetic Vectors ; Integrases ; Luminescent Proteins/genetics ; Neurons/metabolism ; Promoter Regions, Genetic ; RNA, Guide ; Rats ; Rats, Transgenic ; Tyrosine 3-Monooxygenase/genetics ; },
abstract = {Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.},
}

Adult Germline Stem Cells/*metabolism
Animals
Brain/*metabolism
CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
Deoxyribonuclease I/genetics
Dependovirus
Disease Models, Animal
Dopamine Plasma Membrane Transport Proteins/genetics
Dopaminergic Neurons/*metabolism
Gene Editing/*methods
Gene Knock-In Techniques/methods
Gene Knockout Techniques
Gene Targeting/*methods
Genetic Vectors
Integrases
Luminescent Proteins/genetics
Neurons/metabolism
Promoter Regions, Genetic
RNA, Guide
Rats
Rats, Transgenic
Tyrosine 3-Monooxygenase/genetics

@article {pmid30595385,
year = {2019},
author = {Islam, Z and Inui, T and Ishibashi, O},
title = {Gpr137b is an orphan G-protein-coupled receptor associated with M2 macrophage polarization.},
journal = {Biochemical and biophysical research communications},
volume = {509},
number = {3},
pages = {657-663},
doi = {10.1016/j.bbrc.2018.12.140},
pmid = {30595385},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Polarity ; Gene Expression ; Interleukin-4/*immunology ; Lipopolysaccharides/*immunology ; Macrophages/cytology/*immunology/metabolism ; Mice ; RAW 264.7 Cells ; },
abstract = {Macrophages are classified mainly into two subtypes, M1 and M2, which exhibit distinct phenotypes, based on their microenvironment. Although recent studies have suggested that G-protein-coupled receptors (GPCRs) are associated with M1/M2 macrophage polarization, available information on GPCR-mediated macrophage polarization is still limited. In the present study, we identified Gpr137b as an orphan GPCR abundantly expressed in RAW264, a mouse macrophage cell line, and illuminated its role in M2 macrophage polarization. We generated Gpr137b-knockout (Gpr137b-KO) clones of RAW264 cells using the CRISPR/Cas9 genome editing system. Two independent Gpr137b-KO clones were isolated, which were demonstrated to have frameshifting 188-nucleotide deletions at a region containing the ATG start codon of Gpr137b. Consistently, qRT-PCR analysis revealed that the deleted region is not transcribed. We then treated the Gpr137b-KO and wildtype RAW264 cells with interleukin-4 (IL-4) to induce M2 macrophage polarization. Microarray analysis revealed that the IL-4-induced gene expression of representative M2 macrophage markers was significantly reduced in the Gpr137b-KO cells, and this was validated by qRT-PCR analysis. By contrast, M1 macrophage marker gene expression induced by lipopolysaccharide was unaffected by Gpr137b-KO. Collectively, the current study shows that Gpr137b is a possible regulator of M2 macrophage polarization.},
}

Animals
CRISPR-Cas Systems
Cell Polarity
Gene Expression
Interleukin-4/*immunology
Lipopolysaccharides/*immunology
Macrophages/cytology/*immunology/metabolism
Mice
RAW 264.7 Cells

@article {pmid30391781,
year = {2019},
author = {Zhang, P and Kang, B and Xie, G and Li, S and Gu, Y and Shen, Y and Zhao, X and Ma, Y and Li, F and Si, J and Wang, J and Chen, J and Yang, H and Xu, X and Yang, Y},
title = {Genomic sequencing and editing revealed the GRM8 signaling pathway as potential therapeutic targets of squamous cell lung cancer.},
journal = {Cancer letters},
volume = {442},
number = {},
pages = {53-67},
doi = {10.1016/j.canlet.2018.10.035},
pmid = {30391781},
issn = {1872-7980},
mesh = {Animals ; Antineoplastic Agents/therapeutic use ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Carcinoma, Squamous Cell/drug therapy/*genetics/metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cyclic AMP/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/drug therapy/*genetics/metabolism/pathology ; Mice, Inbred NOD ; Mice, SCID ; Mitogen-Activated Protein Kinases/metabolism ; Molecular Targeted Therapy ; Mutation ; Receptors, Metabotropic Glutamate/*genetics/metabolism ; Signal Transduction ; Transcriptional Activation ; Tumor Burden ; Tumor Cells, Cultured ; *Whole Exome Sequencing ; },
abstract = {The study sought to explore novel genetic aberration driving squamous cell lung carcinoma (LUSC). The whole exome (WES), whole genome (WGS) and target region (TS) sequencings and CRISPR-Cas9 genome editing techniques were integrated to explore and validate novel targeting candidates from LUSC primary tumors and corresponding patient-derived xenografts (PDXs). Seven genes (FGFR2, GRM1,PIK3CG, PIK3CA,ZFHX4, CSMD3, GRM8) with high frequencies of both single nucleotide variants (SNVs) and copy number variants (CNVs), and two genes (CLDN1 and RIT1) only with CNVs were identified by bioinformatics analysis. The functions of these candidates were validated through CRISPR-Cas9 system in primary PDX cells. Furthermore, we focused on the genetic and functional analysis of Metabotropic glutamate receptor 8 (GRM8), whose transcriptional activation was elucidated to promote the survival of LUSC tumor cell through inhibiting cAMP pathway and activating MAPK pathway. The SNV identified in GRM8, A112G, activated downstream signaling pathway and induced cell proliferation, which could be reversed by cAMP stimulator and MEK inhibitor. In conclusion, the components of GRM8 signaling pathway could serve as potential targets of squamous cell lung cancer carrying GRM8 activating variants.},
}

Animals
Antineoplastic Agents/therapeutic use
CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems
Carcinoma, Squamous Cell/drug therapy/*genetics/metabolism/pathology
Cell Line, Tumor
Cell Proliferation
Cell Survival
Clustered Regularly Interspaced Short Palindromic Repeats
Cyclic AMP/metabolism
Gene Editing/*methods
Gene Expression Regulation, Neoplastic
Humans
Lung Neoplasms/drug therapy/*genetics/metabolism/pathology
Mice, Inbred NOD
Mice, SCID
Mitogen-Activated Protein Kinases/metabolism
Molecular Targeted Therapy
Mutation
Receptors, Metabotropic Glutamate/*genetics/metabolism
Signal Transduction
Transcriptional Activation
Tumor Burden
Tumor Cells, Cultured
*Whole Exome Sequencing

@article {pmid29696735,
year = {2018},
author = {Li, X and Wang, Z and Tong, H and Yan, Y and Li, S},
title = {Effects of COL8A1 on the proliferation of muscle-derived satellite cells.},
journal = {Cell biology international},
volume = {42},
number = {9},
pages = {1132-1140},
doi = {10.1002/cbin.10979},
pmid = {29696735},
issn = {1095-8355},
support = {2014ZX08007-002//High-quality new varieties of genetically modified bovines from the National Major Transgenic Project/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; Cell Proliferation/drug effects ; Cells, Cultured ; Chromones/pharmacology ; Collagen Type VIII/genetics/*metabolism ; Cyclin B1/metabolism ; Early Growth Response Protein 1/metabolism ; Gene Editing/methods ; Morpholines/pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Satellite Cells, Skeletal Muscle/cytology/drug effects/*metabolism ; Signal Transduction/drug effects ; Transcription Factors/metabolism ; },
abstract = {Collagen type VIII alpha 1 chain (COL8A1) is a component of the extracellular matrix. Our previous studies suggested that COL8A1 is associated with the proliferation of muscle-derived satellite cells (MDSCs). Additionally, it has been demonstrated that COL8A1 promotes the proliferation of smooth muscle cells and liver cancer cells. Therefore, we predicted that COL8A1 is associated with the proliferation of bovine MDSCs, which have potential applications in research. In this study, we constructed vectors to activate and repress COL8A1 in bovine MDSCs using the CRISPR/Cas9 technique and determined the effects of COL8A1 modulation by EdU labeling, Western blotting, and dual-luciferase reporter assays. The results showed that activation of COL8A1 increased the number of EdU-positive cells and expression of the proliferation markers cyclin B1 (CCNB1) and P-AKT. The expression of P-Akt was unchanged after addition of LY294002 (a protein kinase inhibitor capable of blocking the signal transduction pathway of the phosphoinositide 3-kinase). In contrast, repression of COL8A1 reduced the number of EdU-positive cells and expression of CCNB1 and P-AKT. We also observed upregulation and downregulation of COL8A1 following the overexpression and repression of EGR1, respectively. The dual-luciferase reporter assay revealed that EGR1 regulates the promoter activity of COL8A1. To our knowledge, this is the first study demonstrating that EGR1 positively regulates the expression of COL8A1, which in turn promotes the proliferation of bovine MDSCs via the PI3 K/AKT signaling pathway.},
}

Animals
CRISPR-Cas Systems
Cattle
Cell Proliferation/drug effects
Cells, Cultured
Chromones/pharmacology
Collagen Type VIII/genetics/*metabolism
Cyclin B1/metabolism
Early Growth Response Protein 1/metabolism
Gene Editing/methods
Morpholines/pharmacology
Phosphatidylinositol 3-Kinases/metabolism
Proto-Oncogene Proteins c-akt/metabolism
Satellite Cells, Skeletal Muscle/cytology/drug effects/*metabolism
Signal Transduction/drug effects
Transcription Factors/metabolism

@article {pmid31672965,
year = {2019},
author = {Jillette, N and Du, M and Zhu, JJ and Cardoz, P and Cheng, AW},
title = {Split selectable markers.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4968},
doi = {10.1038/s41467-019-12891-2},
pmid = {31672965},
issn = {2041-1723},
support = {1R01HG009900//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; P30CA034196//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
abstract = {Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a "chaining" design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.},
}

@article {pmid31671185,
year = {2019},
author = {Swarts, DC},
title = {Making the cut(s): how Cas12a cleaves target and non-target DNA.},
journal = {Biochemical Society transactions},
volume = {47},
number = {5},
pages = {1499-1510},
doi = {10.1042/BST20190564},
pmid = {31671185},
issn = {1470-8752},
abstract = {CRISPR-Cas12a (previously named Cpf1) is a prokaryotic deoxyribonuclease that can be programmed with an RNA guide to target complementary DNA sequences. Upon binding of the target DNA, Cas12a induces a nick in each of the target DNA strands, yielding a double-stranded DNA break. In addition to inducing cis-cleavage of the targeted DNA, target DNA binding induces trans-cleavage of non-target DNA. As such, Cas12a-RNA guide complexes can provide sequence-specific immunity against invading nucleic acids such as bacteriophages and plasmids. Akin to CRISPR-Cas9, Cas12a has been repurposed as a genetic tool for programmable genome editing and transcriptional control in both prokaryotic and eukaryotic cells. In addition, its trans-cleavage activity has been applied for high-sensitivity nucleic acid detection. Despite the demonstrated value of Cas12a for these applications, the exact molecular mechanisms of both cis- and trans-cleavage of DNA were not completely understood. Recent studies have revealed mechanistic details of Cas12a-mediates DNA cleavage: base pairing of the RNA guide and the target DNA induces major conformational changes in Cas12a. These conformational changes render Cas12a in a catalytically activated state in which it acts as deoxyribonuclease. This deoxyribonuclease activity mediates cis-cleavage of the displaced target DNA strand first, and the RNA guide-bound target DNA strand second. As Cas12a remains in the catalytically activated state after cis-cleavage, it subsequently demonstrates trans-cleavage of non-target DNA. Here, I review the mechanistic details of Cas12a-mediated cis- and trans-cleavage of DNA. In addition, I discuss how bacteriophage-derived anti-CRISPR proteins can inhibit Cas12a activity.},
}

@article {pmid31666940,
year = {2019},
author = {Yang, G and Huang, X},
title = {Methods and applications of CRISPR/Cas system for genome editing in stem cells.},
journal = {Cell regeneration (London, England)},
volume = {8},
number = {2},
pages = {33-41},
doi = {10.1016/j.cr.2019.08.001},
pmid = {31666940},
issn = {2045-9769},
abstract = {Genome editing technology holds great promise for genome manipulation and gene therapy. While widespread utilization, genome editing has been used to unravel the roles of specific genes in differentiation and pluripotency of stem cells, and reinforce the stem cell-based applications. In this review, we summarize the advances of genome editing technology, as well as the derivative technologies from CRISPR/Cas system, which show tremendous potential in various fields. We also highlight the key findings in the studies of stem cells and regeneration by genome editing technology.},
}

@article {pmid31603849,
year = {2019},
author = {Ortez, C and Natera de Benito, D and Carrera García, L and Expósito, J and Nolasco, G and Nascimento, A},
title = {[Advances in the treatment of Duchenne muscular dystrophy].},
journal = {Medicina},
volume = {79 Suppl 3},
number = {},
pages = {77-81},
pmid = {31603849},
issn = {1669-9106},
mesh = {Animals ; CRISPR-Cas Systems ; Dystrophin/genetics ; Genetic Therapy/*methods ; Genotype ; Humans ; Mice ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne/genetics/*therapy ; Phenotype ; },
abstract = {Duchenne muscular dystrophy is a genetically determined disease, linked to the X chromosome, c haracterized clinically by producing progressive muscle weakness, with an incidence of 1 per 3500-6000 males born. It is caused by the mutation of the DMD gene, which encodes dystrophin, a sub-sarcolemmal protein essential for structural muscle stability. The genetic defects in the DMD gene are divided into: deletions (65%) duplications (5.10%) and point mutations (10-15%). At present there is no curative treatment, the only drug that has been shown to modify the natural history of the disease (independently of the genetic mutation) are corticosteroids, currently indicated in early stages of the disease. In relation to clinical trials, in the last ten years, has experienced great advances in the field of therapeutic options, divided into two major therapeutic targets: 1) the area of gene therapies and 2) trying to reverse or block the pathophysiological processes of the disease, such as inflammation, fibrosis, muscle regeneration, etc. It is likely that an effective treatment for Duchenne muscular dystrophy requires combinations of therapies that address both the primary defect and its secondary pathophysiological consequences.},
}

Animals
CRISPR-Cas Systems
Dystrophin/genetics
Genetic Therapy/*methods
Genotype
Humans
Mice
Mice, Inbred mdx
Muscular Dystrophy, Duchenne/genetics/*therapy
Phenotype

@article {pmid31048459,
year = {2019},
author = {Chung, HK and Zou, X and Bajar, BT and Brand, VR and Huo, Y and Alcudia, JF and Ferrell, JE and Lin, MZ},
title = {A compact synthetic pathway rewires cancer signaling to therapeutic effector release.},
journal = {Science (New York, N.Y.)},
volume = {364},
number = {6439},
pages = {},
doi = {10.1126/science.aat6982},
pmid = {31048459},
issn = {1095-9203},
support = {R01 GM098734/GM/NIGMS NIH HHS/United States ; P50 GM107615/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenoviridae ; Apoptosis/*genetics ; Bioengineering/*methods ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line, Tumor ; Endopeptidases/genetics ; Humans ; Models, Theoretical ; Neoplasms/*genetics/pathology/*therapy ; Protein Stability ; Proteolysis ; Receptor, ErbB-2/*antagonists & inhibitors/*genetics/metabolism ; Signal Transduction ; Synthetic Biology ; Transcription, Genetic ; Viral Nonstructural Proteins/genetics ; },
abstract = {An important goal in synthetic biology is to engineer biochemical pathways to address unsolved biomedical problems. One long-standing problem in molecular medicine is the specific identification and ablation of cancer cells. Here, we describe a method, named Rewiring of Aberrant Signaling to Effector Release (RASER), in which oncogenic ErbB receptor activity, instead of being targeted for inhibition as in existing treatments, is co-opted to trigger therapeutic programs. RASER integrates ErbB activity to specifically link oncogenic states to the execution of desired outputs. A complete mathematical model of RASER and modularity in design enable rational optimization and output programming. Using RASER, we induced apoptosis and CRISPR-Cas9-mediated transcription of endogenous genes specifically in ErbB-hyperactive cancer cells. Delivery of apoptotic RASER by adeno-associated virus selectively ablated ErbB-hyperactive cancer cells while sparing ErbB-normal cells. RASER thus provides a new strategy for oncogene-specific cancer detection and treatment.},
}

Adenoviridae
Apoptosis/*genetics
Bioengineering/*methods
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Cell Line, Tumor
Endopeptidases/genetics
Humans
Models, Theoretical
Neoplasms/*genetics/pathology/*therapy
Protein Stability
Proteolysis
Receptor, ErbB-2/*antagonists & inhibitors/*genetics/metabolism
Signal Transduction
Synthetic Biology
Transcription, Genetic
Viral Nonstructural Proteins/genetics

@article {pmid30944478,
year = {2019},
author = {Pluvinage, JV and Haney, MS and Smith, BAH and Sun, J and Iram, T and Bonanno, L and Li, L and Lee, DP and Morgens, DW and Yang, AC and Shuken, SR and Gate, D and Scott, M and Khatri, P and Luo, J and Bertozzi, CR and Bassik, MC and Wyss-Coray, T},
title = {CD22 blockade restores homeostatic microglial phagocytosis in ageing brains.},
journal = {Nature},
volume = {568},
number = {7751},
pages = {187-192},
doi = {10.1038/s41586-019-1088-4},
pmid = {30944478},
issn = {1476-4687},
support = {R01 GM059907/GM/NIGMS NIH HHS/United States ; T32 GM007365/GM/NIGMS NIH HHS/United States ; R01 AG045034/AG/NIA NIH HHS/United States ; S10 OD020141/OD/NIH HHS/United States ; DP1 AG053015/AG/NIA NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; F30 AG060638/AG/NIA NIH HHS/United States ; },
mesh = {Aging/drug effects/genetics/*physiology ; Animals ; Brain/*cytology/drug effects/physiology ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cognition/drug effects/physiology ; Female ; Homeostasis/*drug effects/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Microglia/cytology/*drug effects ; N-Acetylneuraminic Acid/chemistry/*pharmacology ; Phagocytosis/*drug effects/genetics ; Sequence Analysis, RNA ; Sialic Acid Binding Ig-like Lectin 2/*antagonists & inhibitors/genetics/metabolism ; },
abstract = {Microglia maintain homeostasis in the central nervous system through phagocytic clearance of protein aggregates and cellular debris. This function deteriorates during ageing and neurodegenerative disease, concomitant with cognitive decline. However, the mechanisms of impaired microglial homeostatic function and the cognitive effects of restoring this function remain unknown. We combined CRISPR-Cas9 knockout screens with RNA sequencing analysis to discover age-related genetic modifiers of microglial phagocytosis. These screens identified CD22, a canonical B cell receptor, as a negative regulator of phagocytosis that is upregulated on aged microglia. CD22 mediates the anti-phagocytic effect of α2,6-linked sialic acid, and inhibition of CD22 promotes the clearance of myelin debris, amyloid-β oligomers and α-synuclein fibrils in vivo. Long-term central nervous system delivery of an antibody that blocks CD22 function reprograms microglia towards a homeostatic transcriptional state and improves cognitive function in aged mice. These findings elucidate a mechanism of age-related microglial impairment and a strategy to restore homeostasis in the ageing brain.},
}

Aging/drug effects/genetics/*physiology
Animals
Brain/*cytology/drug effects/physiology
CRISPR-Associated Protein 9/metabolism
CRISPR-Cas Systems/genetics
Cognition/drug effects/physiology
Female
Homeostasis/*drug effects/genetics
Male
Mice
Mice, Inbred C57BL
Microglia/cytology/*drug effects
N-Acetylneuraminic Acid/chemistry/*pharmacology
Phagocytosis/*drug effects/genetics
Sequence Analysis, RNA
Sialic Acid Binding Ig-like Lectin 2/*antagonists & inhibitors/genetics/metabolism

@article {pmid30667081,
year = {2019},
author = {Tang, F and Min, L and Seebacher, NA and Li, X and Zhou, Y and Hornicek, FJ and Wei, Y and Tu, C and Duan, Z},
title = {Targeting mutant TP53 as a potential therapeutic strategy for the treatment of osteosarcoma.},
journal = {Journal of orthopaedic research : official publication of the Orthopaedic Research Society},
volume = {37},
number = {3},
pages = {789-798},
doi = {10.1002/jor.24227},
pmid = {30667081},
issn = {1554-527X},
support = {U01 CA151452/CA/NCI NIH HHS/United States ; },
mesh = {Antibiotics, Antineoplastic/therapeutic use ; Bone Neoplasms/*genetics/therapy ; CRISPR-Cas Systems ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Doxorubicin/therapeutic use ; Drug Resistance, Neoplasm ; Drug Synergism ; *Genes, p53 ; *Genetic Therapy ; Humans ; Mutation ; Nitrofurans/pharmacology/*therapeutic use ; Osteosarcoma/*genetics/therapy ; Piperazines/pharmacology/*therapeutic use ; Tumor Suppressor Protein p53/antagonists & inhibitors/genetics ; },
abstract = {Mutant TP53 is a promising therapeutic target in cancers. Considering the current challenges facing the clinical treatment of cancer, as well as the urgent need to identify novel therapeutic targets in osteosarcomas, we aimed to evaluate the clinical significance of mutant TP53 in osteosarcoma patients and to explore the therapeutic effect of targeting mutant TP53 in osteosarcomas. We performed a meta-analysis to investigate the relationship between mutant TP53 and the overall survival of patients with osteosarcoma. A CRISPR-Cas9 system and a TP53 inhibitor, NSC59984, were also used to specifically knock-out and inhibit mutant TP53 in the human osteosarcoma cell lines, KHOS, and KHOSR2. The meta-analysis demonstrated that mutations in the TP53 gene could be used to predict a poor 2-year survival in osteosarcoma patients. We also demonstrated that the expression of mutant TP53 in human osteosarcoma cell lines can be efficiently knocked-out using CRISPR-Cas9, and this decreased the proliferation, migration, and tumor formation activity of these osteosarcoma cells. Moreover, drug sensitivity to doxorubicin was increased in these TP53 knock-out osteosarcoma cells. NSC59984 also showed similar anti-tumor effects as CRISPR-Cas9 targeted TP53 in the osteosarcoma cells in vitro. We have also demonstrated that the knock-out or inhibition of mutant TP53 decreased the expression of the oncogene IGF-1R, anti-apoptotic proteins Bcl-2, and Survivin in osteosarcoma cells. Collectively, these results suggest that mutant TP53 is a promising therapeutic target in osteosarcomas. Therefore, further studies exploring novel strategies to target mutant TP53 may help improve the treatment outcomes of osteosarcoma patients in the clinic. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.},
}

Antibiotics, Antineoplastic/therapeutic use
Bone Neoplasms/*genetics/therapy
CRISPR-Cas Systems
Cell Movement/drug effects
Cell Proliferation/drug effects
Doxorubicin/therapeutic use
Drug Resistance, Neoplasm
Drug Synergism
*Genes, p53
*Genetic Therapy
Humans
Mutation
Nitrofurans/pharmacology/*therapeutic use
Osteosarcoma/*genetics/therapy
Piperazines/pharmacology/*therapeutic use
Tumor Suppressor Protein p53/antagonists & inhibitors/genetics

@article {pmid30451898,
year = {2018},
author = {Santofimia-Castaño, P and Lan, W and Bintz, J and Gayet, O and Carrier, A and Lomberk, G and Neira, JL and González, A and Urrutia, R and Soubeyran, P and Iovanna, J},
title = {Inactivation of NUPR1 promotes cell death by coupling ER-stress responses with necrosis.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {16999},
pmid = {30451898},
issn = {2045-2322},
mesh = {Acinar Cells ; Adenosine Triphosphate/metabolism ; Animals ; Apoptosis ; Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors/genetics/metabolism ; CRISPR-Cas Systems ; *Cell Death ; Cells, Cultured ; DNA-Binding Proteins/*physiology ; *Endoplasmic Reticulum Stress ; Humans ; Mice ; Mice, Knockout ; Mitochondria/metabolism/*pathology ; Mitochondrial Degradation ; *Necrosis ; Neoplasm Proteins/antagonists & inhibitors/genetics/metabolism/*physiology ; Oxidative Phosphorylation ; Pancreas/metabolism/pathology ; Pancreatic Neoplasms/metabolism/*pathology ; Pancreatitis/metabolism/*pathology ; },
abstract = {It was already described that genetic inhibition of NUPR1 induces tumor growth arrest. In this paper we studied the metabolism changes after NUPR1 downregulation in pancreatic cancer cells, which results in a significant decrease of OXPHOS activity with a concomitant lower ATP production which precedes the necrotic cell death. We demonstrated that NUPR1 downregulation induces a mitochondrial failure with a loss of the mitochondrial membrane potential, a strong increase in ROS production and a concomitant relocalization of mitochondria to the vicinity of the endoplasmic reticulum (ER). In addition, the transcriptomic analysis of NUPR1-deficient cells shows a decrease in the expression of some ER stress response-associated genes. Indeed, in ER stressors-treated cells with thapsigargin, brefeldin A or tunicamycin, a greater increase in necrosis and decrease of ATP content was observed in NUPR1-defficent cells. Finally, in vivo experiments, using acute pancreatitis which induces ER stress as well as NUPR1 activation, we observed that NUPR1 expression protects acinar cells from necrosis in mice. Importantly, we also report that the cell death observed after knocking-down NUPR1 expression is completely reversed by incubation with Necrostatin-1, but not by inhibiting caspase activity with Z-VAD-FMK. Altogether, these data enable us to describe a model in which inactivation of NUPR1 in pancreatic cancer cells results in an ER stress that induces a mitochondrial malfunction, a deficient ATP production and, as consequence, the cell death mediated by a programmed necrosis.},
}

Acinar Cells
Adenosine Triphosphate/metabolism
Animals
Apoptosis
Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors/genetics/metabolism
CRISPR-Cas Systems
*Cell Death
Cells, Cultured
DNA-Binding Proteins/*physiology
*Endoplasmic Reticulum Stress
Humans
Mice
Mice, Knockout
Mitochondria/metabolism/*pathology
Mitochondrial Degradation
*Necrosis
Neoplasm Proteins/antagonists & inhibitors/genetics/metabolism/*physiology
Oxidative Phosphorylation
Pancreas/metabolism/pathology
Pancreatic Neoplasms/metabolism/*pathology
Pancreatitis/metabolism/*pathology

@article {pmid30254056,
year = {2018},
author = {Luttrell, LM and Wang, J and Plouffe, B and Smith, JS and Yamani, L and Kaur, S and Jean-Charles, PY and Gauthier, C and Lee, MH and Pani, B and Kim, J and Ahn, S and Rajagopal, S and Reiter, E and Bouvier, M and Shenoy, SK and Laporte, SA and Rockman, HA and Lefkowitz, RJ},
title = {Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9.},
journal = {Science signaling},
volume = {11},
number = {549},
pages = {},
doi = {10.1126/scisignal.aat7650},
pmid = {30254056},
issn = {1937-9145},
support = {R01 HL118369/HL/NHLBI NIH HHS/United States ; I01 BX003188/BX/BLRD VA/United States ; R01 DK055524/DK/NIDDK NIH HHS/United States ; R01 HL016037/HL/NHLBI NIH HHS/United States ; R35 GM126955/GM/NIGMS NIH HHS/United States ; K08 HL114643/HL/NHLBI NIH HHS/United States ; P01 HL075443/HL/NHLBI NIH HHS/United States ; R01 HL056687/HL/NHLBI NIH HHS/United States ; R01 GM122798/GM/NIGMS NIH HHS/United States ; 14843//CIHR/Canada ; MOP-74603//CIHR/Canada ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Membrane/metabolism ; Enzyme Activation ; Gene Deletion ; Gene Editing ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; MAP Kinase Signaling System ; Phosphorylation ; RNA, Small Interfering/*metabolism ; Receptors, Adrenergic, beta-2/metabolism ; Receptors, G-Protein-Coupled/*metabolism ; *Signal Transduction ; beta-Arrestins/*metabolism ; },
abstract = {G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β2 adrenergic receptors (β2ARs) and β1ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein-deleted cells to GPCR behavior in native systems.},
}

*CRISPR-Cas Systems
Cell Membrane/metabolism
Enzyme Activation
Gene Deletion
Gene Editing
Gene Knockdown Techniques
HEK293 Cells
Humans
MAP Kinase Signaling System
Phosphorylation
RNA, Small Interfering/*metabolism
Receptors, Adrenergic, beta-2/metabolism
Receptors, G-Protein-Coupled/*metabolism
*Signal Transduction
beta-Arrestins/*metabolism

@article {pmid29851281,
year = {2018},
author = {Jia, YL and Guo, X and Lu, JT and Wang, XY and Qiu, LL and Wang, TY},
title = {CRISPR/Cas9-mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long-term stability.},
journal = {Journal of cellular and molecular medicine},
volume = {22},
number = {9},
pages = {4106-4116},
pmid = {29851281},
issn = {1582-4934},
mesh = {Animals ; Base Sequence ; CHO Cells ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; CpG Islands ; Cricetulus ; Cytomegalovirus/genetics/metabolism ; DNA (Cytosine-5-)-Methyltransferases/deficiency/*genetics ; DNA Methylation ; Gene Editing/*methods ; Gene Expression ; Gene Knockout Techniques ; Promoter Regions, Genetic ; RNA, Guide/*genetics/metabolism ; *Transgenes ; },
abstract = {CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a-deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a-deficent CHO cell line based on Dnmt3a KO displayed an enhanced long-term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a-deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a-deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.},
}

Animals
Base Sequence
CHO Cells
CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
CpG Islands
Cricetulus
Cytomegalovirus/genetics/metabolism
DNA (Cytosine-5-)-Methyltransferases/deficiency/*genetics
DNA Methylation
Gene Editing/*methods
Gene Expression
Gene Knockout Techniques
Promoter Regions, Genetic
RNA, Guide/*genetics/metabolism
*Transgenes

@article {pmid29851245,
year = {2018},
author = {Tran, MH and Seo, E and Min, S and Nguyen, QT and Choi, J and Lee, UJ and Hong, SS and Kang, H and Mansukhani, A and Jou, I and Lee, SY},
title = {NEDD4-induced degradative ubiquitination of phosphatidylinositol 4-phosphate 5-kinase α and its implication in breast cancer cell proliferation.},
journal = {Journal of cellular and molecular medicine},
volume = {22},
number = {9},
pages = {4117-4129},
pmid = {29851245},
issn = {1582-4934},
mesh = {Breast Neoplasms/genetics/metabolism/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Membrane/chemistry/drug effects/*metabolism ; Cell Proliferation ; Epidermal Growth Factor/pharmacology ; Female ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Humans ; Mutation ; Nedd4 Ubiquitin Protein Ligases/*genetics/metabolism ; Phosphatidylinositol 3-Kinases/genetics/metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Phosphorylation/drug effects ; Phosphotransferases (Alcohol Group Acceptor)/*genetics/metabolism ; Proteasome Endopeptidase Complex/metabolism ; Proteolysis ; Proto-Oncogene Proteins c-akt/genetics/*metabolism ; Signal Transduction ; Ubiquitination/drug effects ; },
abstract = {Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family members generate phosphatidylinositol 4,5-bisphosphate (PIP2), a critical lipid regulator of diverse physiological processes. The PIP5K-dependent PIP2 generation can also act upstream of the oncogenic phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Many studies have demonstrated various mechanisms of spatiotemporal regulation of PIP5K catalytic activity. However, there are few studies on regulation of PIP5K protein stability. Here, we examined potential regulation of PIP5Kα, a PIP5K isoform, via ubiquitin-proteasome system, and its implication for breast cancer. Our results showed that the ubiquitin ligase NEDD4 (neural precursor cell expressed, developmentally down-regulated gene 4) mediated ubiquitination and proteasomal degradation of PIP5Kα, consequently reducing plasma membrane PIP2 level. NEDD4 interacted with the C-terminal region and ubiquitinated the N-terminal lysine 88 in PIP5Kα. In addition, PIP5Kα gene disruption inhibited epidermal growth factor (EGF)-induced Akt activation and caused significant proliferation defect in breast cancer cells. Notably, PIP5Kα K88R mutant that was resistant to NEDD4-mediated ubiquitination and degradation showed more potentiating effects on Akt activation by EGF and cell proliferation than wild-type PIP5Kα. Collectively, these results suggest that PIP5Kα is a novel degradative substrate of NEDD4 and that the PIP5Kα-dependent PIP2 pool contributing to breast cancer cell proliferation through PI3K/Akt activation is negatively controlled by NEDD4.},
}

Breast Neoplasms/genetics/metabolism/pathology
CRISPR-Cas Systems
Cell Line, Tumor
Cell Membrane/chemistry/drug effects/*metabolism
Cell Proliferation
Epidermal Growth Factor/pharmacology
Female
Gene Editing
*Gene Expression Regulation, Neoplastic
Humans
Mutation
Nedd4 Ubiquitin Protein Ligases/*genetics/metabolism
Phosphatidylinositol 3-Kinases/genetics/metabolism
Phosphatidylinositol 4,5-Diphosphate/metabolism
Phosphorylation/drug effects
Phosphotransferases (Alcohol Group Acceptor)/*genetics/metabolism
Proteasome Endopeptidase Complex/metabolism
Proteolysis
Proto-Oncogene Proteins c-akt/genetics/*metabolism
Signal Transduction
Ubiquitination/drug effects

@article {pmid31665284,
year = {2019},
author = {Deaner, M and Alper, HS},
title = {Enhanced scale and scope of genome engineering and regulation using CRISPR/Cas in Saccharomyces cerevisiae.},
journal = {FEMS yeast research},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsyr/foz076},
pmid = {31665284},
issn = {1567-1364},
abstract = {Although only 6 years old, the CRISPR system has blossomed into a tool for rapid, on-demand genome engineering and gene regulation in Saccharomyces cerevisiae. In this mini-review, we discuss fundamental CRISPR technologies, tools to improve the efficiency and capabilities of gene targeting, and cutting edge techniques to explore gene editing and transcriptional regulation at genome-scale using pooled approaches. The focus is applications to metabolic engineering with topics including development of techniques to edit the genome in multiplex, tools to enable large numbers of genetic modifications using pooled sgRNA libraries, and efforts to enable programmable transcriptional regulation using endonuclease-null Cas enzymes.},
}

@article {pmid31663848,
year = {2019},
author = {Li, W and Zhang, Y and Han, B and Li, L and Li, M and Lu, X and Chen, C and Lu, M and Zhang, Y and Jia, X and Zhu, Z and Tong, X and Zhang, B},
title = {One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
doi = {10.7554/eLife.48081},
pmid = {31663848},
issn = {2050-084X},
support = {2018YFA0801000//National Key Research and Development Program of China/ ; 2016YFA0100500//National Key Research and Development Program of China/ ; 2015CB942803//National Key Basic Research Program of China/ ; 31671500//National Natural Science Foundation of China/ ; 31871458//National Natural Science Foundation of China/ ; 81371264//National Natural Science Foundation of China/ ; },
abstract = {CRISPR/Cas systems are widely used to knockout genes by inducing indel mutations, which are prone to genetic compensation. Complex genome modifications such as knockin (KI) might bypass compensation, though difficult to practice due to low efficiency. Moreover, no 'two-in-one' KI strategy combining conditional knockout (CKO) with fluorescent gene-labeling or further allele-labeling has been reported. Here, we developed a dual-cassette-donor strategy and achieved one-step and efficient generation of dual-function KI alleles at tbx5a and kctd10 loci in zebrafish via targeted insertion. These alleles display fluorescent gene-tagging and CKO effects before and after Cre induction, respectively. By introducing a second fluorescent reporter, geno-tagging effects were achieved at tbx5a and sox10 loci, exhibiting CKO coupled with fluorescent reporter switch upon Cre induction, enabling tracing of three distinct genotypes. We found that LiCl purification of gRNA is critical for highly efficient KI, and preselection of founders allows the efficient germline recovery of KI events.},
}

@article {pmid31471004,
year = {2019},
author = {The Lancet Haematology, },
title = {CRISPR-Cas9 gene editing for patients with haemoglobinopathies.},
journal = {The Lancet. Haematology},
volume = {6},
number = {9},
pages = {e438},
doi = {10.1016/S2352-3026(19)30169-3},
pmid = {31471004},
issn = {2352-3026},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Carrier Proteins/genetics ; Gene Editing ; Hemoglobinopathies/genetics/*pathology ; Humans ; Mice ; Nuclear Proteins/genetics ; },
}

Animals
CRISPR-Cas Systems/*genetics
Carrier Proteins/genetics
Gene Editing
Hemoglobinopathies/genetics/*pathology
Humans
Mice
Nuclear Proteins/genetics

@article {pmid31159699,
year = {2018},
author = {Feeney, O and Cockbain, J and Morrison, M and Diependaele, L and Van Assche, K and Sterckx, S},
title = {Patenting Foundational Technologies: Lessons From CRISPR and Other Core Biotechnologies.},
journal = {The American journal of bioethics : AJOB},
volume = {18},
number = {12},
pages = {36-48},
doi = {10.1080/15265161.2018.1531160},
pmid = {31159699},
issn = {1536-0075},
mesh = {Biotechnology/economics/ethics/*legislation & jurisprudence ; *CRISPR-Cas Systems ; Gene Editing ; Genetic Research/legislation & jurisprudence ; Genetics, Medical/economics/ethics/*legislation & jurisprudence ; Health Policy/legislation & jurisprudence ; Humans ; Licensure/legislation & jurisprudence ; Patents as Topic/ethics/*legislation & jurisprudence ; Social Justice ; },
abstract = {In 2012, a new and promising gene manipulation technique, CRISPR-Cas9, was announced that seems likely to be a foundational technique in health care and agriculture. However, patents have been granted. As with other technological developments, there are concerns of social justice regarding inequalities in access. Given the technologies' "foundational" nature and societal impact, it is vital for such concerns to be translated into workable recommendations for policymakers and legislators. Colin Farrelly has proposed a moral justification for the use of patents to speed up the arrival of technology by encouraging innovation and investment. While sympathetic to his argument, this article highlights a number of problems. By examining the role of patents in CRISPR and in two previous foundational technologies, we make some recommendations for realistic and workable guidelines for patenting and licensing.},
}

Biotechnology/economics/ethics/*legislation & jurisprudence
*CRISPR-Cas Systems
Gene Editing
Genetic Research/legislation & jurisprudence
Genetics, Medical/economics/ethics/*legislation & jurisprudence
Health Policy/legislation & jurisprudence
Humans
Licensure/legislation & jurisprudence
Patents as Topic/ethics/*legislation & jurisprudence
Social Justice

@article {pmid31010727,
year = {2019},
author = {Easmin, F and Hassan, N and Sasano, Y and Ekino, K and Taguchi, H and Harashima, S},
title = {gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing.},
journal = {Journal of bioscience and bioengineering},
volume = {128},
number = {3},
pages = {373-378},
doi = {10.1016/j.jbiosc.2019.02.009},
pmid = {31010727},
issn = {1347-4421},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; *Gene Transfer Techniques ; Genetic Engineering/methods ; Genome, Fungal ; Organisms, Genetically Modified ; Plasmids ; Polymerase Chain Reaction/methods ; Promoter Regions, Genetic ; RNA, Guide/*genetics ; Saccharomyces cerevisiae/genetics/metabolism ; Transformation, Genetic ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is one of the most powerful tools for genome engineering. However, some of the steps are laborious, reducing its usability. In this study, we have developed a simplified method, called the guide RNA-transient expression system (gRNA-TES), to deliver gRNA in yeast. In gRNA-TES, a DNA fragment containing the promoter and gRNA is prepared by two simple PCR steps and co-transformed with a DNA module into the host strain; all steps including PCR steps and yeast transformation are completed within 5-6 h in a single day, in contrast to conventional plasmid-based gRNA delivery systems, which require at least 3-4 days to construct and verify the gRNA-expressing plasmids. The performance of gRNA-TES was evaluated by the replacement of 150-kb, 200-kb, 300-kb, 400-kb, and 500-kb regions of yeast chromosome 4 with a DNA module. Increased numbers of transformants with a high frequency of expected replacement of even the 500-kb region were obtained with gRNA-TES as compared with transformation without gRNA-TES. In addition, the integrity of the replaced region was verified in 67%-100% of transformants tested by colony PCR. We believe that gRNA-TES will vastly increase the accessibility of CRISPR/Cas9 technology to biologists and biotechnologists by offering a simple, fast, and cost-effective tool to deliver gRNA in genome engineering. Furthermore, it might be applied to plant and animal systems if appropriate gene promoters are incorporated in the technology.},
}

Animals
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Editing/*methods
Gene Expression Regulation, Fungal
*Gene Transfer Techniques
Genetic Engineering/methods
Genome, Fungal
Organisms, Genetically Modified
Plasmids
Polymerase Chain Reaction/methods
Promoter Regions, Genetic
RNA, Guide/*genetics
Saccharomyces cerevisiae/genetics/metabolism
Transformation, Genetic

@article {pmid30967334,
year = {2019},
author = {Zhu, M and Sun, L and Lu, X and Zong, H and Zhuge, B},
title = {Establishment of a transient CRISPR-Cas9 genome editing system in Candida glycerinogenes for co-production of ethanol and xylonic acid.},
journal = {Journal of bioscience and bioengineering},
volume = {128},
number = {3},
pages = {283-289},
doi = {10.1016/j.jbiosc.2019.03.009},
pmid = {30967334},
issn = {1347-4421},
mesh = {Aldehyde Reductase/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Candida/*genetics/metabolism ; Cloning, Molecular/methods ; Ethanol/*metabolism ; Gene Editing/*methods ; Gene Knockout Techniques ; Glucose/metabolism ; Metabolic Engineering/*methods ; Organisms, Genetically Modified ; Xylose/*analogs & derivatives/metabolism ; },
abstract = {Candida glycerinogenes, an industrial yeast with excellent multi-stress tolerance, has been applied to glycerol production for decades. However, its genetic manipulation was limited by the absence of meiosis, the diploid genome, and the lack of molecular tools. We described here the implementation of a transient CRISPR-Cas9 system for efficient genome editing in C. glycerinogenes. By targeting the counterselectable marker genes (TRP1, URA3), single and double gene knock-outs were achieved and the auxotroph obtained can be used as a background for targeting other gene (HOG1) at a mutation efficiency of 80%. Further, a xylonic acid producing C. glycerinogenes strain was constructed by knock-in of the xylose dehydrogenase gene, which produced up to 28 g/L ethanol and 9 g/L xylonic acid simultaneously from simulated lignocellulosic hydrolysate (contained 70 g/L glucose and 24 g/L xylose). These results indicated that the CRSIPR-Cas9 system developed here can facilitate the study of gene functions and metabolic pathways in C. glycerinogenes.},
}

Aldehyde Reductase/genetics/metabolism
CRISPR-Cas Systems/*genetics
Candida/*genetics/metabolism
Cloning, Molecular/methods
Ethanol/*metabolism
Gene Editing/*methods
Gene Knockout Techniques
Glucose/metabolism
Metabolic Engineering/*methods
Organisms, Genetically Modified
Xylose/*analogs & derivatives/metabolism

@article {pmid30301924,
year = {2018},
author = {Leidy-Davis, T and Cheng, K and Goodwin, LO and Morgan, JL and Juan, WC and Roca, X and Ong, ST and Bergstrom, DE},
title = {Viable Mice with Extensive Gene Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {15028},
pmid = {30301924},
issn = {2045-2322},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Bcl-2-Like Protein 11/*genetics ; Blastocyst/cytology/*metabolism ; CRISPR-Cas Systems ; Embryonic Stem Cells/cytology/*metabolism ; Gene Editing ; Humans ; Introns/genetics ; Mice ; Microinjections ; RNA, Guide/genetics ; Zygote/growth & development/*metabolism ; },
abstract = {Here, we describe an expansion of the typical DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus by zygotic injection. Driving our efforts was the desire to create a whole animal model that would replace 17 kilobase pairs (kbp) of the mouse Bcl2l11 gene with the corresponding 25-kbp segment of human BCL2L11, including a conditionally removable segment (2.9-kbp) of intron 2, a cryptic human exon immediately 3' of this, and a native human exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic embryonic stem cell (ESC) targeting, dual selection, and recombinase-driven cassette removal (ESC/Blastocyst Approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with redundant single guide RNAs (sgRNAs) and Cas9 mRNA into mouse zygotes (CRISPR/Zygote Approach). In both instances, we were able to achieve humanization of Bcl2l11 to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, 2.9-kbp intronic segment.},
}

Animals
Bcl-2-Like Protein 11/*genetics
Blastocyst/cytology/*metabolism
CRISPR-Cas Systems
Embryonic Stem Cells/cytology/*metabolism
Gene Editing
Humans
Introns/genetics
Mice
Microinjections
RNA, Guide/genetics
Zygote/growth & development/*metabolism

@article {pmid30254203,
year = {2018},
author = {Foster, AJ and Martin-Urdiroz, M and Yan, X and Wright, HS and Soanes, DM and Talbot, NJ},
title = {CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {14355},
pmid = {30254203},
issn = {2045-2322},
support = {294702//European Research Council/International ; BB/ BB/N009959/1//Biotechnology and Biological Sciences Research Council (BBSRC)/International ; },
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Magnaporthe/*genetics/metabolism/*physiology ; Melanins/biosynthesis ; Mutation ; Oryza/*microbiology ; Plant Diseases/*microbiology ; Polymorphism, Single Nucleotide ; Ribonucleoproteins/*metabolism ; },
abstract = {The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.},
}

Base Sequence
CRISPR-Cas Systems/*genetics
Gene Editing/*methods
Magnaporthe/*genetics/metabolism/*physiology
Melanins/biosynthesis
Mutation
Oryza/*microbiology
Plant Diseases/*microbiology
Polymorphism, Single Nucleotide
Ribonucleoproteins/*metabolism

@article {pmid29717174,
year = {2018},
author = {Wang, Q and He, G and Hou, M and Chen, L and Chen, S and Xu, A and Fu, Y},
title = {Cell Cycle Regulation by Alternative Polyadenylation of CCND1.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {6824},
pmid = {29717174},
issn = {2045-2322},
mesh = {3' Untranslated Regions/physiology ; CRISPR-Cas Systems/physiology ; Cell Cycle Checkpoints/*physiology ; Cell Proliferation/physiology ; Cyclin D1/genetics/*metabolism ; Genetic Loci ; Genetic Vectors ; HEK293 Cells ; Humans ; Mutation ; Open Reading Frames/physiology ; Poly A/metabolism ; Polyadenylation/*physiology ; Protein Isoforms/metabolism ; RNA, Messenger/metabolism ; Untranslated Regions/physiology ; },
abstract = {Global shortening of 3'UTRs by alternative polyadenylation (APA) has been observed in cancer cells. However, the role of APA in cancer remains unknown. CCND1 is a proto-oncogene that regulates progression through the G1-S phase of the cell cycle; moreover, it has been observed to be switching to proximal APA sites in cancer cells. To investigate the biological function of the APA of CCND1, we edited the weak poly(A) signal (PAS) of the proximal APA site to a canonical PAS using the CRISPR/Cas9 method, which can force the cells to use a proximal APA site. Cell cycle profiling and proliferation assays revealed that the proximal APA sites of CCND1 accelerated the cell cycle and promoted cell proliferation, but UTR-APA and CR-APA act via different molecular mechanisms. These results indicate that PAS editing with CRISPR/Cas9 provides a good method by which to study the biological function of APA.},
}

3' Untranslated Regions/physiology
CRISPR-Cas Systems/physiology
Cell Cycle Checkpoints/*physiology
Cell Proliferation/physiology
Cyclin D1/genetics/*metabolism
Genetic Loci
Genetic Vectors
HEK293 Cells
Humans
Mutation
Open Reading Frames/physiology
Poly A/metabolism
Polyadenylation/*physiology
Protein Isoforms/metabolism
RNA, Messenger/metabolism
Untranslated Regions/physiology

@article {pmid31658261,
year = {2019},
author = {Liu, Q and He, D and Xie, L},
title = {Prediction of off-target specificity and cell-specific fitness of CRISPR-Cas System using attention boosted deep learning and network-based gene feature.},
journal = {PLoS computational biology},
volume = {15},
number = {10},
pages = {e1007480},
doi = {10.1371/journal.pcbi.1007480},
pmid = {31658261},
issn = {1553-7358},
abstract = {CRISPR-Cas is a powerful genome editing technology and has a great potential for in vivo gene therapy. Successful translational application of CRISPR-Cas to biomedicine still faces many safety concerns, including off-target side effect, cell fitness problem after CRISPR-Cas treatment, and on-target genome editing side effect in undesired tissues. To solve these issues, it is needed to design sgRNA with high cell-specific efficacy and specificity. Existing single-guide RNA (sgRNA) design tools mainly depend on a sgRNA sequence and the local information of the targeted genome, thus are not sufficient to account for the difference in the cellular response of the same gene in different cell types. To incorporate cell-specific information into the sgRNA design, we develop novel interpretable machine learning models, which integrate features learned from advanced transformer-based deep neural network with cell-specific gene property derived from biological network and gene expression profile, for the prediction of CRISPR-Cas9 and CRISPR-Cas12a efficacy and specificity. In benchmark studies, our models significantly outperform state-of-the-art algorithms. Furthermore, we find that the network-based gene property is critical for the prediction of cell-specific post-treatment cellular response. Our results suggest that the design of efficient and safe CRISPR-Cas needs to consider cell-specific information of genes. Our findings may bolster developing more accurate predictive models of CRISPR-Cas across a broad spectrum of biological conditions as well as provide new insight into developing efficient and safe CRISPR-based gene therapy.},
}

@article {pmid31641267,
year = {2019},
author = {Ledford, H},
title = {Super-precise new CRISPR tool could tackle a plethora of genetic diseases.},
journal = {Nature},
volume = {574},
number = {7779},
pages = {464-465},
doi = {10.1038/d41586-019-03164-5},
pmid = {31641267},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *Gene Editing ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA
*Gene Editing

@article {pmid31272828,
year = {2019},
author = {Gao, Y and Hisey, E and Bradshaw, TWA and Erata, E and Brown, WE and Courtland, JL and Uezu, A and Xiang, Y and Diao, Y and Soderling, SH},
title = {Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering.},
journal = {Neuron},
volume = {103},
number = {4},
pages = {583-597.e8},
doi = {10.1016/j.neuron.2019.05.047},
pmid = {31272828},
issn = {1097-4199},
mesh = {Animals ; Brain/cytology/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Dependovirus/genetics ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; Genetic Vectors/genetics ; Genomics/*methods ; Humans ; Immunochemistry/methods ; Inteins ; Mice ; Mutagenesis, Insertional ; Nerve Tissue Proteins/chemistry/genetics ; Protein Engineering/*methods ; *Protein Processing, Post-Translational ; Proteomics ; RNA, Guide/genetics ; Recombinant Fusion Proteins/genetics ; Sequence Homology, Nucleic Acid ; },
abstract = {Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.},
}

Animals
Brain/cytology/metabolism
CRISPR-Cas Systems
Cells, Cultured
Dependovirus/genetics
Gene Editing/methods
Gene Knock-In Techniques/*methods
Genetic Vectors/genetics
Genomics/*methods
Humans
Immunochemistry/methods
Inteins
Mice
Mutagenesis, Insertional
Nerve Tissue Proteins/chemistry/genetics
Protein Engineering/*methods
*Protein Processing, Post-Translational
Proteomics
RNA, Guide/genetics
Recombinant Fusion Proteins/genetics
Sequence Homology, Nucleic Acid

@article {pmid31021228,
year = {2019},
author = {Russell, A},
title = {CRISPR Screens: The Right Tool for the Job.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {9-11},
doi = {10.1089/crispr.2019.29045.adr},
pmid = {31021228},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Cell Differentiation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome ; T-Lymphocytes, Helper-Inducer ; },
}

CRISPR-Cas Systems
Cell Differentiation
*Clustered Regularly Interspaced Short Palindromic Repeats
*Genome
T-Lymphocytes, Helper-Inducer

@article {pmid30767015,
year = {2019},
author = {Lu, Z and Yang, S and Yuan, X and Shi, Y and Ouyang, L and Jiang, S and Yi, L and Zhang, G},
title = {CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis.},
journal = {Nucleic acids research},
volume = {47},
number = {7},
pages = {e40},
doi = {10.1093/nar/gkz072},
pmid = {30767015},
issn = {1362-4962},
mesh = {Bacillus subtilis/*genetics ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Gene Expression Regulation, Bacterial ; Genes, Bacterial/genetics ; Molecular Chaperones/metabolism ; Mutation ; Promoter Regions, Genetic/genetics ; Protein Folding ; RNA, Guide/genetics ; Transcription, Genetic ; Transformation, Genetic ; },
abstract = {Fine-tuning of gene expression is crucial for protein expression and pathway construction, but it still faces formidable challenges due to the hierarchical gene regulation at multiple levels in a context-dependent manner. In this study, we defined the optimal targeting windows for CRISPRa and CRISPRi of the dCas9-α/ω system, and demonstrated that this system could act as a single master regulator to simultaneously activate and repress the expression of different genes by designing position-specific gRNAs. The application scope of dCas9-ω was further expanded by a newly developed CRISPR-assisted Oligonucleotide Annealing based Promoter Shuffling (OAPS) strategy, which could generate a high proportion of functional promoter mutants and facilitate the construction of effective promoter libraries in microorganisms with low transformation efficiency. Combing OAPS and dCas9-ω, the influences of promoter-based transcription, molecular chaperone-assisted protein folding and protease-mediated degradation on the expression of amylase BLA in Bacillus subtilis were systematically evaluated, and a 260-fold enhancement of BLA production was obtained. The success of the OAPS strategy and dCas9-ω for BLA production in this study thus demonstrated that it could serve as a powerful tool kit to regulate the expression of multiple genes multi-directionally and multi-dimensionally in bacteria.},
}

Bacillus subtilis/*genetics
CRISPR-Associated Protein 9/metabolism
CRISPR-Cas Systems/*genetics
*Gene Editing
*Gene Expression Regulation, Bacterial
Genes, Bacterial/genetics
Molecular Chaperones/metabolism
Mutation
Promoter Regions, Genetic/genetics
Protein Folding
RNA, Guide/genetics
Transcription, Genetic
Transformation, Genetic

@article {pmid30595383,
year = {2019},
author = {Mojumder, S and Sawamura, R and Murayama, Y and Ogura, T and Yamanaka, K},
title = {Functional characterization of UBXN-6, a C-terminal cofactor of CDC-48, in C. elegans.},
journal = {Biochemical and biophysical research communications},
volume = {509},
number = {2},
pages = {462-468},
doi = {10.1016/j.bbrc.2018.12.155},
pmid = {30595383},
issn = {1090-2104},
mesh = {Animal Nutritional Physiological Phenomena ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics/*physiology ; Eating ; Gene Deletion ; Hunger ; Lysosomes/genetics/metabolism ; Male ; Protein Interaction Maps ; },
abstract = {CDC-48 is a AAA (ATPases associated with diverse cellular activities) chaperone and participates in a wide range of cellular activities. Its functional diversity is determined by differential binding of a variety of cofactors. In this study, we analyzed the physiological role of a CDC-48 cofactor UBXN-6 in Caenorhabditis elegans. The amount of UBXN-6 was markedly increased upon starvation, but not with the treatment of tunicamycin and rapamycin. The induction upon starvation is a unique characteristic for UBXN-6 among C-terminal cofactors of CDC-48. During starvation, lysosomal activity is triggered for rapid clearance of cellular materials. We observed the lysosomal activity by monitoring GLO-1::GFP, a marker for lysosome-related organelles. We found that more puncta of GLO-1::GFP were observed in the ubxn-6 deletion mutant after 12 h starvation compared with the wild-type strain. Taken together, we propose that UBXN-6 is involved in clearance of cellular materials upon starvation in C. elegans.},
}

Animal Nutritional Physiological Phenomena
Animals
CRISPR-Cas Systems
Caenorhabditis elegans/genetics/*physiology
Eating
Gene Deletion
Hunger
Lysosomes/genetics/metabolism
Male
Protein Interaction Maps

@article {pmid30310080,
year = {2018},
author = {Idoko-Akoh, A and Taylor, L and Sang, HM and McGrew, MJ},
title = {High fidelity CRISPR/Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {15126},
pmid = {30310080},
issn = {2045-2322},
support = {BB/P013732/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P013759/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Alleles ; Animals ; Base Sequence ; Binding Sites ; *CRISPR-Cas Systems ; Chickens ; *Gene Editing ; Gene Order ; Genetic Vectors/genetics ; Germ Cells/cytology/*metabolism ; Heterozygote ; INDEL Mutation ; Mutation ; Protein Binding ; Sequence Analysis, DNA ; },
abstract = {Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chicken PGCs can be precise edited using donors containing CRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM). However, targeting was more efficient using SpCas9-HF1 when mutations were introduced only into the gRNA target sequence. We subsequently employed an eGFP-to-BFP conversion assay, to directly compare HDR mediated by SpCas9-WT and SpCas9-HF1 and discovered that SpCas9-HF1 increases HDR while reducing INDEL formation. Furthermore, SpCas9-HF1 increases the frequency of single allele editing in comparison to SpCas9-WT. We used SpCas9-HF1 to demonstrate the introduction of monoallelic and biallelic point mutations into the FGF20 gene and generate clonal populations of edited PGCs with defined homozygous and heterozygous genotypes. Our results demonstrate the use of oligonucleotide donors and high fidelity CRISPR/Cas9 variants to perform precise genome editing with high efficiency in PGCs.},
}

*Alleles
Animals
Base Sequence
Binding Sites
*CRISPR-Cas Systems
Chickens
*Gene Editing
Gene Order
Genetic Vectors/genetics
Germ Cells/cytology/*metabolism
Heterozygote
INDEL Mutation
Mutation
Protein Binding
Sequence Analysis, DNA

@article {pmid30291308,
year = {2018},
author = {Chanock, SJ},
title = {Gene editing reveals the effect of thousands of variants in a key cancer gene.},
journal = {Nature},
volume = {562},
number = {7726},
pages = {201-202},
doi = {10.1038/d41586-018-06022-y},
pmid = {30291308},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Oncogenes ; },
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
Oncogenes

@article {pmid30232365,
year = {2018},
author = {Hikita, T and Miyata, M and Watanabe, R and Oneyama, C},
title = {Sensitive and rapid quantification of exosomes by fusing luciferase to exosome marker proteins.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {14035},
pmid = {30232365},
issn = {2045-2322},
support = {JP1005457//Japan Science and Technology Agency (JST)/International ; },
mesh = {A549 Cells ; Animals ; CRISPR-Cas Systems ; Cell Line ; Exosomes/chemistry/*genetics ; HCT116 Cells ; HT29 Cells ; Humans ; Mice ; Nanoparticles ; Nanotechnology/*methods ; Tetraspanin 30/*genetics ; },
abstract = {Exosomes have emerged as important mediators of intercellular communication. Although their modes of action have been elucidated, the molecular mechanisms underlying their secretion, sorting of molecules, uptake into recipient cells, and biological distribution in vivo remain elusive. Here, we present a novel system for quantifying secreted exosomes by introducing ectopic or CRISPR/Cas9-mediated knock-in of luciferase-fusion exosome markers such as CD63. This luciferase-based method makes it possible to measure exosomes secreted into the culture medium with high linearity and wide dynamic range in a high-throughput manner. We demonstrate that data obtained by luminescent quantification are well correlated with data obtained by conventional nanoparticle tracking analysis under multiple conditions. In addition, our system is capable of evaluating the recipient cells or tissues that take up exosomes, as well as visualizing exosomes in vivo. The proposed system represents a powerful tool for understanding the molecular mechanisms underlying exosome production, uptake, and long-term distribution.},
}

A549 Cells
Animals
CRISPR-Cas Systems
Cell Line
Exosomes/chemistry/*genetics
HCT116 Cells
HT29 Cells
Humans
Mice
Nanoparticles
Nanotechnology/*methods
Tetraspanin 30/*genetics

@article {pmid30190522,
year = {2018},
author = {Shiraki, T and Kawakami, K},
title = {A tRNA-based multiplex sgRNA expression system in zebrafish and its application to generation of transgenic albino fish.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {13366},
pmid = {30190522},
issn = {2045-2322},
support = {JP15H02370//Japan Society for the Promotion of Science (JSPS)/International ; JP18H04988//Japan Society for the Promotion of Science (JSPS)/International ; NBRP//Japan Agency for Medical Research and Development (AMED)/International ; },
mesh = {*Albinism/genetics/metabolism ; Animals ; *Animals, Genetically Modified/genetics/metabolism ; CRISPR-Cas Systems ; *Phenotype ; *RNA, Transfer/genetics/metabolism ; *Zebrafish/genetics/metabolism ; },
abstract = {The CRISPR/Cas9 system can be introduced into zebrafish as transgenes. Namely, expression of single-guide RNA (sgRNA) and controlled expression of Cas9 in transgenic zebrafish enables the study of gene functions in specific cell types. This transgenic CRISPR/Cas9 approach would be more useful if multiple sgRNAs could be expressed simultaneously since we could knock-out a gene more efficiently or disrupt multiple genes simultaneously. Here we describe a novel system to express multiple sgRNAs efficiently in zebrafish, that relies on the endogenous tRNA processing machinery. We cloned nine endogenous zebrafish tRNA genes, fused them to sgRNAs, and demonstrated that an active sgRNA can be produced from a precursor transcript containing either of these tRNAs. To show a proof of principle, we constructed transgenic fish expressing Cas9 under the control of the ubiquitin promoter and a single transcript containing three distinct sgRNAs, that targeted the slc45a2 (albino) gene, fused to tRNAs under the control of the U6 promoter. We found that the Tg(ubb:SpCas9,u6c:3xslc45a2-sgRNA) harbored mutations in all of the target sites in the albino gene and showed nearly complete albino phenotypes, which were amenable to imaging experiments. Thus, the tRNA-based multiplex sgRNA expression system should facilitate gene knock-out studies in transgenic zebrafish.},
}

*Albinism/genetics/metabolism
Animals
*Animals, Genetically Modified/genetics/metabolism
CRISPR-Cas Systems
*Phenotype
*RNA, Transfer/genetics/metabolism
*Zebrafish/genetics/metabolism

@article {pmid30108345,
year = {2018},
author = {Johnson, MJ and Laoharawee, K and Lahr, WS and Webber, BR and Moriarity, BS},
title = {Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {12144},
pmid = {30108345},
issn = {2045-2322},
support = {R21 AI128087/AI/NIAID NIH HHS/United States ; },
mesh = {Antigens, CD19/genetics/metabolism ; B-Lymphocytes/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Engineering/*methods ; Cells, Cultured ; Electroporation ; Endonucleases/*genetics ; Gene Knockout Techniques/methods ; Genetic Engineering/*methods ; Healthy Volunteers ; Humans ; Primary Cell Culture ; RNA, Guide/genetics ; },
abstract = {B cells offer unique opportunities for gene therapy because of their ability to secrete large amounts of protein in the form of antibody and persist for the life of the organism as plasma cells. Here, we report optimized CRISPR/Cas9 based genome engineering of primary human B cells. Our procedure involves enrichment of CD19+ B cells from PBMCs followed by activation, expansion, and electroporation of CRISPR/Cas9 reagents. We are able expand total B cells in culture 10-fold and outgrow the IgD+ IgM+ CD27- naïve subset from 35% to over 80% of the culture. B cells are receptive to nucleic acid delivery via electroporation 3 days after stimulation, peaking at Day 7 post stimulation. We tested chemically modified sgRNAs and Alt-R gRNAs targeting CD19 with Cas9 mRNA or Cas9 protein. Using this system, we achieved genetic and protein knockout of CD19 at rates over 70%. Finally, we tested sgRNAs targeting the AAVS1 safe harbor site using Cas9 protein in combination with AAV6 to deliver donor template encoding a splice acceptor-EGFP cassette, which yielded site-specific integration frequencies up to 25%. The development of methods for genetically engineered B cells opens the door to a myriad of applications in basic research, antibody production, and cellular therapeutics.},
}

Antigens, CD19/genetics/metabolism
B-Lymphocytes/*metabolism
CRISPR-Cas Systems/genetics
Cell Engineering/*methods
Cells, Cultured
Electroporation
Endonucleases/*genetics
Gene Knockout Techniques/methods
Genetic Engineering/*methods
Healthy Volunteers
Humans
Primary Cell Culture
RNA, Guide/genetics

@article {pmid30104738,
year = {2018},
author = {Zaini, MN and Patel, SA and Syafruddin, SE and Rodrigues, P and Vanharanta, S},
title = {Endogenous HIF2A reporter systems for high-throughput functional screening.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {12063},
pmid = {30104738},
issn = {2045-2322},
support = {MC_UU_12022/7//Medical Research Council/United Kingdom ; MR/N501876/1//Medical Research Council/United Kingdom ; },
mesh = {Basic Helix-Loop-Helix Transcription Factors/*genetics ; CRISPR-Cas Systems/genetics ; Carcinogenesis/genetics ; Carcinoma, Renal Cell/genetics ; Cell Line, Tumor ; Feasibility Studies ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Neoplastic/genetics ; Gene Knock-In Techniques ; Gene Regulatory Networks/genetics ; Genes, Reporter/*genetics ; Genetic Testing/*methods ; HEK293 Cells ; High-Throughput Screening Assays/*methods ; Humans ; Kidney Neoplasms/genetics ; Transcription, Genetic ; },
abstract = {Tissue-specific transcriptional programs control most biological phenotypes, including disease states such as cancer. However, the molecular details underlying transcriptional specificity is largely unknown, hindering the development of therapeutic approaches. Here, we describe novel experimental reporter systems that allow interrogation of the endogenous expression of HIF2A, a critical driver of renal oncogenesis. Using a focused CRISPR-Cas9 library targeting chromatin regulators, we provide evidence that these reporter systems are compatible with high-throughput screening. Our data also suggests redundancy in the control of cancer type-specific transcriptional traits. Reporter systems such as those described here could facilitate large-scale mechanistic dissection of transcriptional programmes underlying cancer phenotypes, thus paving the way for novel therapeutic approaches.},
}

Basic Helix-Loop-Helix Transcription Factors/*genetics
CRISPR-Cas Systems/genetics
Carcinogenesis/genetics
Carcinoma, Renal Cell/genetics
Cell Line, Tumor
Feasibility Studies
Gene Expression Profiling/*methods
Gene Expression Regulation, Neoplastic/genetics
Gene Knock-In Techniques
Gene Regulatory Networks/genetics
Genes, Reporter/*genetics
Genetic Testing/*methods
HEK293 Cells
High-Throughput Screening Assays/*methods
Humans
Kidney Neoplasms/genetics
Transcription, Genetic

@article {pmid30104681,
year = {2018},
author = {Takabayashi, S and Aoshima, T and Kabashima, K and Aoto, K and Ohtsuka, M and Sato, M},
title = {i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {12059},
pmid = {30104681},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Associated Protein 9/administration & dosage/genetics ; CRISPR-Cas Systems/*genetics ; Electroporation/*methods ; Embryo, Mammalian ; *Fallopian Tubes ; Female ; Gene Editing/*methods ; Male ; Mutation ; PAX6 Transcription Factor/genetics ; Pregnancy ; RNA, Guide/administration & dosage/genetics ; Rats ; Rats, Inbred Lew ; Rats, Sprague-Dawley ; Rats, Transgenic ; },
abstract = {Zygote-microinjection or in vitro electroporation of isolated zygotes are now widely used methods to produce genome-edited mice. However, these technologies require laborious and time-consuming ex vivo handling of fertilized eggs, including zygote isolation, gene delivery into zygotes and embryo transfer into recipients. We recently developed an alternative method called improved genome-editing via oviductal nucleic acids delivery (i-GONAD), which does not require the above-mentioned ex vivo handing of zygotes, but instead involves intraoviductal instillation of genome-editing components, Cas9 protein and synthetic gRNAs, into the oviducts of pregnant females at the late 1-cell embryo stage under a dissecting microscope and subsequent electroporation. With this method, we succeeded in generating genome-edited mice at relatively high efficiencies (for example, knockout alleles were produced at ~97% efficiency). Here, we extended this improved technology to rats, and found that i-GONAD can create genome-edited rats in various strains, including Sprague Dawley and Lewis, and F1 hybrids (between Sprague Dawley and Brown Norway), with efficiencies of ~62% for indel mutations and ~9% for knock-ins. Thus, i-GONAD will be especially useful for the production of genome-edited rats in small laboratories where expensive micromanipulator systems and highly skilled personnel for embryo manipulation are unavailable.},
}

Animals
CRISPR-Associated Protein 9/administration & dosage/genetics
CRISPR-Cas Systems/*genetics
Electroporation/*methods
Embryo, Mammalian
*Fallopian Tubes
Female
Gene Editing/*methods
Male
Mutation
PAX6 Transcription Factor/genetics
Pregnancy
RNA, Guide/administration & dosage/genetics
Rats
Rats, Inbred Lew
Rats, Sprague-Dawley
Rats, Transgenic

@article {pmid30006631,
year = {2018},
author = {Holme, H and Gulati, A and Brough, R and Fleuren, EDG and Bajrami, I and Campbell, J and Chong, IY and Costa-Cabral, S and Elliott, R and Fenton, T and Frankum, J and Jones, SE and Menon, M and Miller, R and Pemberton, HN and Postel-Vinay, S and Rafiq, R and Selfe, JL and von Kriegsheim, A and Munoz, AG and Rodriguez, J and Shipley, J and van der Graaf, WTA and Williamson, CT and Ryan, CJ and Pettitt, S and Ashworth, A and Strauss, SJ and Lord, CJ},
title = {Chemosensitivity profiling of osteosarcoma tumour cell lines identifies a model of BRCAness.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {10614},
pmid = {30006631},
issn = {2045-2322},
support = {P30 CA082103/CA/NCI NIH HHS/United States ; C347/A8363//Cancer Research UK/United Kingdom ; },
mesh = {BRCA1 Protein/*genetics ; BRCA2 Protein/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Nucleus/drug effects/genetics/metabolism ; DNA Damage/drug effects/genetics ; DNA Repair/drug effects/genetics ; Datasets as Topic ; Drug Resistance, Neoplasm/*genetics ; Drug Screening Assays, Antitumor/methods ; High-Throughput Screening Assays/methods ; Humans ; Mutagenesis ; Mutation ; Osteosarcoma/*drug therapy/genetics ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/*therapeutic use ; Rad51 Recombinase/metabolism ; },
abstract = {Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS.},
}

BRCA1 Protein/*genetics
BRCA2 Protein/*genetics
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Cell Nucleus/drug effects/genetics/metabolism
DNA Damage/drug effects/genetics
DNA Repair/drug effects/genetics
Datasets as Topic
Drug Resistance, Neoplasm/*genetics
Drug Screening Assays, Antitumor/methods
High-Throughput Screening Assays/methods
Humans
Mutagenesis
Mutation
Osteosarcoma/*drug therapy/genetics
Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/*therapeutic use
Rad51 Recombinase/metabolism

@article {pmid29993199,
year = {2018},
author = {Li, J and Peng, W and Du, L and Yang, Q and Wang, C and Mo, YY},
title = {The oncogenic potentials and diagnostic significance of long non-coding RNA LINC00310 in breast cancer.},
journal = {Journal of cellular and molecular medicine},
volume = {22},
number = {9},
pages = {4486-4495},
pmid = {29993199},
issn = {1582-4934},
support = {R01 CA154989/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Breast Neoplasms/diagnosis/*drug therapy/genetics/mortality ; CRISPR-Cas Systems ; Carcinogenesis/*genetics/metabolism/pathology ; Case-Control Studies ; Cell Line, Tumor ; Cell Proliferation ; Female ; *Gene Expression Regulation, Neoplastic ; Humans ; Mice ; Mice, Nude ; Oligoribonucleotides/genetics/metabolism ; Prognosis ; Proto-Oncogene Proteins c-myc/*genetics/metabolism ; RNA, Long Noncoding/antagonists & inhibitors/*genetics/metabolism ; Signal Transduction ; Survival Analysis ; Xenograft Model Antitumor Assays ; },
abstract = {Recent studies have revealed that long non-coding RNAs (lncRNAs) are involved in different physiological processes and human diseases. However, to date, the function and overall clinical significance of the vast majority of lncRNAs in breast cancer remain largely unexplored. Here, we focused on LINC00310 by interrogating the breast invasive carcinoma data set of the Cancer Genome Atlas (TCGA). The results showed that LINC00310 was increased as breast cancer progressed, and the deregulation of LINC00310 was significantly associated with patients' survival. Experiments with knockout (KO) approach by CRISPR/Cas9 system and the subsequent rescue experiments revealed that LINC00310 promoted cell proliferation by regulating c-Myc expression in vitro. Nude mouse xenograft assay demonstrated that LINC00310 KO significantly suppressed tumour growth in vivo. Furthermore, we found that serum LINC00310 expression was significantly up-regulated in patients with breast cancer, and receiver operating characteristic (ROC) curve analysis indicated that LINC00310 had a powerful capability of distinguishing patients with breast cancer from healthy individuals (the area under curve 0.828). Taken together, these results provide a more intuitive approach to explore the clinical relevance and functional roles of lncRNAs. As a result, lncRNAs, such as LINC00310, may be used in clinical applications as circulating markers for breast cancer.},
}

Animals
Breast Neoplasms/diagnosis/*drug therapy/genetics/mortality
CRISPR-Cas Systems
Carcinogenesis/*genetics/metabolism/pathology
Case-Control Studies
Cell Line, Tumor
Cell Proliferation
Female
*Gene Expression Regulation, Neoplastic
Humans
Mice
Mice, Nude
Oligoribonucleotides/genetics/metabolism
Prognosis
Proto-Oncogene Proteins c-myc/*genetics/metabolism
RNA, Long Noncoding/antagonists & inhibitors/*genetics/metabolism
Signal Transduction
Survival Analysis
Xenograft Model Antitumor Assays

@article {pmid29785489,
year = {2018},
author = {Kim, EY and Yazdizadeh Shotorbani, P and Dryer, SE},
title = {Trpc6 inactivation confers protection in a model of severe nephrosis in rats.},
journal = {Journal of molecular medicine (Berlin, Germany)},
volume = {96},
number = {7},
pages = {631-644},
pmid = {29785489},
issn = {1432-1440},
support = {R01 DK104708/DK/NIDDK NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Gene Knockout Techniques ; *Gene Silencing ; Gene Targeting ; *Genetic Association Studies ; *Genetic Predisposition to Disease ; Kidney/metabolism/pathology/ultrastructure ; Kidney Function Tests ; Kidney Glomerulus/metabolism/pathology/ultrastructure ; Mice, Transgenic ; Nephrosis/*etiology/metabolism/pathology/urine ; Rats ; Severity of Illness Index ; TRPC6 Cation Channel/*genetics/metabolism ; },
abstract = {Mutations in canonical transient receptor potential-6 (TRPC6) channels give rise to rare familial forms of focal and segmental glomerulosclerosis (FSGS). Here we examined a possible role for TRPC6 in the progression of chronic puromycin aminonucleoside (PAN) nephrosis in Sprague-Dawley rats, a classic model of acquired nephrotic syndromes. We used CRISPR/Cas9 technology to delete a 239-bp region within exon 2 of the Trpc6 gene (Trpc6del allele). Trpc6del/del rats expressed detectable Trpc6 transcripts missing exon 2, and TRPC6 proteins could be detected by immunoblot of renal cortex. However, the abundance of Trpc6 transcripts and TRPC6 protein in renal cortex was much lower than in Trpc6wt/wt littermates, and functional TRPC6 channels could not be detected in whole-cell recordings from glomerular cells cultured from Trpc6del/del animals, possibly because of disruption of ankyrin repeats 1 and 2. During the chronic phase of PAN nephrosis, Trpc6del/del rats had reduced urine albumin excretion, reduced serum cholesterol and triglycerides, and improved azotemia compared to wild-type Trpc6wt/wt littermates. Glomerulosclerosis was severe during chronic PAN nephrosis in Trpc6wt/wt rats but was markedly reduced in Trpc6del/del littermates. Trpc6del/del animals also had less severe tubulointerstitial fibrosis as assessed by several biochemical and histological analyses, as well as reduced foot process effacement and glomerular basement thickening compared to Trpc6wtt/wt controls. None of the manipulations in this study affected the abundance of TRPC5 channels in renal cortex. TRPC3 was increased in PAN nephrosis and in Trpc6del/del rats. These data support a role for TRPC6 channels in driving an acquired form of secondary FSGS.

KEY MESSAGES: We examined aminonucleoside nephrosis in rats with wild type and inactivated TRPC6. TRPC6 channels were inactivated by CRISPR/Cas9 editing of the Trpc6 gene. TRPC6 inactivation reduced albuminuria in the chronic but not the acute phase. TRPC6 inactivation reduced glomerulosclerosis and ultrastructural changes. TRPC6 inactivation also reduced interstitial changes and renal fibrosis.},
}

Alleles
Animals
CRISPR-Cas Systems
Disease Models, Animal
Gene Knockout Techniques
*Gene Silencing
Gene Targeting
*Genetic Association Studies
*Genetic Predisposition to Disease
Kidney/metabolism/pathology/ultrastructure
Kidney Function Tests
Kidney Glomerulus/metabolism/pathology/ultrastructure
Mice, Transgenic
Nephrosis/*etiology/metabolism/pathology/urine
Rats
Severity of Illness Index
TRPC6 Cation Channel/*genetics/metabolism

@article {pmid29507322,
year = {2018},
author = {Cova, M and López-Gutiérrez, B and Artigas-Jerónimo, S and González-Díaz, A and Bandini, G and Maere, S and Carretero-Paulet, L and Izquierdo, L},
title = {The Apicomplexa-specific glucosamine-6-phosphate N-acetyltransferase gene family encodes a key enzyme for glycoconjugate synthesis with potential as therapeutic target.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {4005},
pmid = {29507322},
issn = {2045-2322},
support = {R01 AI110638/AI/NIAID NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Base Sequence ; CRISPR-Cas Systems ; Crystallography, X-Ray ; Evolution, Molecular ; Glucosamine 6-Phosphate N-Acetyltransferase/antagonists & inhibitors/chemistry/*genetics/metabolism ; Glycoconjugates/*biosynthesis ; Mutation ; Plasmodium falciparum/*enzymology ; Protein Structure, Secondary ; Substrate Specificity ; Trypanosoma brucei brucei/*enzymology ; },
abstract = {Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans.},
}

Amino Acid Sequence
Base Sequence
CRISPR-Cas Systems
Crystallography, X-Ray
Evolution, Molecular
Glucosamine 6-Phosphate N-Acetyltransferase/antagonists & inhibitors/chemistry/*genetics/metabolism
Glycoconjugates/*biosynthesis
Mutation
Plasmodium falciparum/*enzymology
Protein Structure, Secondary
Substrate Specificity
Trypanosoma brucei brucei/*enzymology

@article {pmid31654436,
year = {2019},
author = {Yang, JW and Jiang, JH and Wang, HC and Li, CY},
title = {The Extra Domain A of fibronectin facilitates osteoclastogenesis in radicular cysts through Vascular Endothelial Growth Factor.},
journal = {International endodontic journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/iej.13241},
pmid = {31654436},
issn = {1365-2591},
abstract = {AIM: To analyse the effects of the alternatively spliced fibronectin (FN) gene and its isoforms on osteoclastogenesis in radicular cysts.

METHODOLOGY: Specimens of radicular cysts were collected surgically from 22 patients whose radiolucent periapical areas were measured on digital panoramic radiographs before surgery. The associations between the radiolucent areas and FN isoforms, Vascular Endothelial Growth Factor (VEGF) expression or micro-vessel density, as well as the relationships among them, were analysed by immunohistochemical staining using the antibodies IST-9, BC-1, P1F11, VEGF, and CD34. Fibroblasts isolated from those specimens were used to induce Trap+MNCs, and the effects of induction were assessed by blocking FN containing extra domain A (EDA+FN), COX-2, or VEGF in vitro. The effects of EDA exon knockout using CRISPR/Cas system was also assessed. Quantitative PCR was used to analyse relative expression of FN isoforms and osteoclastogenic genes. Data were analysed using Linear regression, Spearman's rank correlation analysis, Chi-square test and Student's t-test; P < 0.05 was considered significant.

RESULTS: Micro-vessel density and EDA+FN staining were positively associated with the size of radiolucent periapical areas (mm2) (P<0.05), consistent with a positive association between Trap+MNCs and VEGF expression in fibroblasts (P<0.05). Blocking the interaction between EDA+FN and fibroblasts inhibited Trap+MNC formation. In addition, EDA exon knockout decreased VEGF expression, and inhibited Trap+MNC formation to the extent of blocking VEGF by bevacizumab, but osteoclastogenic induction was restored by recombinant VEGF. Using retrospective clinicopathological data, VEGF staining was shown to be positively associated with EDA+FN staining, micro-vessel density, and the size of radiolucent areas (P<0.05).

CONCLUSION: In fibrous capsules of radicular cysts, the alternatively spliced isoform EDA+FN generated by fibroblasts stimulated VEGF expression via an autocrine effect, and then facilitated osteoclastogenesis. Both blockage of VEGF and EDA exon knockout could be used to inhibit bone destruction.},
}

@article {pmid31653696,
year = {2019},
author = {Andreeva, A and Bekkhozhin, Z and Omertassova, N and Baizhumanov, T and Yeltay, G and Akhmetali, M and Toibazar, D and Utepbergenov, D},
title = {Apparent deglycase activity of DJ-1 results from the conversion of free methylglyoxal present in fast equilibrium with hemithioacetals and hemiaminals.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA119.011237},
pmid = {31653696},
issn = {1083-351X},
abstract = {Loss of function mutations in the gene encoding human protein DJ-1 cause early onset of Parkinson's disease, suggesting that DJ-1 protects dopaminergic neurons. The molecular mechanisms underlying this neuroprotection are unclear; however DJ-1 has been suggested to be a glutathione-independent glyoxalase that detoxifies methylglyoxal (MGO) by converting it into lactate. It has also been suggested that DJ-1 serves as a deglycase that catalyzes hydrolysis of hemithioacetals and hemiaminals formed by reactions of MGO with the thiol and amino groups of proteins. In this report, we demonstrate that the equilibrium constant of reaction of MGO with thiols is ~500 M-1 at 37 °C and that the half-life of the resulting hemithioacetal is only 12 sec. These thermodynamic parameters would dictate that a significant fraction of free MGO will be present in a fast equilibrium with hemithioacetals in solution. We found that removal of free MGO by DJ-1's glyoxalase activity forces immediate spontaneous decomposition of hemithioacetals due to the shift in equilibrium position. This spontaneous decomposition of hemithioacetals could be mistaken for deglycase activity of DJ-1. Further, we demonstrate that higher initial concentrations of hemithioacetals are associated with lower rates of DJ-1-mediated conversion of MGO, ruling out the possibility that hemithioacetals are DJ-1 substrates. Experiments with CRISPR/Cas generated DJ-1 knockout HEK293 cells revealed that DJ-1 does not protect against acute MGO toxicity or carboxymethylation of lysine residues in cells. Combined, our results suggest that DJ-1 does not possess protein deglycase activity.},
}

@article {pmid31349852,
year = {2019},
author = {Liu, G and Yin, K and Zhang, Q and Gao, C and Qiu, JL},
title = {Modulating chromatin accessibility by transactivation and targeting proximal dsgRNAs enhances Cas9 editing efficiency in vivo.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {145},
doi = {10.1186/s13059-019-1762-8},
pmid = {31349852},
issn = {1474-760X},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems ; Chromatin/*chemistry ; *Gene Editing ; Oryza/genetics ; RNA/genetics ; Trans-Activators/genetics ; *Transcriptional Activation ; },
abstract = {The CRISPR/Cas9 system is unable to edit all targetable genomic sites with full efficiency in vivo. We show that Cas9-mediated editing is more efficient in open chromatin regions than in closed chromatin regions in rice. A construct (Cas9-TV) formed by fusing a synthetic transcription activation domain to Cas9 edits target sites more efficiently, even in closed chromatin regions. Moreover, combining Cas9-TV with a proximally binding dead sgRNA (dsgRNA) further improves editing efficiency up to several folds. The use of Cas9-TV/dsgRNA thus provides a novel strategy for obtaining efficient genome editing in vivo, especially at nuclease-refractory target sites.},
}

CRISPR-Associated Protein 9/genetics/*metabolism
CRISPR-Cas Systems
Chromatin/*chemistry
*Gene Editing
Oryza/genetics
RNA/genetics
Trans-Activators/genetics
*Transcriptional Activation

@article {pmid31296872,
year = {2019},
author = {Santos-Pereira, JM and Gallardo-Fuentes, L and Neto, A and Acemel, RD and Tena, JJ},
title = {Pioneer and repressive functions of p63 during zebrafish embryonic ectoderm specification.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3049},
doi = {10.1038/s41467-019-11121-z},
pmid = {31296872},
issn = {2041-1723},
support = {IJCI-2016-29884//Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness)/International ; BFU2014-58449-JIN//Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness)/International ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Chromatin/metabolism ; Down-Regulation ; Ectoderm/*embryology/metabolism ; Embryo, Nonmammalian ; Embryonic Development/*genetics ; Enhancer Elements, Genetic/genetics ; Epidermis/embryology/metabolism ; *Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Models, Animal ; Neural Plate/*embryology/metabolism ; Phosphoproteins/genetics/*metabolism ; Protein Binding/genetics ; SOXB1 Transcription Factors/genetics/metabolism ; Trans-Activators/genetics/*metabolism ; Zebrafish/embryology ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {The transcription factor p63 is a master regulator of ectoderm development. Although previous studies show that p63 triggers epidermal differentiation in vitro, the roles of p63 in developing embryos remain poorly understood. Here, we use zebrafish embryos to analyze in vivo how p63 regulates gene expression during development. We generate tp63-knock-out mutants that recapitulate human phenotypes and show down-regulated epidermal gene expression. Following p63-binding dynamics, we find two distinct functions clearly separated in space and time. During early development, p63 binds enhancers associated to neural genes, limiting Sox3 binding and reducing neural gene expression. Indeed, we show that p63 and Sox3 are co-expressed in the neural plate border. On the other hand, p63 acts as a pioneer factor by binding non-accessible chromatin at epidermal enhancers, promoting their opening and epidermal gene expression in later developmental stages. Therefore, our results suggest that p63 regulates cell fate decisions during vertebrate ectoderm specification.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems/genetics
Cell Differentiation/genetics
Chromatin/metabolism
Down-Regulation
Ectoderm/*embryology/metabolism
Embryo, Nonmammalian
Embryonic Development/*genetics
Enhancer Elements, Genetic/genetics
Epidermis/embryology/metabolism
*Gene Expression Regulation, Developmental
Gene Knockout Techniques
Models, Animal
Neural Plate/*embryology/metabolism
Phosphoproteins/genetics/*metabolism
Protein Binding/genetics
SOXB1 Transcription Factors/genetics/metabolism
Trans-Activators/genetics/*metabolism
Zebrafish/embryology
Zebrafish Proteins/genetics/*metabolism

@article {pmid31021231,
year = {2019},
author = {Swarts, DC},
title = {Stirring Up the Type V Alphabet Soup.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {14-16},
doi = {10.1089/crispr.2019.29044.dcs},
pmid = {31021231},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Food ; },
}

*CRISPR-Cas Systems
*Food

@article {pmid30662440,
year = {2018},
author = {Lloyd, KA and Wigerblad, G and Sahlström, P and Garimella, MG and Chemin, K and Steen, J and Titcombe, PJ and Marklein, B and Zhou, D and Stålesen, R and Ossipova, E and Lundqvist, C and Ekwall, O and Rönnelid, J and Mueller, DL and Karlsson, MCI and Kaplan, MJ and Skriner, K and Klareskog, L and Wermeling, F and Malmström, V and Grönwall, C},
title = {Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {3033},
doi = {10.3389/fimmu.2018.03033},
pmid = {30662440},
issn = {1664-3224},
support = {T32 GM008244/GM/NIGMS NIH HHS/United States ; ZIA AR041199/AR/NIAMS NIH HHS/United States ; },
mesh = {Acetylation ; Adult ; Aged ; Animals ; Anti-Citrullinated Protein Antibodies/*immunology/metabolism ; Antigens, Nuclear/*immunology/metabolism ; Apoptosis/immunology ; Arthritis, Rheumatoid/blood/*immunology ; Autoantigens/*immunology/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Cross Reactions ; Extracellular Traps/immunology/metabolism ; Female ; Gene Knockout Techniques ; Histones/immunology/metabolism ; Humans ; Male ; Mice ; Mice, Knockout ; Middle Aged ; Neutrophils/immunology/metabolism ; Primary Cell Culture ; Protein Processing, Post-Translational/immunology ; Protein-Arginine Deiminases/genetics/immunology/*metabolism ; },
abstract = {Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACPA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal ACPA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NET reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis.},
}

Acetylation
Adult
Aged
Animals
Anti-Citrullinated Protein Antibodies/*immunology/metabolism
Antigens, Nuclear/*immunology/metabolism
Apoptosis/immunology
Arthritis, Rheumatoid/blood/*immunology
Autoantigens/*immunology/metabolism
CRISPR-Cas Systems
Cells, Cultured
Cross Reactions
Extracellular Traps/immunology/metabolism
Female
Gene Knockout Techniques
Histones/immunology/metabolism
Humans
Male
Mice
Mice, Knockout
Middle Aged
Neutrophils/immunology/metabolism
Primary Cell Culture
Protein Processing, Post-Translational/immunology
Protein-Arginine Deiminases/genetics/immunology/*metabolism

@article {pmid30662438,
year = {2018},
author = {Cheng, Y and Lun, M and Liu, Y and Wang, H and Yan, Y and Sun, J},
title = {CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {3010},
doi = {10.3389/fimmu.2018.03010},
pmid = {30662438},
issn = {1664-3224},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Chick Embryo ; Chickens/genetics/*immunology/metabolism ; Fibroblasts ; Gene Knockout Techniques/methods ; *Immunity, Innate ; Interferon-beta/immunology/*metabolism ; Membrane Proteins/immunology/*metabolism ; Primary Cell Culture ; Protein-Serine-Threonine Kinases/genetics/*immunology ; RNA, Guide/genetics ; },
abstract = {TANK-binding kinase 1 (TBK1) is involved in innate immunity, prompting transcriptional induction of type I interferons in response to pathogenic infection. Many studies have focused on mammals but the function of TBK1 in chickens remains poorly defined. CRISPR/Cas9 system has made gene-knockout easy to accomplish. Although CRISPR/Cas9 has been used in chicken cells, low mutation efficiency limits its wide application in chickens. In this study, an effective gene-knockout system was developed based on the CRISPR/Cas9 system in chicken embryonic fibroblast DF-1. Two CRISPR/Cas9 plasmids were constructed, TBK1-g1 and TBK1-g2, which express gRNAs targeting different sequences of the chicken TBK1 gene. After transfection and enrichment with puromycin screening, the mutation rates as assessed via T7E1 assay were 88.05 and 89.55%, respectively, and subsequent sequence analysis showed mutation efficiencies of 86.67 and 93.33%. With the limiting-dilution method, a chTBK1 gene-deficiency monoclonal cell line was obtained and was named DF-1-TBK1-C3. The DF-1-TBK1-C3 cells exhibited normal morphology and maintained stable proliferation ability compared to wild-type cells. The gene-overexpression system and luciferase reporter assay showed that IFN-β induction induced by chSTING was almost completely blocked in DF-1-TBK1-C3 cells. With quantitative real-time PCR, we further confirmed the essential role of chTBK1 in the chSTING-mediated IFN-β induction. At last, the study demonstrated that the chTBK1 knockout system is also applicable in primary chick embryo fibroblasts (CEFs). In this study, an effective gene-knockout system was applied in chickens, a TBK1 gene-deleted DF-1 cell line was successfully created using this system, and with the chTBK1 knockout cells, chTBK1 was revealed to be indispensable in STING-mediated IFN-β activation in chicken cells.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Line
Chick Embryo
Chickens/genetics/*immunology/metabolism
Fibroblasts
Gene Knockout Techniques/methods
*Immunity, Innate
Interferon-beta/immunology/*metabolism
Membrane Proteins/immunology/*metabolism
Primary Cell Culture
Protein-Serine-Threonine Kinases/genetics/*immunology
RNA, Guide/genetics