@article {pmid31520072,
year = {2019},
author = {Shmakov, SA and Faure, G and Makarova, KS and Wolf, YI and Severinov, KV and Koonin, EV},
title = {Systematic prediction of functionally linked genes in bacterial and archaeal genomes.},
journal = {Nature protocols},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41596-019-0211-1},
pmid = {31520072},
issn = {1750-2799},
abstract = {Functionally linked genes in bacterial and archaeal genomes are often organized into operons. However, the composition and architecture of operons are highly variable and frequently differ even among closely related genomes. Therefore, to efficiently extract reliable functional predictions for uncharacterized genes from comparative analyses of the rapidly growing genomic databases, dedicated computational approaches are required. We developed a protocol to systematically and automatically identify genes that are likely to be functionally associated with a 'bait' gene or locus by using relevance metrics. Given a set of bait loci and a genomic database defined by the user, this protocol compares the genomic neighborhoods of the baits to identify genes that are likely to be functionally linked to the baits by calculating the abundance of a given gene within and outside the bait neighborhoods and the distance to the bait. We exemplify the performance of the protocol with three test cases, namely, genes linked to CRISPR-Cas systems using the 'CRISPRicity' metric, genes associated with archaeal proviruses and genes linked to Argonaute genes in halobacteria. The protocol can be run by users with basic computational skills. The computational cost depends on the sizes of the genomic dataset and the list of reference loci and can vary from one CPU-hour to hundreds of hours on a supercomputer.},
}

@article {pmid31519756,
year = {2019},
author = {Hoffman, HK and Fernandez, MV and Groves, NS and Freed, EO and van Engelenburg, SB},
title = {Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA119.009372},
pmid = {31519756},
issn = {1083-351X},
abstract = {The endosomal sorting complexes required for transport (ESCRT) machinery drives membrane scission for diverse cellular functions that require budding away from the cytosol, including cell division and transmembrane receptor trafficking and degradation. The ESCRT machinery is also hijacked by retroviruses, such as HIV-1, to release virions from infected cells. The crucial roles of the ESCRTs in cellular physiology and viral disease make it imperative to understand the membrane scission mechanism. Current methodological limitations, namely, artifacts caused by overexpression of ESCRT subunits, obstruct our understanding of the spatiotemporal organization of the endogenous human ESCRT machinery. Here, we used CRISPR/Cas9-mediated knock-in to tag the critical ESCRT-I component tumor susceptibility 101 (Tsg101) with GFP at its native locus in two widely used human cell types, HeLa epithelial cells and Jurkat T cells. We validated this approach by assessing the function of these knock-in cell lines in cytokinesis, receptor degradation, and virus budding. Using this probe, we measured the incorporation of endogenous Tsg101 in released HIV-1 particles, supporting the notion that the ESCRT machinery initiates virus abscission by scaffolding early-acting ESCRT-I within the head of the budding virus. We anticipate that these validated cell lines will be a valuable tool for interrogating dynamics of the native human ESCRT machinery.},
}

@article {pmid31519690,
year = {2019},
author = {Robertson, FL and Marqués-Torrejón, MA and Morrison, GM and Pollard, SM},
title = {Experimental models and tools to tackle glioblastoma.},
journal = {Disease models & mechanisms},
volume = {12},
number = {9},
pages = {},
doi = {10.1242/dmm.040386},
pmid = {31519690},
issn = {1754-8411},
abstract = {Glioblastoma multiforme (GBM) is one of the deadliest human cancers. Despite increasing knowledge of the genetic and epigenetic changes that underlie tumour initiation and growth, the prognosis for GBM patients remains dismal. Genome analysis has failed to lead to success in the clinic. Fresh approaches are needed that can stimulate new discoveries across all levels: cell-intrinsic mechanisms (transcriptional/epigenetic and metabolic), cell-cell signalling, niche and microenvironment, systemic signals, immune regulation, and tissue-level physical forces. GBMs are inherently extremely challenging: tumour detection occurs too late, and cells infiltrate widely, hiding in quiescent states behind the blood-brain barrier. The complexity of the brain tissue also provides varied and complex microenvironments that direct cancer cell fates. Phenotypic heterogeneity is therefore superimposed onto pervasive genetic heterogeneity. Despite this bleak outlook, there are reasons for optimism. A myriad of complementary, and increasingly sophisticated, experimental approaches can now be used across the research pipeline, from simple reductionist models devised to delineate molecular and cellular mechanisms, to complex animal models required for preclinical testing of new therapeutic approaches. No single model can cover the breadth of unresolved questions. This Review therefore aims to guide investigators in choosing the right model for their question. We also discuss the recent convergence of two key technologies: human stem cell and cancer stem cell culture, as well as CRISPR/Cas tools for precise genome manipulations. New functional genetic approaches in tailored models will likely fuel new discoveries, new target identification and new therapeutic strategies to tackle GBM.},
}

@article {pmid31519332,
year = {2019},
author = {Dorman, CJ and Ní Bhriain, N},
title = {CRISPR-Cas, DNA Supercoiling, and Nucleoid-Associated Proteins.},
journal = {Trends in microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tim.2019.08.004},
pmid = {31519332},
issn = {1878-4380},
abstract = {In this opinion article we highlight links between the H-NS nucleoid-associated protein, variable DNA topology, the regulation of CRISPR-cas locus expression, CRISPR-Cas activity, and the recruitment of novel genetic information by the CRISPR array. We propose that the requirement that the invading mobile genetic element be negatively supercoiled limits effective CRISPR action to a window in the bacterial growth cycle when DNA topology is optimal, and that this same window is used for the efficient integration of new spacer sequences at the CRISPR array. H-NS silences CRISPR promoters, and we propose that antagonists of H-NS, such as the LeuO transcription factor, provide a basis for a stochastic genetic switch that acts at random in each cell in the bacterial population. In addition, we wish to propose a mechanism by which mobile genetic elements can suppress CRISPR-cas transcription using H-NS homologues. Although the individual components of this network are known, we propose a new model in which they are integrated and linked to the physiological state of the bacterium. The model provides a basis for cell-to-cell variation in the expression and performance of CRISPR systems in bacterial populations.},
}

@article {pmid31516887,
year = {2019},
author = {Bozorg Qomi, S and Asghari, A and Mojarrad, M},
title = {An Overview of the CRISPR-Based Genomic- and Epigenome-Editing System: Function, Applications, and Challenges.},
journal = {Advanced biomedical research},
volume = {8},
number = {},
pages = {49},
doi = {10.4103/abr.abr_41_19},
pmid = {31516887},
issn = {2277-9175},
abstract = {Developing a new strategy for an efficient targeted genome editing has always been a great perspective in biology. Although different approaches have been suggested in the last three decades, each one is confronting with limitations. CRISPR-Cas complex is a bacterial-derived system which made a breakthrough in the area of genome editing. This paper presents a brief history of CRISPR genome editing and discusses thoroughly how it works in bacteria and mammalians. At the end, some applications and challenges of this growing research area are also reviewed. In addition to moving the boundaries of genetics, CRISPR-Cas can also provide the ground for fundamental advances in other fields of biological sciences.},
}

@article {pmid30962569,
year = {2019},
author = {Lee, HH and Ostrov, N and Wong, BG and Gold, MA and Khalil, AS and Church, GM},
title = {Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi.},
journal = {Nature microbiology},
volume = {4},
number = {7},
pages = {1105-1113},
doi = {10.1038/s41564-019-0423-8},
pmid = {30962569},
issn = {2058-5276},
mesh = {Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Chromosomes, Bacterial/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Culture Media ; Gene Knockdown Techniques ; Gene Library ; Genome, Bacterial/*genetics ; Genomics ; Vibrio/*genetics/growth & development ; },
abstract = {The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time1,2 and metabolic prowess3,4. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.},
}

Bacterial Proteins/genetics/metabolism
*CRISPR-Cas Systems
Chromosomes, Bacterial/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Culture Media
Gene Knockdown Techniques
Gene Library
Genome, Bacterial/*genetics
Genomics
Vibrio/*genetics/growth & development

@article {pmid30254054,
year = {2018},
author = {Gurevich, VV and Gurevich, EV},
title = {Arrestins and G proteins in cellular signaling: The coin has two sides.},
journal = {Science signaling},
volume = {11},
number = {549},
pages = {},
doi = {10.1126/scisignal.aav1646},
pmid = {30254054},
issn = {1937-9145},
support = {R01 EY011500/EY/NEI NIH HHS/United States ; R01 NS065868/NS/NINDS NIH HHS/United States ; R21 DA030103/DA/NIDA NIH HHS/United States ; R35 GM122491/GM/NIGMS NIH HHS/United States ; },
mesh = {*Arrestins ; *CRISPR-Cas Systems ; Phosphorylation ; RNA, Small Interfering ; beta-Arrestins ; },
abstract = {Several studies have suggested that arrestin-mediated signaling by GPCRs requires G protein activation; however, in this issue of Science Signaling, Luttrell et al. documented arrestin-dependent activation of ERK1/2 by a number of GPCRs. These studies do not contradict each other, but illustrate the complexity of cellular signaling that cannot and should not be reduced to simplistic models.},
}

*Arrestins
*CRISPR-Cas Systems
Phosphorylation
RNA, Small Interfering
beta-Arrestins

@article {pmid30030150,
year = {2018},
author = {Van Gils, M and Willaert, A and De Vilder, EYG and Coucke, PJ and Vanakker, OM},
title = {Generation and Validation of a Complete Knockout Model of abcc6a in Zebrafish.},
journal = {The Journal of investigative dermatology},
volume = {138},
number = {11},
pages = {2333-2342},
doi = {10.1016/j.jid.2018.06.183},
pmid = {30030150},
issn = {1523-1747},
mesh = {ATP-Binding Cassette Transporters/*genetics ; Animals ; CRISPR-Cas Systems ; Calcification, Physiologic/genetics ; Disease Models, Animal ; Gene Knockout Techniques ; Humans ; Morpholinos/genetics ; Mutation/*genetics ; Phenotype ; Pseudoxanthoma Elasticum/*genetics ; Ribs/*physiology ; Scoliosis/*genetics ; Spine/*physiology ; Zebrafish ; Zebrafish Proteins/*genetics ; },
abstract = {Pseudoxanthoma elasticum is an ectopic mineralization disease due to biallelic ABCC6 mutations. As no treatment options are currently available, a reliable zebrafish model is invaluable for high throughput compound screening. However, data from previously reported knockdown and mutant zebrafish models for abcc6a, the functional orthologue of ABCC6, showed phenotypic discrepancies. To address this, we developed a complete abcc6a knockout model using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 and compared its phenotype to that of a mutant model (Sa963) and a splice junction morpholino model. Our data showed that abcc6a is not required for embryonic survival, but rather that it has an essential role in controlling mineralization. The three models developed very similar hypermineralization of spine and ribs starting embryonically and progressing in adulthood with development of scoliosis. Our results indicate a direct relation between loss of abcc6a expression and dysregulated osteogenesis. As such, our models recapitulate part of the human phenotype in which ectopic mineralization and pro-osteogenic signaling have been reported. Because of its reproducibility in three models and its ease of quantification, we consider this phenotype to be unequivocally the result of abcc6 deficiency and, as such, an excellent readout for drug screening purposes and multiplex mutagene analysis.},
}

ATP-Binding Cassette Transporters/*genetics
Animals
CRISPR-Cas Systems
Calcification, Physiologic/genetics
Disease Models, Animal
Gene Knockout Techniques
Humans
Morpholinos/genetics
Mutation/*genetics
Phenotype
Pseudoxanthoma Elasticum/*genetics
Ribs/*physiology
Scoliosis/*genetics
Spine/*physiology
Zebrafish
Zebrafish Proteins/*genetics

@article {pmid31513626,
year = {2019},
author = {Wu, YW and Yang, SH and Hwangbo, M and Chu, KH},
title = {Analysis of Zobellella denitrificans ZD1 draft genome: Genes and gene clusters responsible for high polyhydroxybutyrate (PHB) production from glycerol under saline conditions and its CRISPR-Cas system.},
journal = {PloS one},
volume = {14},
number = {9},
pages = {e0222143},
doi = {10.1371/journal.pone.0222143},
pmid = {31513626},
issn = {1932-6203},
abstract = {Polyhydroxybutyrate (PHB) is biodegradable and renewable and thus considered as a promising alternative to petroleum-based plastics. However, PHB production is costly due to expensive carbon sources for culturing PHB-accumulating microorganisms under sterile conditions. We discovered a hyper PHB-accumulating denitrifying bacterium, Zobellella denitrificans ZD1 (referred as strain ZD1 hereafter) capable of using non-sterile crude glycerol (a waste from biodiesel production) and nitrate to produce high PHB yield under saline conditions. Nevertheless, the underlying genetic mechanisms of PHB production in strain ZD1 have not been elucidated. In this study, we discovered a complete pathway of glycerol conversion to PHB, a novel PHB synthesis gene cluster, a salt-tolerant gene cluster, denitrifying genes, and an assimilatory nitrate reduction gene cluster in the ZD1 genome. Interestingly, the novel PHB synthesis gene cluster was found to be conserved among marine Gammaproteobacteria. Higher levels of PHB accumulation were linked to higher expression levels of the PHB synthesis gene cluster in ZD1 grown with glycerol and nitrate under saline conditions. Additionally, a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas type-I-E antiviral system was found in the ZD1 genome along with a long spacer list, in which most of the spacers belong to either double-stranded DNA viruses or unknown phages. The results of the genome analysis revealed strain ZD1 used the novel PHB gene cluster to produce PHB from non-sterile crude glycerol under saline conditions.},
}

@article {pmid30859452,
year = {2019},
author = {Zhang, J and Zhu, Z and Yue, W and Li, J and Chen, Q and Yan, Y and Lei, A and Hua, J},
title = {Establishment of CRISPR/Cas9-Mediated Knock-in System for Porcine Cells with High Efficiency.},
journal = {Applied biochemistry and biotechnology},
volume = {189},
number = {1},
pages = {26-36},
doi = {10.1007/s12010-019-02984-5},
pmid = {30859452},
issn = {1559-0291},
support = {31572399//Major Research Plan/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Swine ; Transgenes ; },
abstract = {Since the birth of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, the new genome engineering technology has become a hot topic in the scientific community. However, for swine, the system of pig cells' homology directed repair (HDR) is generally unstable and costly. Here, we aim to make knock-in of porcine cells more realizable. The Rosa26 locus was chosen for gene editing. Through the optimization of strategy, an efficient sgRNA was selected by TIDE analysis. Correspondingly, a vector system was constructed for gene insertion in pRosa26 locus by homologous recombination. A large percentage of cells whose gene is edited easily result in apoptosis. To improve the positive rate, culturing systems have been optimized. Sequence alignment and nuclear transfer confirmed that we got two knock-in cell lines and transgene primary porcine fetal fibroblasts (PFFs) successfully. Results showed that the gene editing platform we used can obtain genetically modified pig cells stably and efficiently. This system can contribute to pig gene research and production of transgenic pigs.},
}

Animals
*CRISPR-Cas Systems
Cell Line
Gene Editing
Swine
Transgenes

@article {pmid30096351,
year = {2018},
author = {Fenini, G and Grossi, S and Contassot, E and Biedermann, T and Reichmann, E and French, LE and Beer, HD},
title = {Genome Editing of Human Primary Keratinocytes by CRISPR/Cas9 Reveals an Essential Role of the NLRP1 Inflammasome in UVB Sensing.},
journal = {The Journal of investigative dermatology},
volume = {138},
number = {12},
pages = {2644-2652},
doi = {10.1016/j.jid.2018.07.016},
pmid = {30096351},
issn = {1523-1747},
mesh = {Adaptor Proteins, Signal Transducing/*metabolism ; Apoptosis Regulatory Proteins/*metabolism ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cells, Cultured ; Gene Editing/*methods ; Humans ; Inflammasomes/*metabolism ; Interleukin-18/metabolism ; Interleukin-1beta/metabolism ; Keratinocytes/*physiology ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism ; Nigericin/pharmacology ; Primary Cell Culture ; Radiodermatitis/*genetics/metabolism ; Ultraviolet Rays/*adverse effects ; },
abstract = {By forming a protective barrier, epidermal keratinocytes represent the first line of defense against environmental insults. UVB radiation of the sun is a major challenge for the skin and can induce inflammation, aging, and eventually skin cancer. UVB induces an immune response in human keratinocytes resulting in activation and secretion of the proinflammatory cytokines proIL-1β and -18. This is mediated by an assembly of protein complexes, termed inflammasomes. However, the mechanisms underlying sensing of UVB by keratinocytes, and particularly the types of inflammasomes required for cytokine secretion, are a matter of debate. To address these questions, we established a protocol that allows the generation of CRISPR/Cas9-targeted human primary keratinocytes. Our experiments showed an essential role of the NLRP1 rather than the NLRP3 inflammasome in UVB sensing and subsequent IL-1β and -18 secretion by keratinocytes. Moreover, NLRP1 but not NLRP3 was required for inflammasome activation in response to nigericin, a potassium ionophore and well-established NLRP3 activator in immune cells. Because the CRISPR/Cas9-targeted cells retained their full differentiation capacity, genome editing of human primary keratinocytes might be useful for numerous research and medical applications.},
}

Adaptor Proteins, Signal Transducing/*metabolism
Apoptosis Regulatory Proteins/*metabolism
CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
Cells, Cultured
Gene Editing/*methods
Humans
Inflammasomes/*metabolism
Interleukin-18/metabolism
Interleukin-1beta/metabolism
Keratinocytes/*physiology
NLR Family, Pyrin Domain-Containing 3 Protein/metabolism
Nigericin/pharmacology
Primary Cell Culture
Radiodermatitis/*genetics/metabolism
Ultraviolet Rays/*adverse effects

@article {pmid29614266,
year = {2018},
author = {Wu, CM and Roth, TL and Baglaenko, Y and Ferri, DM and Brauer, P and Zuniga-Pflucker, JC and Rosbe, KW and Wither, JE and Marson, A and Allen, CDC},
title = {Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins.},
journal = {Journal of immunological methods},
volume = {457},
number = {},
pages = {33-40},
pmid = {29614266},
issn = {1872-7905},
support = {P30 DK063720/DK/NIDDK NIH HHS/United States ; R01 AI130470/AI/NIAID NIH HHS/United States ; T32 DK007418/DK/NIDDK NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; PJT-148948//CIHR/Canada ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adolescent ; Adult ; B-Lymphocytes/*cytology/immunology ; *CRISPR-Cas Systems ; Cell Line ; Gene Knockout Techniques ; *Genetic Engineering ; Humans ; Mutation ; Palatine Tonsil/cytology ; Recombinational DNA Repair ; Ribonucleoproteins/*genetics ; Sialic Acid Binding Ig-like Lectin 2/genetics ; Young Adult ; },
abstract = {Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics.},
}

Adolescent
Adult
B-Lymphocytes/*cytology/immunology
*CRISPR-Cas Systems
Cell Line
Gene Knockout Techniques
*Genetic Engineering
Humans
Mutation
Palatine Tonsil/cytology
Recombinational DNA Repair
Ribonucleoproteins/*genetics
Sialic Acid Binding Ig-like Lectin 2/genetics
Young Adult

@article {pmid31507572,
year = {2019},
author = {Gao, NJ and Al-Bassam, MM and Poudel, S and Wozniak, JM and Gonzalez, DJ and Olson, J and Zengler, K and Nizet, V and Valderrama, JA},
title = {Functional and Proteomic Analysis of Streptococcus pyogenes Virulence Upon Loss of Its Native Cas9 Nuclease.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {1967},
doi = {10.3389/fmicb.2019.01967},
pmid = {31507572},
issn = {1664-302X},
abstract = {The public health impact of Streptococcus pyogenes (group A Streptococcus, GAS) as a top 10 cause of infection-related mortality in humans contrasts with its benefit to biotechnology as the main natural source of Cas9 nuclease, the key component of the revolutionary CRISPR-Cas9 gene editing platform. Despite widespread knowledge acquired in the last decade on the molecular mechanisms by which GAS Cas9 achieves precise DNA targeting, the functions of Cas9 in the biology and pathogenesis of its native organism remain unknown. In this study, we generated an isogenic serotype M1 GAS mutant deficient in Cas9 protein and compared its behavior and phenotypes to the wild-type parent strain. Absence of Cas9 was linked to reduced GAS epithelial cell adherence, reduced growth in human whole blood ex vivo, and attenuation of virulence in a murine necrotizing skin infection model. Virulence defects of the GAS Δcas9 strain were explored through quantitative proteomic analysis, revealing a significant reduction in the abundance of key GAS virulence determinants. Similarly, deletion of cas9 affected the expression of several known virulence regulatory proteins, indicating that Cas9 impacts the global architecture of GAS gene regulation.},
}

@article {pmid31506266,
year = {2019},
author = {Yin, Y and Yang, B and Entwistle, S},
title = {Bioinformatics Identification of Anti-CRISPR Loci by Using Homology, Guilt-by-Association, and CRISPR Self-Targeting Spacer Approaches.},
journal = {mSystems},
volume = {4},
number = {5},
pages = {},
doi = {10.1128/mSystems.00455-19},
pmid = {31506266},
issn = {2379-5077},
abstract = {Anti-CRISPR (Acr) loci/operons encode Acr proteins and Acr-associated (Aca) proteins. Forty-five Acr families have been experimentally characterized inhibiting seven subtypes of CRISPR-Cas systems. We have developed a bioinformatics pipeline to identify genomic loci containing Acr homologs and/or Aca homologs by combining three computational approaches: homology, guilt-by-association, and self-targeting spacers. Homology search found thousands of Acr homologs in bacterial and viral genomes, but most are homologous to AcrIIA7 and AcrIIA9. Investigating the gene neighborhood of these Acr homologs revealed that only a small percentage (23.0% in bacteria and 8.2% in viruses) of them have neighboring Aca homologs and thus form Acr-Aca operons. Surprisingly, although a self-targeting spacer is a strong indicator of the presence of Acr genes in a genome, a large percentage of Acr-Aca loci are found in bacterial genomes without self-targeting spacers or even without complete CRISPR-Cas systems. Additionally, for Acr homologs from genomes with self-targeting spacers, homology-based Acr family assignments do not always agree with the self-targeting CRISPR-Cas subtypes. Last, by investigating Acr genomic loci coexisting with self-targeting spacers in the same genomes, five known subtypes (I-C, I-E, I-F, II-A, and II-C) and five new subtypes (I-B, III-A, III-B, IV-A, and V-U4) of Acrs were inferred. Based on these findings, we conclude that the discovery of new anti-CRISPRs should not be restricted to genomes with self-targeting spacers and loci with Acr homologs. The evolutionary arms race of CRISPR-Cas systems and anti-CRISPR systems may have driven the adaptive and rapid gain and loss of these elements in closely related genomes.IMPORTANCE As a naturally occurring adaptive immune system, CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated genes) systems are widely found in bacteria and archaea to defend against viruses. Since 2013, the application of various bacterial CRISPR-Cas systems has become very popular due to their development into targeted and programmable genome engineering tools with the ability to edit almost any genome. As the natural off-switch of CRISPR-Cas systems, anti-CRISPRs have a great potential to serve as regulators of CRISPR-Cas tools and enable safer and more controllable genome editing. This study will help understand the relative usefulness of the three bioinformatics approaches for new Acr discovery, as well as guide the future development of new bioinformatics tools to facilitate anti-CRISPR research. The thousands of Acr homologs and hundreds of new anti-CRISPR loci identified in this study will be a valuable data resource for genome engineers to search for new CRISPR-Cas regulators.},
}

@article {pmid31504832,
year = {2019},
author = {González-Delgado, A and Mestre, MR and Martínez-Abarca, F and Toro, N},
title = {Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated with a type III-D CRISPR-Cas system in Vibrio vulnificus.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkz746},
pmid = {31504832},
issn = {1362-4962},
abstract = {The association of reverse transcriptases (RTs) with CRISPR-Cas system has recently attracted interest because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. However, our understanding of this spacer acquisition process remains limited. We characterized the in vivo acquisition of spacers mediated by an RT-Cas1 fusion protein linked to a type III-D system from Vibrio vulnificus strain YJ016, and showed that the adaptation module, consisting of the RT-Cas1 fusion, two different Cas2 proteins (A and B) and one of the two CRISPR arrays, was completely functional in a heterologous host. We found that mutations of the active site of the RT domain significantly decreased the acquisition of new spacers and showed that this RT-Cas1-associated adaptation module was able to incorporate spacers from RNA molecules into the CRISPR array. We demonstrated that the two Cas2 proteins of the adaptation module were required for spacer acquisition. Furthermore, we found that several sequence-specific features were required for the acquisition and integration of spacers derived from any region of the genome, with no bias along the 5'and 3'ends of coding sequences. This study provides new insight into the RT-Cas1 fusion protein-mediated acquisition of spacers from RNA molecules.},
}

@article {pmid31502713,
year = {2019},
author = {Peters, JE},
title = {Targeted transposition with Tn7 elements: Safe sites, mobile plasmids, CRISPR/Cas and beyond.},
journal = {Molecular microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/mmi.14383},
pmid = {31502713},
issn = {1365-2958},
abstract = {Transposon Tn7 is notable for the control it exercises over where transposition events are directed. One Tn7 integration pathways recognizes a highly conserved attachment (att) site in the chromosome, while a second pathway specifically recognizes mobile plasmids that facilitate transfer of the element to new hosts. In this review, I discuss newly discovered families of Tn7-like elements with different targeting pathways. Perhaps the most exciting examples are multiple instances where Tn7-like elements have repurposed CRISPR/Cas systems. In these cases, the CRISPR/Cas systems have lost their canonical defensive function to destroy incoming mobile elements; instead, the systems have been naturally adapted to use guide RNAs to specifically direct transposition into these mobile elements. The new families of Tn7-like elements also include a variety of novel att sites in bacterial chromosomes where genome islands can form. Interesting families have also been revealed where proteins described in the prototypic Tn7 element are fused or otherwise repurposed for new dual activities. This expanded understanding of Tn7-like elements broadens our view of how genetic systems are repurposed and provides potentially exciting new tools for genome modification and genomics. Future opportunities and challenges to understanding the impact of the new families of Tn7-like elements are discussed.},
}

@article {pmid31502535,
year = {2019},
author = {Forsberg, KJ and Bhatt, IV and Schmidtke, DT and Javanmardi, K and Dillard, KE and Stoddard, BL and Finkelstein, IJ and Kaiser, BK and Malik, HS},
title = {Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
doi = {10.7554/eLife.46540},
pmid = {31502535},
issn = {2050-084X},
support = {R01 GM105691/GM/NIGMS NIH HHS/United States ; F31 GM125201/GM/NIGMS NIH HHS/United States ; R01 GM124131/GM/NIGMS NIH HHS/United States ; F-1808//Welch Foundation/ ; },
abstract = {CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because Acrs are challenging to identify, their natural diversity and impact on microbial ecosystems are underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection to isolate ten DNA fragments from human oral and fecal metagenomes that inhibit Streptococcus pyogenes Cas9 (SpyCas9) in Escherichia coli. The most potent Acr from this set, AcrIIA11, was recovered from a Lachnospiraceae phage. We found that AcrIIA11 inhibits SpyCas9 in bacteria and in human cells. AcrIIA11 homologs are distributed across diverse bacteria; many distantly-related homologs inhibit both SpyCas9 and a divergent Cas9 from Treponema denticola. We find that AcrIIA11 antagonizes SpyCas9 using a different mechanism than other previously characterized Type II-A Acrs. Our study highlights the power of functional selection to uncover widespread Cas9 inhibitors within diverse microbiomes.},
}

@article {pmid31501243,
year = {2019},
author = {Callies, LK and Tadeo, D and Simper, J and Bugge, TH and Szabo, R},
title = {Iterative, multiplexed CRISPR-mediated gene editing for functional analysis of complex protease gene clusters.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA119.009773},
pmid = {31501243},
issn = {1083-351X},
abstract = {Elucidation of gene function by reverse genetics in animal models frequently is complicated by the functional redundancy of homologous genes. This obstacle often is compounded by the tight clustering of homologous genes, which precludes the generation of multi-gene-deficient animals through standard interbreeding of single-deficient animals. Here, we describe an iterative, multiplexed CRISPR-based approach for simultaneous gene editing in the complex seven-member HAT/DESC cluster of membrane-anchored serine proteases. Through four cycles of targeting, we generated a library of 18 unique congenic mouse strains lacking combinations of HAT/DESC proteases, including a mouse strain deficient in all seven proteases. Using this library, we demonstrate that HAT/DESC proteases are dispensable for term development, postnatal health, and fertility, and that the recently described function of the HAT-like 4 protease in epidermal barrier formation is unique among all HAT/DESC proteases. The study demonstrates the potential of iterative, multiplexed CRISPR-mediated gene editing for functional analysis of multi-gene clusters, and it provides a large array of new congenic mouse strains for the study of HAT/DESC proteases in physiological and in pathophysiological processes.},
}

@article {pmid31273196,
year = {2019},
author = {Buchmuller, BC and Herbst, K and Meurer, M and Kirrmaier, D and Sass, E and Levy, ED and Knop, M},
title = {Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2960},
doi = {10.1038/s41467-019-10816-7},
pmid = {31273196},
issn = {2041-1723},
support = {KN498/12-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/International ; INST 35/1314-1 FUGG//Deutsche Forschungsgemeinschaft (German Research Foundation)/International ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clone Cells ; Gene Library ; Genetic Engineering ; *Genetic Techniques ; Genome, Fungal ; Green Fluorescent Proteins/metabolism ; Nuclear Proteins/metabolism ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Clone collections of modified strains ("libraries") are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.},
}

CRISPR-Cas Systems/*genetics
Clone Cells
Gene Library
Genetic Engineering
*Genetic Techniques
Genome, Fungal
Green Fluorescent Proteins/metabolism
Nuclear Proteins/metabolism
Saccharomyces cerevisiae/*genetics

@article {pmid31036063,
year = {2019},
author = {Zhang, Y and Qi, Y},
title = {CRISPR enables directed evolution in plants.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {83},
doi = {10.1186/s13059-019-1693-4},
pmid = {31036063},
issn = {1474-760X},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Oryza/*genetics ; RNA Splicing ; Spliceosomes ; },
abstract = {A proof-of-concept study has demonstrated the application of CRISPR-Cas9 for directed evolution in rice, engineering crops for desired traits.},
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Oryza/*genetics
RNA Splicing
Spliceosomes

@article {pmid30470168,
year = {2018},
author = {Gao, Z and Herrera-Carrillo, E and Berkhout, B},
title = {Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA.},
journal = {RNA biology},
volume = {15},
number = {12},
pages = {1458-1467},
doi = {10.1080/15476286.2018.1551703},
pmid = {30470168},
issn = {1555-8584},
mesh = {Bacterial Proteins/*metabolism ; Base Sequence ; CRISPR-Cas Systems ; Cell Line ; Chromosomes/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/*metabolism ; *Gene Editing ; Gene Expression Regulation ; Genes, Reporter ; Humans ; RNA, Catalytic/chemistry/*genetics ; },
abstract = {The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system expands the genome editing toolbox. This system exhibits several distinct features compared to the widely used CRISPR-Cas9 system, but has reduced gene editing efficiency. To optimize the CRISPR-Cpf1 (Cas12a) system, we report the inclusion of self-cleaving ribozymes that facilitate processing of the crRNA transcript to produce the precise guide molecule. Insertion of the 3'-terminal HDV ribozyme boosted the gene editing activity of the CRISPR-Cpf1 system ranging from 1.1 to 5.2 fold. We also demonstrate that this design can enhance CRISPR-based gene activation. We thus generated an improved CRISPR-Cpf1 system for more efficient gene editing and gene regulation.},
}

Bacterial Proteins/*metabolism
Base Sequence
CRISPR-Cas Systems
Cell Line
Chromosomes/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Endonucleases/*metabolism
*Gene Editing
Gene Expression Regulation
Genes, Reporter
Humans
RNA, Catalytic/chemistry/*genetics

@article {pmid30417100,
year = {2018},
author = {Wang, L and Ozark, PA and Smith, ER and Zhao, Z and Marshall, SA and Rendleman, EJ and Piunti, A and Ryan, C and Whelan, AL and Helmin, KA and Morgan, MA and Zou, L and Singer, BD and Shilatifard, A},
title = {TET2 coactivates gene expression through demethylation of enhancers.},
journal = {Science advances},
volume = {4},
number = {11},
pages = {eaau6986},
pmid = {30417100},
issn = {2375-2548},
support = {K08 HL128867/HL/NHLBI NIH HHS/United States ; R35 CA197569/CA/NCI NIH HHS/United States ; R50 CA211428/CA/NCI NIH HHS/United States ; T32 CA070085/CA/NCI NIH HHS/United States ; },
mesh = {Breast Neoplasms/genetics/*metabolism/pathology ; CRISPR-Cas Systems ; Cell Differentiation ; Cohort Studies ; DNA Methylation ; DNA-Binding Proteins/antagonists & inhibitors/genetics/*metabolism ; *Demethylation ; *Enhancer Elements, Genetic ; Epigenesis, Genetic ; Estrogen Receptor alpha/antagonists & inhibitors/genetics/*metabolism ; Female ; *Gene Expression Regulation, Neoplastic ; Humans ; Proto-Oncogene Proteins/antagonists & inhibitors/genetics/*metabolism ; Survival Rate ; Tumor Cells, Cultured ; },
abstract = {The tet methylcytosine dioxygenase 2 (TET2) enzyme catalyzes the conversion of the modified DNA base 5-methylcytosine to 5-hydroxymethylcytosine. TET2 is frequently mutated or dysregulated in multiple human cancers, and loss of TET2 is associated with changes in DNA methylation patterns. Here, using newly developed TET2-specific antibodies and the estrogen response as a model system for studying the regulation of gene expression, we demonstrate that endogenous TET2 occupies active enhancers and facilitates the proper recruitment of estrogen receptor α (ERα). Knockout of TET2 by CRISPR-CAS9 leads to a global increase of DNA methylation at enhancers, resulting in attenuation of the estrogen response. We further identified a positive feedback loop between TET2 and ERα, which further requires MLL3 COMPASS at these enhancers. Together, this study reveals an epigenetic axis coordinating a transcriptional program through enhancer activation via DNA demethylation.},
}

Breast Neoplasms/genetics/*metabolism/pathology
CRISPR-Cas Systems
Cell Differentiation
Cohort Studies
DNA Methylation
DNA-Binding Proteins/antagonists & inhibitors/genetics/*metabolism
*Demethylation
*Enhancer Elements, Genetic
Epigenesis, Genetic
Estrogen Receptor alpha/antagonists & inhibitors/genetics/*metabolism
Female
*Gene Expression Regulation, Neoplastic
Humans
Proto-Oncogene Proteins/antagonists & inhibitors/genetics/*metabolism
Survival Rate
Tumor Cells, Cultured

@article {pmid30217531,
year = {2019},
author = {Spille, JH and Hecht, M and Grube, V and Cho, WK and Lee, C and Cissé, II},
title = {A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells.},
journal = {Methods (San Diego, Calif.)},
volume = {153},
number = {},
pages = {35-45},
doi = {10.1016/j.ymeth.2018.09.004},
pmid = {30217531},
issn = {1095-9130},
support = {DP2 CA195769/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Gene Editing/*methods ; Mice ; Mouse Embryonic Stem Cells/*metabolism ; RNA, Messenger/*analysis/chemistry/metabolism ; Single Molecule Imaging/*methods ; },
abstract = {The MS2 system is a powerful tool for investigating transcription dynamics at the single molecule directly in live cells. In the past, insertion of the RNA-labelling cassette at specific gene loci has been a major hurdle. Here, we present a CRISPR/Cas9-based approach to insert an MS2 cassette with selectable marker at the start of the 3' untranslated region of any coding gene. We demonstrate applicability of our approach by tagging RNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells. Using quantitative fluorescence microscopy we determine the number of nascent transcripts at the Esrrb locus and the fraction of cells expressing the gene. We find that upon differentiation towards epiblast-like cells, expression of Esrrb is down-regulated in an increasing fraction of cells in a binary manner.},
}

Animals
*CRISPR-Cas Systems
Cells, Cultured
Gene Editing/*methods
Mice
Mouse Embryonic Stem Cells/*metabolism
RNA, Messenger/*analysis/chemistry/metabolism
Single Molecule Imaging/*methods

@article {pmid30193003,
year = {2018},
author = {Dunbar, KL and Büttner, H and Molloy, EM and Dell, M and Kumpfmüller, J and Hertweck, C},
title = {Genome Editing Reveals Novel Thiotemplated Assembly of Polythioamide Antibiotics in Anaerobic Bacteria.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {57},
number = {43},
pages = {14080-14084},
doi = {10.1002/anie.201807970},
pmid = {30193003},
issn = {1521-3773},
mesh = {Anti-Bacterial Agents/*chemistry/pharmacology ; Bacteria, Anaerobic/*chemistry/genetics ; CRISPR-Cas Systems ; Carbon-13 Magnetic Resonance Spectroscopy ; Chromatography, High Pressure Liquid ; Clostridiales/*chemistry/genetics ; DNA Gyrase/drug effects ; *Gene Editing ; Genes, Bacterial ; Introns ; Mass Spectrometry ; Multigene Family ; Peptide Synthases/chemistry ; Proton Magnetic Resonance Spectroscopy ; Thioamides/*chemistry/pharmacology ; },
abstract = {Closthioamide (CTA) is a unique symmetric nonribosomal peptide with six thioamide moieties that is produced by the Gram-positive obligate anaerobe Ruminiclostridium cellulolyticum. CTA displays potent inhibitory activity against important clinical pathogens, making it a promising drug candidate. Yet, the biosynthesis of this DNA gyrase-targeting antibiotic has remained enigmatic. Using a combination of genome mining, genome editing (targeted group II intron, CRISPR/Cas9), and heterologous expression, we show that CTA biosynthesis involves specialized enzymes for starter unit biosynthesis, amide bond formation, thionation, and dimerization. Surprisingly, CTA biosynthesis involves a novel thiotemplated peptide assembly line that markedly differs from known nonribosomal peptide synthetases. These findings provide the first insights into the biosynthesis of thioamide-containing nonribosomal peptides and offer a starting point for the discovery of related natural products.},
}

Anti-Bacterial Agents/*chemistry/pharmacology
Bacteria, Anaerobic/*chemistry/genetics
CRISPR-Cas Systems
Carbon-13 Magnetic Resonance Spectroscopy
Chromatography, High Pressure Liquid
Clostridiales/*chemistry/genetics
DNA Gyrase/drug effects
*Gene Editing
Genes, Bacterial
Introns
Mass Spectrometry
Multigene Family
Peptide Synthases/chemistry
Proton Magnetic Resonance Spectroscopy
Thioamides/*chemistry/pharmacology

@article {pmid30063096,
year = {2018},
author = {Kong, J and Wang, Y and Zhang, J and Qi, W and Su, R and He, Z},
title = {Rationally Designed Peptidyl Virus-Like Particles Enable Targeted Delivery of Genetic Cargo.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {57},
number = {43},
pages = {14032-14036},
doi = {10.1002/anie.201805868},
pmid = {30063096},
issn = {1521-3773},
mesh = {CRISPR-Cas Systems ; Cell Line ; Electrophoretic Mobility Shift Assay ; *Gene Transfer Techniques ; HIV/chemistry/physiology ; Humans ; Membrane Fusion ; Microscopy, Atomic Force ; Microscopy, Electron, Transmission ; Nanoparticles/chemistry ; Peptides/*chemistry ; Simian virus 40/chemistry/physiology ; Virion/*chemistry ; },
abstract = {We report a strategy to construct peptidyl virus-like particles (pVLPs) by mimicking the human immunodeficiency virus and simian virus 40. We designed two viral peptides with cell/nucleus-targeting capabilities that can co-assemble in their active conformations into well-defined nanoparticles. The self-assembled nanoparticles can encapsulate the DNA of clustered regularly interspaced short palindromic repeat associated proteins 9 (CRISPR/Cas9) to form biodegradable pVLPs with excellent cell-targeting ability and biocompatibility. The pVLPs can penetrate the cellular membrane and deliver genetic cargos into the nucleus through the viral entry route. The results provide a promising pathway for engineering artificial viruses with desired functions.},
}

CRISPR-Cas Systems
Cell Line
Electrophoretic Mobility Shift Assay
*Gene Transfer Techniques
HIV/chemistry/physiology
Humans
Membrane Fusion
Microscopy, Atomic Force
Microscopy, Electron, Transmission
Nanoparticles/chemistry
Peptides/*chemistry
Simian virus 40/chemistry/physiology
Virion/*chemistry

@article {pmid29843775,
year = {2018},
author = {Shapiro, E},
title = {On the journey from nematode to human, scientists dive by the zebrafish cell lineage tree.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {63},
pmid = {29843775},
issn = {1474-760X},
support = {670535//European Research Council ()/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Lineage ; Gene Editing ; Humans ; *Nematoda ; RNA, Guide ; Trees ; Zebrafish/*genetics ; },
abstract = {Three recent single-cell papers use novel CRISPR-Cas9-sgRNA genome editing methods to shed light on the zebrafish cell lineage tree.},
}

Animals
CRISPR-Cas Systems
Cell Lineage
Gene Editing
Humans
*Nematoda
RNA, Guide
Trees
Zebrafish/*genetics

@article {pmid29708508,
year = {2018},
author = {Ribierre, T and Deleuze, C and Bacq, A and Baldassari, S and Marsan, E and Chipaux, M and Muraca, G and Roussel, D and Navarro, V and Leguern, E and Miles, R and Baulac, S},
title = {Second-hit mosaic mutation in mTORC1 repressor DEPDC5 causes focal cortical dysplasia-associated epilepsy.},
journal = {The Journal of clinical investigation},
volume = {128},
number = {6},
pages = {2452-2458},
pmid = {29708508},
issn = {1558-8238},
support = {322721//European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Dendrites/metabolism/pathology ; *Epilepsies, Partial/genetics/metabolism/pathology ; Female ; *GTPase-Activating Proteins/genetics/metabolism ; *Germ-Line Mutation ; Humans ; Male ; *Malformations of Cortical Development/genetics/metabolism/pathology ; *Mechanistic Target of Rapamycin Complex 1/genetics/metabolism ; Mice ; Mice, Mutant Strains ; Neurons/metabolism/pathology ; *Repressor Proteins/genetics/metabolism ; Spine/metabolism/pathology ; },
abstract = {DEP domain-containing 5 protein (DEPDC5) is a repressor of the recently recognized amino acid-sensing branch of the mTORC1 pathway. So far, its function in the brain remains largely unknown. Germline loss-of-function mutations in DEPDC5 have emerged as a major cause of familial refractory focal epilepsies, with case reports of sudden unexpected death in epilepsy (SUDEP). Remarkably, a fraction of patients also develop focal cortical dysplasia (FCD), a neurodevelopmental cortical malformation. We therefore hypothesized that a somatic second-hit mutation arising during brain development may support the focal nature of the dysplasia. Here, using postoperative human tissue, we provide the proof of concept that a biallelic 2-hit - brain somatic and germline - mutational mechanism in DEPDC5 causes focal epilepsy with FCD. We discovered a mutation gradient with a higher rate of mosaicism in the seizure-onset zone than in the surrounding epileptogenic zone. Furthermore, we demonstrate the causality of a Depdc5 brain mosaic inactivation using CRISPR-Cas9 editing and in utero electroporation in a mouse model recapitulating focal epilepsy with FCD and SUDEP-like events. We further unveil a key role of Depdc5 in shaping dendrite and spine morphology of excitatory neurons. This study reveals promising therapeutic avenues for treating drug-resistant focal epilepsies with mTORC1-targeting molecules.},
}

Animals
CRISPR-Cas Systems
Dendrites/metabolism/pathology
*Epilepsies, Partial/genetics/metabolism/pathology
Female
*GTPase-Activating Proteins/genetics/metabolism
*Germ-Line Mutation
Humans
Male
*Malformations of Cortical Development/genetics/metabolism/pathology
*Mechanistic Target of Rapamycin Complex 1/genetics/metabolism
Mice
Mice, Mutant Strains
Neurons/metabolism/pathology
*Repressor Proteins/genetics/metabolism
Spine/metabolism/pathology

@article {pmid29531313,
year = {2018},
author = {Li, SY and Cheng, QX and Liu, JK and Nie, XQ and Zhao, GP and Wang, J},
title = {CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA.},
journal = {Cell research},
volume = {28},
number = {4},
pages = {491-493},
pmid = {29531313},
issn = {1748-7838},
mesh = {CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/*genetics ; Gene Editing ; RNA, Guide/genetics ; },
}

CRISPR-Associated Proteins/genetics
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA, Single-Stranded/*genetics
Gene Editing
RNA, Guide/genetics

@article {pmid29522311,
year = {2018},
author = {De Silva Feelixge, HS and Stone, D and Roychoudhury, P and Aubert, M and Jerome, KR},
title = {CRISPR/Cas9 and Genome Editing for Viral Disease-Is Resistance Futile?.},
journal = {ACS infectious diseases},
volume = {4},
number = {6},
pages = {871-880},
pmid = {29522311},
issn = {2373-8227},
support = {R21 AI117519/AI/NIAID NIH HHS/United States ; UM1 AI126623/AI/NIAID NIH HHS/United States ; P30 AI027757/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Carrier State ; Combined Modality Therapy ; Drug Resistance, Viral ; *Gene Editing ; Genetic Therapy ; Genetic Variation ; *Genome, Viral ; Humans ; Quasispecies/genetics ; RNA, Guide ; RNA, Viral ; Virus Diseases/*therapy/*virology ; Virus Replication/genetics ; Viruses/drug effects/*genetics ; },
abstract = {Chronic viral infections remain a major public health issue affecting millions of people worldwide. Highly active antiviral treatments have significantly improved prognosis and infection-related morbidity and mortality but have failed to eliminate persistent viral forms. Therefore, new strategies to either eradicate or control these viral reservoirs are paramount to allow patients to stop antiretroviral therapy and realize a cure. Viral genome disruption based on gene editing by programmable endonucleases is one promising curative gene therapy approach. Recent findings on RNA-guided human immunodeficiency virus 1 (HIV-1) genome cleavage by Cas9 and other gene-editing enzymes in latently infected cells have shown high levels of site-specific genome disruption and potent inhibition of virus replication. However, HIV-1 can readily develop resistance to genome editing at a single antiviral target site. Current data suggest that cellular repair associated with DNA double-strand breaks can accelerate the emergence of resistance. On the other hand, a combination antiviral target strategy can exploit the same repair mechanism to functionally cure HIV-1 infection in vitro while avoiding the development of resistance. This perspective summarizes recent findings on the biology of resistance to genome editing and discusses the significance of viral genetic diversity on the application of gene editing strategies toward cure.},
}

Animals
*CRISPR-Cas Systems
Carrier State
Combined Modality Therapy
Drug Resistance, Viral
*Gene Editing
Genetic Therapy
Genetic Variation
*Genome, Viral
Humans
Quasispecies/genetics
RNA, Guide
RNA, Viral
Virus Diseases/*therapy/*virology
Virus Replication/genetics
Viruses/drug effects/*genetics

@article {pmid29453406,
year = {2018},
author = {Borca, MV and Holinka, LG and Berggren, KA and Gladue, DP},
title = {CRISPR-Cas9, a tool to efficiently increase the development of recombinant African swine fever viruses.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {3154},
pmid = {29453406},
issn = {2045-2322},
mesh = {African Swine Fever Virus/*genetics ; CRISPR-Cas Systems/*genetics ; DNA, Recombinant/*genetics ; Genetic Engineering/*methods ; High-Throughput Nucleotide Sequencing ; },
abstract = {African swine fever virus (ASFV) causes a highly contagious disease called African swine fever. This disease is often lethal for domestic pigs, causing extensive losses for the swine industry. ASFV is a large and complex double stranded DNA virus. Currently there is no commercially available treatment or vaccine to prevent this devastating disease. Development of recombinant ASFV for producing live-attenuated vaccines or studying the involvement of specific genes in virus virulence has relied on the relatively rare event of homologous recombination in primary swine macrophages, causing difficulty to purify the recombinant virus from the wild-type parental ASFV. Here we present the use of the CRISPR-Cas9 gene editing system as a more robust and efficient system to produce recombinant ASFVs. Using CRISPR-Cas9 a recombinant virus was efficiently developed by deleting the non-essential gene 8-DR from the genome of the highly virulent field strain Georgia07 using swine macrophages as cell substrate.},
}

African Swine Fever Virus/*genetics
CRISPR-Cas Systems/*genetics
DNA, Recombinant/*genetics
Genetic Engineering/*methods
High-Throughput Nucleotide Sequencing

@article {pmid29445193,
year = {2018},
author = {Celestini, V and Tezil, T and Russo, L and Fasano, C and Sanese, P and Forte, G and Peserico, A and Lepore Signorile, M and Longo, G and De Rasmo, D and Signorile, A and Gadaleta, RM and Scialpi, N and Terao, M and Garattini, E and Cocco, T and Villani, G and Moschetta, A and Grossi, V and Simone, C},
title = {Uncoupling FoxO3A mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy.},
journal = {Cell death & disease},
volume = {9},
number = {2},
pages = {231},
pmid = {29445193},
issn = {2041-4889},
mesh = {AMP-Activated Protein Kinases/genetics/metabolism ; Animals ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Nucleus/drug effects/*metabolism ; Cell Survival ; Cisplatin/pharmacology ; Extracellular Signal-Regulated MAP Kinases/genetics/metabolism ; Fluorouracil/pharmacology ; Forkhead Box Protein O3/*genetics/metabolism ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Genome, Mitochondrial ; HEK293 Cells ; Humans ; Irinotecan/pharmacology ; MAP Kinase Kinase Kinases/genetics/metabolism ; Metformin/pharmacology ; Mice ; Mice, Inbred C57BL ; Mitochondria/drug effects/genetics/*metabolism ; NIH 3T3 Cells ; Phosphorylation ; Signal Transduction ; Stress, Physiological/drug effects/genetics ; },
abstract = {While aberrant cancer cell growth is frequently associated with altered biochemical metabolism, normal mitochondrial functions are usually preserved and necessary for full malignant transformation. The transcription factor FoxO3A is a key determinant of cancer cell homeostasis, playing a dual role in survival/death response to metabolic stress and cancer therapeutics. We recently described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells upon nutrient shortage. Here, we show that in metabolically stressed cancer cells, FoxO3A is recruited to the mitochondria through activation of MEK/ERK and AMPK, which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminal domain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression of the mitochondrial genome to support mitochondrial metabolism. Using FoxO3A-/- cancer cells generated with the CRISPR/Cas9 genome editing system and reconstituted with FoxO3A mutants being impaired in their nuclear or mitochondrial subcellular localization, we show that mitochondrial FoxO3A promotes survival in response to metabolic stress. In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into the mitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required for apoptosis induction by metformin. Elucidation of FoxO3A mitochondrial vs. nuclear functions in cancer cell homeostasis might help devise novel therapeutic strategies to selectively disable FoxO3A prosurvival activity.},
}

AMP-Activated Protein Kinases/genetics/metabolism
Animals
Antineoplastic Agents/*pharmacology
Apoptosis/drug effects/genetics
CRISPR-Cas Systems
Cell Line, Tumor
Cell Nucleus/drug effects/*metabolism
Cell Survival
Cisplatin/pharmacology
Extracellular Signal-Regulated MAP Kinases/genetics/metabolism
Fluorouracil/pharmacology
Forkhead Box Protein O3/*genetics/metabolism
Gene Editing
*Gene Expression Regulation, Neoplastic
Genome, Mitochondrial
HEK293 Cells
Humans
Irinotecan/pharmacology
MAP Kinase Kinase Kinases/genetics/metabolism
Metformin/pharmacology
Mice
Mice, Inbred C57BL
Mitochondria/drug effects/genetics/*metabolism
NIH 3T3 Cells
Phosphorylation
Signal Transduction
Stress, Physiological/drug effects/genetics

@article {pmid29362972,
year = {2018},
author = {Ferreira, R and David, F and Nielsen, J},
title = {Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape.},
journal = {Journal of industrial microbiology & biotechnology},
volume = {45},
number = {7},
pages = {467-480},
pmid = {29362972},
issn = {1476-5535},
mesh = {Bacteriophages/genetics ; *Biotechnology ; *CRISPR-Cas Systems/genetics/physiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Gene Expression Regulation ; Genetic Engineering/*methods ; Patents as Topic ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) is poised to become one of the key scientific discoveries of the twenty-first century. Originating from prokaryotic and archaeal immune systems to counter phage invasions, CRISPR-based applications have been tailored for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology.},
}

Bacteriophages/genetics
*Biotechnology
*CRISPR-Cas Systems/genetics/physiology
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
*Gene Expression Regulation
Genetic Engineering/*methods
Patents as Topic

@article {pmid31497003,
year = {2019},
author = {Spencer, BL and Deng, L and Patras, KA and Burcham, ZM and Sanches, GF and Nagao, PE and Doran, KS},
title = {Cas9 Contributes to Group B Streptococcal Colonization and Disease.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {1930},
doi = {10.3389/fmicb.2019.01930},
pmid = {31497003},
issn = {1664-302X},
abstract = {Group B Streptococcus (GBS) is a major opportunistic pathogen in certain adult populations, including pregnant women, and remains a leading etiologic agent of newborn disease. During pregnancy, GBS asymptomatically colonizes the vaginal tract of 20-30% of healthy women, but can be transmitted to the neonate in utero or during birth resulting in neonatal pneumonia, sepsis, meningitis, and subsequently 10-15% mortality regardless of antibiotic treatment. While various GBS virulence factors have been implicated in vaginal colonization and invasive disease, the regulation of many of these factors remains unclear. Recently, CRISPR-associated protein-9 (Cas9), an endonuclease known for its role in CRISPR/Cas immunity, has also been observed to modulate virulence in a number of bacterial pathogens. However, the role of Cas9 in GBS colonization and disease pathogenesis has not been well-studied. We performed allelic replacement of cas9 in GBS human clinical isolates of the hypervirulent sequence-type 17 strain lineage to generate isogenic Δcas9 mutants. Compared to parental strains, Δcas9 mutants were attenuated in murine models of hematogenous meningitis and vaginal colonization and exhibited significantly decreased invasion of human brain endothelium and adherence to vaginal epithelium. To determine if Cas9 alters transcription in GBS, we performed RNA-Seq analysis and found that 353 genes (>17% of the GBS genome) were differentially expressed between the parental WT and Δcas9 mutant strain. Significantly dysregulated genes included those encoding predicted virulence factors, metabolic factors, two-component systems (TCS), and factors important for cell wall formation. These findings were confirmed by qRT-PCR and suggest that Cas9 may regulate a significant portion of the GBS genome. We studied one of the TCS regulators, CiaR, that was significantly downregulated in the Δcas9 mutant strain. RNA-Seq analysis of the WT and ΔciaR strains demonstrated that almost all CiaR-regulated genes were also significantly regulated by Cas9, suggesting that Cas9 may modulate GBS gene expression through other regulators. Further we show that CiaR contributes to GBS vaginal colonization and persistence. Altogether, these data highlight the potential complexity and importance of the non-canonical function of Cas9 in GBS colonization and disease.},
}

@article {pmid31266936,
year = {2019},
author = {Dash, PK and Kaminski, R and Bella, R and Su, H and Mathews, S and Ahooyi, TM and Chen, C and Mancuso, P and Sariyer, R and Ferrante, P and Donadoni, M and Robinson, JA and Sillman, B and Lin, Z and Hilaire, JR and Banoub, M and Elango, M and Gautam, N and Mosley, RL and Poluektova, LY and McMillan, J and Bade, AN and Gorantla, S and Sariyer, IK and Burdo, TH and Young, WB and Amini, S and Gordon, J and Jacobson, JM and Edagwa, B and Khalili, K and Gendelman, HE},
title = {Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2753},
doi = {10.1038/s41467-019-10366-y},
pmid = {31266936},
issn = {2041-1723},
support = {P01 NS043985/NS/NINDS NIH HHS/United States ; P01 DA028555/DA/NIDA NIH HHS/United States ; R01NS036126//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/International ; P30MH062261//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/International ; R01DA042706//U.S. Department of Health & Human Services | NIH | National Institute on Drug Abuse (NIDA)/International ; P01DA037830//U.S. Department of Health & Human Services | NIH | National Institute on Drug Abuse (NIDA)/International ; R01MH110360//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/International ; P30MH092177//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/International ; R01NS034239//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/International ; },
mesh = {Adoptive Transfer ; Animals ; Anti-HIV Agents/*administration & dosage ; *CRISPR-Cas Systems ; Combined Modality Therapy ; DNA, Viral/genetics/immunology ; Gene Editing ; HIV Infections/drug therapy/immunology/*therapy/virology ; HIV-1/*genetics/immunology/physiology ; Humans ; Mice ; Treatment Outcome ; Virus Latency ; },
abstract = {Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.},
}

Adoptive Transfer
Animals
Anti-HIV Agents/*administration & dosage
*CRISPR-Cas Systems
Combined Modality Therapy
DNA, Viral/genetics/immunology
Gene Editing
HIV Infections/drug therapy/immunology/*therapy/virology
HIV-1/*genetics/immunology/physiology
Humans
Mice
Treatment Outcome
Virus Latency

@article {pmid31037730,
year = {2019},
author = {Kina, H and Yoshitani, T and Hanyu-Nakamura, K and Nakamura, A},
title = {Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing.},
journal = {Development, growth & differentiation},
volume = {61},
number = {4},
pages = {265-275},
doi = {10.1111/dgd.12607},
pmid = {31037730},
issn = {1440-169X},
support = {20112002//Grants-in-Aids for Scientific Research/ ; 26114508//Grants-in-Aids for Scientific Research/ ; 25650088//Grants-in-Aids for Scientific Research/ ; 15K14530//Grants-in-Aids for Scientific Research/ ; 17H03686//Grants-in-Aids for Scientific Research/ ; //Takeda Science Foundation/ ; //Mitsubishi Foundation/ ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Drosophila/*genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Green Fluorescent Proteins/*genetics ; Time Factors ; },
abstract = {The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.},
}

Animals
CRISPR-Cas Systems/*genetics
Drosophila/*genetics
Gene Editing/*methods
Gene Knock-In Techniques/*methods
Green Fluorescent Proteins/*genetics
Time Factors

@article {pmid30323226,
year = {2018},
author = {Beisel, CL},
title = {CRISPR tool puts RNA on the record.},
journal = {Nature},
volume = {562},
number = {7727},
pages = {347-349},
doi = {10.1038/d41586-018-06869-1},
pmid = {30323226},
issn = {1476-4687},
support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *RNA ; },
}

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

@article {pmid30042156,
year = {2018},
author = {Izumi, R and Kusakabe, T and Noguchi, M and Iwakura, H and Tanaka, T and Miyazawa, T and Aotani, D and Hosoda, K and Kangawa, K and Nakao, K},
title = {CRISPR/Cas9-mediated Angptl8 knockout suppresses plasma triglyceride concentrations and adiposity in rats.},
journal = {Journal of lipid research},
volume = {59},
number = {9},
pages = {1575-1585},
pmid = {30042156},
issn = {1539-7262},
mesh = {Adipogenesis/genetics ; Adiposity/*genetics ; Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Diet/adverse effects ; Female ; *Gene Knockout Techniques ; Glucose/metabolism ; Lipid Metabolism/genetics ; Male ; Muscle, Skeletal/metabolism ; Myocardium/metabolism ; Obesity/chemically induced/*genetics ; Oxidation-Reduction ; Rats ; Triglycerides/*blood ; },
abstract = {Angiopoietin-like protein (ANGPTL)8 is a liver- and adipocyte-derived protein that controls plasma triglyceride (TG) levels. Most animal studies have used mouse models. Here, we generated an Angptl8 KO rat model using a clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system to clarify the roles of ANGPTL8 in glucose and lipid metabolism. Compared with WT rats, Angptl8 KO rats had lower body weight and fat content, associated with impaired lipogenesis in adipocytes; no differences existed between the groups in food intake or rectal temperature. Plasma TG levels in both the fasted and refed states were significantly lower in KO than in WT rats, and an oral fat tolerance test showed decreased plasma TG excursion in Angptl8 KO rats. Higher levels of lipase activity in the heart and greater expression of genes related to β-oxidation in heart and skeletal muscle were observed in Angptl8 KO rats. However, there were no significant differences between KO and WT rats in glucose metabolism or the histology of pancreatic β-cells on both standard and high-fat diets. In conclusion, we demonstrated that Angptl8 KO in rats resulted in lower body weight and plasma TG levels without affecting glucose metabolism. ANGPTL8 might be an important therapeutic target for obesity and dyslipidemia.},
}

Adipogenesis/genetics
Adiposity/*genetics
Animals
Base Sequence
CRISPR-Cas Systems/*genetics
Diet/adverse effects
Female
*Gene Knockout Techniques
Glucose/metabolism
Lipid Metabolism/genetics
Male
Muscle, Skeletal/metabolism
Myocardium/metabolism
Obesity/chemically induced/*genetics
Oxidation-Reduction
Rats
Triglycerides/*blood

@article {pmid29981811,
year = {2018},
author = {Karthikeyan, S and Russo, A and Dean, M and Lantvit, DD and Endsley, M and Burdette, JE},
title = {Prolactin signaling drives tumorigenesis in human high grade serous ovarian cancer cells and in a spontaneous fallopian tube derived model.},
journal = {Cancer letters},
volume = {433},
number = {},
pages = {221-231},
doi = {10.1016/j.canlet.2018.07.003},
pmid = {29981811},
issn = {1872-7980},
support = {UG3 ES029073/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/pathology ; Carcinoma, Ovarian Epithelial/*pathology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Fallopian Tube Neoplasms/*pathology ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; Mice ; Mice, Nude ; Ovarian Neoplasms/*pathology ; Prolactin/genetics/*metabolism/pharmacology ; Proto-Oncogene Proteins/metabolism ; Receptors, Prolactin/biosynthesis ; Wnt Proteins/metabolism ; },
abstract = {The pathways responsible for tumorigenesis of high grade serous ovarian cancer (HGSOC) from the fallopian tube epithelium (FTE) are still poorly understood. A human prolactin (PRL) like gene, Prl2c2 was amplified >100 fold in a spontaneous model of FTE-derived ovarian cancer (MOEhigh - murine oviductal epithelium high passage). Prl2c2 stable knockdown in MOEhigh cells demonstrated a significant reduction in cell proliferation, 2-dimensional foci, anchorage independent growth, and blocked tumor formation. The overall survival of ovarian cancer patients from transcriptome analysis of 1868 samples was lower when abundant PRL and prolactin receptors (PRL-R) were expressed. A HGSOC cell line (OVCAR3) and a tumorigenic human FTE cell line (FT33-Tag-Myc) were treated with recombinant PRL and a significant increase in cellular proliferation was detected. A CRISPR/Cas9 mediated PRL-R deletion in OVCAR3 and FT33-Tag-Myc cells demonstrated significant reduction in cell proliferation and eliminated tumor growth using the OVCAR3 model. PRL was found to phosphorylate STAT5, m-TOR and ERK in ovarian cancer cells. This study identified Prl2c2 as a driver of tumorigenesis in a spontaneous model and confirmed that prolactin signaling supports tumorigenesis in high grade serous ovarian cancer.},
}

Animals
CRISPR-Cas Systems
Carcinogenesis/pathology
Carcinoma, Ovarian Epithelial/*pathology
Cell Line, Tumor
Cell Proliferation/drug effects
Fallopian Tube Neoplasms/*pathology
Female
Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
Humans
Mice
Mice, Nude
Ovarian Neoplasms/*pathology
Prolactin/genetics/*metabolism/pharmacology
Proto-Oncogene Proteins/metabolism
Receptors, Prolactin/biosynthesis
Wnt Proteins/metabolism

@article {pmid29964204,
year = {2018},
author = {Xiao, Y and Li, Y and Tao, H and Humphries, B and Li, A and Jiang, Y and Yang, C and Luo, R and Wang, Z},
title = {Integrin α5 down-regulation by miR-205 suppresses triple negative breast cancer stemness and metastasis by inhibiting the Src/Vav2/Rac1 pathway.},
journal = {Cancer letters},
volume = {433},
number = {},
pages = {199-209},
doi = {10.1016/j.canlet.2018.06.037},
pmid = {29964204},
issn = {1872-7980},
support = {P30 CA177558/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Female ; Gene Knockout Techniques ; Humans ; Integrins/*biosynthesis ; Lung Neoplasms/prevention & control/secondary ; MCF-7 Cells ; Mice ; Mice, Nude ; MicroRNAs/*genetics ; Neoplasm Invasiveness/genetics/*prevention & control ; Neoplasm Transplantation ; Neoplastic Stem Cells/*pathology ; Proto-Oncogene Proteins c-vav/antagonists & inhibitors ; Transplantation, Heterologous ; Triple Negative Breast Neoplasms/genetics/*pathology ; rac1 GTP-Binding Protein/antagonists & inhibitors ; src-Family Kinases/antagonists & inhibitors ; },
abstract = {Triple negative breast cancer (TNBC) usually displays more aggressive metastasis, the underlying mechanism is unclear. Previous studies showed that microRNA-205 (miR-205) has controversial roles in cancer, however, its role in TNBC metastasis and the underlying mechanism have not been well-understood. In this study we found that miR-205 expression level is extremely low in basal mesenchymal-like highly migratory and invasive TNBC cells. Stably re-expressing miR-205 in TNBC cells significantly reduced their migration, invasion capability and cancer stem cell (CSC)-like property. Nude mouse orthotopic mammary xenograft tumor model study revealed that miR-205 re-expression greatly decreases TNBC tumor growth and abolishes spontaneous lung metastasis. Mechanistic studies demonstrated that miR-205 inhibits TNBC cell metastatic traits and tumor metastasis by down-regulating integrin α5 (ITGA5). Moreover, ITGA5 knockout using the CRISPR/Cas9 technique achieved the same strong inhibitory effect on TNBC cell CSC-like property and tumor metastasis as re-expressing miR-205 did. Further mechanistic studies indicated that ITGA5 down-regulation by miR-205 re-expression impairs TNBC cell metastatic traits by inhibiting the Src/Vav2/Rac1 pathway. Together, our findings suggest that miR-205 and ITGA5 may serve as potential targets for developing effective therapies for metastatic TNBC.},
}

Animals
CRISPR-Cas Systems
Cell Line, Tumor
Cell Movement
Female
Gene Knockout Techniques
Humans
Integrins/*biosynthesis
Lung Neoplasms/prevention & control/secondary
MCF-7 Cells
Mice
Mice, Nude
MicroRNAs/*genetics
Neoplasm Invasiveness/genetics/*prevention & control
Neoplasm Transplantation
Neoplastic Stem Cells/*pathology
Proto-Oncogene Proteins c-vav/antagonists & inhibitors
Transplantation, Heterologous
Triple Negative Breast Neoplasms/genetics/*pathology
rac1 GTP-Binding Protein/antagonists & inhibitors
src-Family Kinases/antagonists & inhibitors

@article {pmid29957992,
year = {2018},
author = {Zhan, H and Xie, H and Zhou, Q and Liu, Y and Huang, W},
title = {Synthesizing a Genetic Sensor Based on CRISPR-Cas9 for Specifically Killing p53-Deficient Cancer Cells.},
journal = {ACS synthetic biology},
volume = {7},
number = {7},
pages = {1798-1807},
doi = {10.1021/acssynbio.8b00202},
pmid = {29957992},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Humans ; Tumor Suppressor Protein p53/*deficiency/genetics ; },
abstract = {Cancer is still one of the greatest medical challenges in the world. The p53 protein plays an important role in the process of cancer formation. In addition, p53 is found as the most common mutant gene in cancers. Because of the central role of p53 in oncology, it is necessary to construct effective sensors to detect this protein. However, there are few methods to detect wild type p53 protein (WTP53) or to distinguish the wild type and mutant p53 proteins. In our study, we designed and constructed a p53 genetic sensor that detected the expression of WTP53 in cells. Moreover, we combined the p53 sensor with diphtheria toxin using the CRISPR-Cas9 system to construct a p53 genetic sensor that specifically killed p53-deficient cells such as tumor cells. Our study therefore developed a new way to treat cancers by using an available genetic sensor based on p53 protein.},
}

CRISPR-Cas Systems/*genetics
Humans
Tumor Suppressor Protein p53/*deficiency/genetics

@article {pmid29924933,
year = {2018},
author = {Jun, S and Lim, H and Jang, H and Lee, W and Ahn, J and Lee, JH and Bang, D},
title = {Straightforward Delivery of Linearized Double-Stranded DNA Encoding sgRNA and Donor DNA for the Generation of Single Nucleotide Variants Based on the CRISPR/Cas9 System.},
journal = {ACS synthetic biology},
volume = {7},
number = {7},
pages = {1651-1659},
doi = {10.1021/acssynbio.7b00345},
pmid = {29924933},
issn = {2161-5063},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/*genetics ; Gene Editing ; Humans ; Mutagenesis ; RNA Editing/genetics ; },
abstract = {CRISPR/Cas9 for genome editing requires delivery of a guide RNA sequence and donor DNA for targeted homologous recombination. Typically, single-stranded oligodeoxynucleotide, serving as the donor template, and a plasmid encoding guide RNA are delivered as two separate components. However, in the multiplexed generation of single nucleotide variants, this two-component delivery system is limited by difficulty of delivering a matched pair of sgRNA and donor DNA to the target cell. Here, we describe a novel codelivery system called "sgR-DNA" that uses a linearized double-stranded DNA consisting of donor DNA component and a component encoding sgRNA. Our sgR-DNA-based method is simple to implement because it does not require cloning steps. We also report the potential of our delivery system to generate multiplex genomic substitutions in Escherichia coli and human cells.},
}

Animals
CRISPR-Cas Systems/*genetics
DNA/*genetics
Gene Editing
Humans
Mutagenesis
RNA Editing/genetics

@article {pmid29466914,
year = {2018},
author = {Ferreccio, A and Mathieu, J and Detraux, D and Somasundaram, L and Cavanaugh, C and Sopher, B and Fischer, K and Bello, T and M Hussein, A and Levy, S and Cook, S and Sidhu, SB and Artoni, F and Palpant, NJ and Reinecke, H and Wang, Y and Paddison, P and Murry, C and Jayadev, S and Ware, C and Ruohola-Baker, H},
title = {Inducible CRISPR genome editing platform in naive human embryonic stem cells reveals JARID2 function in self-renewal.},
journal = {Cell cycle (Georgetown, Tex.)},
volume = {17},
number = {5},
pages = {535-549},
pmid = {29466914},
issn = {1551-4005},
support = {K02 AG044473/AG/NIA NIH HHS/United States ; P30 DK017047/DK/NIDDK NIH HHS/United States ; T32 CA080416/CA/NCI NIH HHS/United States ; P01 GM081619/GM/NIGMS NIH HHS/United States ; R01 GM097372/GM/NIGMS NIH HHS/United States ; R01 GM083867/GM/NIGMS NIH HHS/United States ; U01 HL099997/HL/NHLBI NIH HHS/United States ; },
mesh = {Blastocyst/cytology/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Self Renewal ; DNA End-Joining Repair ; *Gene Editing ; Genetic Loci ; Histones/metabolism ; Human Embryonic Stem Cells ; Humans ; INDEL Mutation ; Microscopy, Confocal ; Phenotype ; Polycomb Repressive Complex 2/chemistry/deficiency/genetics/*metabolism ; Presenilin-2/genetics/metabolism ; Protein Domains ; },
abstract = {To easily edit the genome of naïve human embryonic stem cells (hESC), we introduced a dual cassette encoding an inducible Cas9 into the AAVS1 site of naïve hESC (iCas9). The iCas9 line retained karyotypic stability, expression of pluripotency markers, differentiation potential, and stability in 5iLA and EPS pluripotency conditions. The iCas9 line induced efficient homology-directed repair (HDR) and non-homologous end joining (NHEJ) based mutations through CRISPR-Cas9 system. We utilized the iCas9 line to study the epigenetic regulator, PRC2 in early human pluripotency. The PRC2 requirement distinguishes between early pluripotency stages, however, what regulates PRC2 activity in these stages is not understood. We show reduced H3K27me3 and pluripotency markers in JARID2 2iL-I-F hESC mutants, indicating JARID2 requirement in maintenance of hESC 2iL-I-F state. These data suggest that JARID2 regulates PRC2 in 2iL-I-F state and the lack of PRC2 function in 5iLA state may be due to lack of sufficient JARID2 protein.},
}

Blastocyst/cytology/metabolism
CRISPR-Cas Systems/*genetics
Cell Self Renewal
DNA End-Joining Repair
*Gene Editing
Genetic Loci
Histones/metabolism
Human Embryonic Stem Cells
Humans
INDEL Mutation
Microscopy, Confocal
Phenotype
Polycomb Repressive Complex 2/chemistry/deficiency/genetics/*metabolism
Presenilin-2/genetics/metabolism
Protein Domains

@article {pmid29388938,
year = {2018},
author = {Liang, Z and Chen, K and Zhang, Y and Liu, J and Yin, K and Qiu, JL and Gao, C},
title = {Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins.},
journal = {Nature protocols},
volume = {13},
number = {3},
pages = {413-430},
pmid = {29388938},
issn = {1750-2799},
mesh = {Biolistics ; Bread ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Oryza ; Ribonucleoproteins ; Triticum/genetics ; },
abstract = {This protocol is an extension to: Nat. Protoc. 9, 2395-2410 (2014); doi:10.1038/nprot.2014.157; published online 18 September 2014In recent years, CRISPR/Cas9 has emerged as a powerful tool for improving crop traits. Conventional plant genome editing mainly relies on plasmid-carrying cassettes delivered by Agrobacterium or particle bombardment. Here, we describe DNA-free editing of bread wheat by delivering in vitro transcripts (IVTs) or ribonucleoprotein complexes (RNPs) of CRISPR/Cas9 by particle bombardment. This protocol serves as an extension of our previously published protocol on genome editing in bread wheat using CRISPR/Cas9 plasmids delivered by particle bombardment. The methods we describe not only eliminate random integration of CRISPR/Cas9 into genomic DNA, but also reduce off-target effects. In this protocol extension article, we present detailed protocols for preparation of IVTs and RNPs; validation by PCR/restriction enzyme (RE) and next-generation sequencing; delivery by biolistics; and recovery of mutants and identification of mutants by pooling methods and Sanger sequencing. To use these protocols, researchers should have basic skills and experience in molecular biology and biolistic transformation. By using these protocols, plants edited without the use of any foreign DNA can be generated and identified within 9-11 weeks.},
}

Biolistics
Bread
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
*Oryza
Ribonucleoproteins
Triticum/genetics

@article {pmid29319470,
year = {2018},
author = {Gingras, H and Dridi, B and Leprohon, P and Ouellette, M},
title = {Coupling next-generation sequencing to dominant positive screens for finding antibiotic cellular targets and resistance mechanisms in Escherichia coli.},
journal = {Microbial genomics},
volume = {4},
number = {2},
pages = {},
pmid = {29319470},
issn = {2057-5858},
mesh = {Anti-Bacterial Agents/*pharmacology ; CRISPR-Cas Systems ; Ceftriaxone/pharmacology ; DNA-Binding Proteins/genetics ; Drug Resistance, Multiple, Bacterial/*genetics ; Escherichia coli/*drug effects/*genetics ; Escherichia coli Proteins/genetics ; Gene Knockdown Techniques ; Genes, Bacterial/genetics ; Gentamicins/pharmacology ; High-Throughput Nucleotide Sequencing/*methods ; Levofloxacin/pharmacology ; Membrane Proteins/genetics ; Microbial Sensitivity Tests ; Plasmids/genetics ; R Factors/genetics ; Tetracycline/pharmacology ; Transcription Factors/genetics ; Trimethoprim/pharmacology ; },
abstract = {In order to expedite the discovery of genes coding for either drug targets or antibiotic resistance, we have developed a functional genomic strategy termed Plas-Seq. This technique involves coupling a multicopy suppressor library to next-generation sequencing. We generated an Escherichia coli plasmid genomic library that was transformed into E. coli. These transformants were selected step by step using 0.25× to 2× minimum inhibitory concentrations for ceftriaxone, gentamicin, levofloxacin, tetracycline or trimethoprim. Plasmids were isolated at each selection step and subjected to Illumina sequencing. By searching for genomic loci whose sequencing coverage increased with antibiotic pressure we were able to detect 48 different genomic loci that were enriched by at least one antibiotic. Fifteen of these loci were studied functionally, and we showed that 13 can decrease the susceptibility of E. coli to antibiotics when overexpressed. These genes coded for drug targets, transcription factors, membrane proteins and resistance factors. The technique of Plas-Seq is expediting the discovery of genes associated with the mode of action or resistance to antibiotics and led to the isolation of a novel gene influencing drug susceptibility. It has the potential for being applied to novel molecules and to other microbial species.},
}

Anti-Bacterial Agents/*pharmacology
CRISPR-Cas Systems
Ceftriaxone/pharmacology
DNA-Binding Proteins/genetics
Drug Resistance, Multiple, Bacterial/*genetics
Escherichia coli/*drug effects/*genetics
Escherichia coli Proteins/genetics
Gene Knockdown Techniques
Genes, Bacterial/genetics
Gentamicins/pharmacology
High-Throughput Nucleotide Sequencing/*methods
Levofloxacin/pharmacology
Membrane Proteins/genetics
Microbial Sensitivity Tests
Plasmids/genetics
R Factors/genetics
Tetracycline/pharmacology
Transcription Factors/genetics
Trimethoprim/pharmacology

@article {pmid31492655,
year = {2019},
author = {Soto-Perez, P and Bisanz, JE and Berry, JD and Lam, KN and Bondy-Denomy, J and Turnbaugh, PJ},
title = {CRISPR-Cas System of a Prevalent Human Gut Bacterium Reveals Hyper-targeting against Phages in a Human Virome Catalog.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2019.08.008},
pmid = {31492655},
issn = {1934-6069},
abstract = {Bacteriophages are abundant within the human gastrointestinal tract, yet their interactions with gut bacteria remain poorly understood, particularly with respect to CRISPR-Cas immunity. Here, we show that the type I-C CRISPR-Cas system in the prevalent gut Actinobacterium Eggerthella lenta is transcribed and sufficient for specific targeting of foreign and chromosomal DNA. Comparative analyses of E. lenta CRISPR-Cas systems across (meta)genomes revealed 2 distinct clades according to cas sequence similarity and spacer content. We assembled a human virome database (HuVirDB), encompassing 1,831 samples enriched for viral DNA, to identify protospacers. This revealed matches for a majority of spacers, a marked increase over other databases, and uncovered "hyper-targeted" phage sequences containing multiple protospacers targeted by several E. lenta strains. Finally, we determined the positional mismatch tolerance of observed spacer-protospacer pairs. This work emphasizes the utility of merging computational and experimental approaches for determining the function and targets of CRISPR-Cas systems.},
}

@article {pmid30664332,
year = {2019},
author = {Pieczynski, JN and Deets, A and McDuffee, A and Lynn Kee, H},
title = {An undergraduate laboratory experience using CRISPR-cas9 technology to deactivate green fluorescent protein expression in Escherichia coli.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {47},
number = {2},
pages = {145-155},
doi = {10.1002/bmb.21206},
pmid = {30664332},
issn = {1539-3429},
mesh = {CRISPR-Cas Systems/*genetics ; Escherichia coli/*genetics ; Gene Expression Regulation, Bacterial/*genetics ; Green Fluorescent Proteins/biosynthesis/*deficiency/*genetics ; *Laboratories ; Software ; Students ; *Universities ; },
abstract = {Undergraduates learn that gene editing in diverse organisms is now possible. How targeted manipulation of genes and genomes is utilized in basic science and biomedicine to address biological questions is challenging for undergraduates to conceptualize. Thus, we developed a lab experience that would allow students to be actively engaged in the full process of design, implementation of a gene editing strategy, and interpretation of results within an 8-week lab period of a Genetics course. The laboratory experience combines two transformative biotechnology tools; the utilization of green fluorescent protein (GFP) as a diagnostic marker of gene expression and the fundamentals and specificity of Clustered Regularly Interspaced Short Palindromic Repeats-cas9 (CRISPR-cas9) gene editing in bacterial cells. The students designed and constructed plasmids that express single guide RNA targeted to GFP, expressed the sgRNA and cas9 in bacteria cells, and successfully deactivated GFP gene expression in the bacterial cells with their designed CRISPR-cas9 tools. Student assessment revealed most students achieved student learning objectives. We conclude this lab experience is an effective and accessible method for engaging students in the scientific practices, knowledge and challenges revolving targeted CRISPR-cas9 gene manipulation. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 145-155, 2019.},
}

CRISPR-Cas Systems/*genetics
Escherichia coli/*genetics
Gene Expression Regulation, Bacterial/*genetics
Green Fluorescent Proteins/biosynthesis/*deficiency/*genetics
*Laboratories
Software
Students
*Universities

@article {pmid30598393,
year = {2018},
author = {Shan, Q and Baltes, NJ and Atkins, P and Kirkland, ER and Zhang, Y and Baller, JA and Lowder, LG and Malzahn, AA and Haugner, JC and Seelig, B and Voytas, DF and Qi, Y},
title = {ZFN, TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis: A disconnect between somatic and germinal cells.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {45},
number = {12},
pages = {681-684},
doi = {10.1016/j.jgg.2018.07.011},
pmid = {30598393},
issn = {1673-8527},
mesh = {Arabidopsis/*genetics/metabolism ; Arabidopsis Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/genetics/*metabolism ; Gene Editing ; Gene Targeting/*methods ; Germ Cells, Plant/metabolism ; Homologous Recombination ; Mutagenesis, Insertional/*methods ; Transcription Activator-Like Effector Nucleases/genetics/*metabolism ; },
}

Arabidopsis/*genetics/metabolism
Arabidopsis Proteins/genetics/metabolism
*CRISPR-Cas Systems
Endodeoxyribonucleases/genetics/*metabolism
Gene Editing
Gene Targeting/*methods
Germ Cells, Plant/metabolism
Homologous Recombination
Mutagenesis, Insertional/*methods
Transcription Activator-Like Effector Nucleases/genetics/*metabolism

@article {pmid30575820,
year = {2019},
author = {Saenz, DT and Fiskus, W and Manshouri, T and Mill, CP and Qian, Y and Raina, K and Rajapakshe, K and Coarfa, C and Soldi, R and Bose, P and Borthakur, G and Kadia, TM and Khoury, JD and Masarova, L and Nowak, AJ and Sun, B and Saenz, DN and Kornblau, SM and Horrigan, S and Sharma, S and Qiu, P and Crews, CM and Verstovsek, S and Bhalla, KN},
title = {Targeting nuclear β-catenin as therapy for post-myeloproliferative neoplasm secondary AML.},
journal = {Leukemia},
volume = {33},
number = {6},
pages = {1373-1386},
doi = {10.1038/s41375-018-0334-3},
pmid = {30575820},
issn = {1476-5551},
support = {R01 CA173877/CA/NCI NIH HHS/United States ; R35 CA197589/CA/NCI NIH HHS/United States ; },
mesh = {Acetanilides/pharmacology ; Animals ; Apoptosis/drug effects ; CRISPR-Cas Systems ; Cell Nucleus/*drug effects/metabolism/pathology ; *Drug Synergism ; Heterocyclic Compounds, 3-Ring/pharmacology ; Humans ; Leukemia, Myeloid, Acute/complications/*drug therapy/metabolism/pathology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Myeloproliferative Disorders/complications/*drug therapy/metabolism/pathology ; Protein Kinase Inhibitors/*pharmacology ; Pyrazoles/pharmacology ; Signal Transduction ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; beta Catenin/*antagonists & inhibitors/genetics/metabolism ; },
abstract = {Transformation of post-myeloproliferative neoplasms into secondary (s) AML exhibit poor clinical outcome. In addition to increased JAK-STAT and PI3K-AKT signaling, post-MPN sAML blast progenitor cells (BPCs) demonstrate increased nuclear β-catenin levels and TCF7L2 (TCF4) transcriptional activity. Knockdown of β-catenin or treatment with BC2059 that disrupts binding of β-catenin to TBL1X (TBL1) depleted nuclear β-catenin levels. This induced apoptosis of not only JAKi-sensitive but also JAKi-persister/resistant post-MPN sAML BPCs, associated with attenuation of TCF4 transcriptional targets MYC, BCL-2, and Survivin. Co-targeting of β-catenin and JAK1/2 inhibitor ruxolitinib (rux) synergistically induced lethality in post-MPN sAML BPCs and improved survival of mice engrafted with human sAML BPCs. Notably, co-treatment with BET protein degrader ARV-771 and BC2059 also synergistically induced apoptosis and improved survival of mice engrafted with JAKi-sensitive or JAKi-persister/resistant post-MPN sAML cells. These preclinical findings highlight potentially promising anti-post-MPN sAML activity of the combination of β-catenin and BETP antagonists against post-MPN sAML BPCs.},
}

Acetanilides/pharmacology
Animals
Apoptosis/drug effects
CRISPR-Cas Systems
Cell Nucleus/*drug effects/metabolism/pathology
*Drug Synergism
Heterocyclic Compounds, 3-Ring/pharmacology
Humans
Leukemia, Myeloid, Acute/complications/*drug therapy/metabolism/pathology
Mice
Mice, Inbred NOD
Mice, SCID
Myeloproliferative Disorders/complications/*drug therapy/metabolism/pathology
Protein Kinase Inhibitors/*pharmacology
Pyrazoles/pharmacology
Signal Transduction
Tumor Cells, Cultured
Xenograft Model Antitumor Assays
beta Catenin/*antagonists & inhibitors/genetics/metabolism

@article {pmid30573379,
year = {2018},
author = {Xing, S and Jia, M and Wei, L and Mao, W and Abbasi, UA and Zhao, Y and Chen, Y and Cao, M and Zhang, K and Dai, Z and Dou, Z and Jia, W and Li, B},
title = {CRISPR/Cas9-introduced single and multiple mutagenesis in strawberry.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {45},
number = {12},
pages = {685-687},
doi = {10.1016/j.jgg.2018.04.006},
pmid = {30573379},
issn = {1673-8527},
mesh = {*CRISPR-Cas Systems ; Fragaria/*genetics/growth & development/metabolism ; Gene Editing ; Genome, Plant ; *Mutagenesis ; Plant Proteins/genetics/metabolism ; },
}

*CRISPR-Cas Systems
Fragaria/*genetics/growth & development/metabolism
Gene Editing
Genome, Plant
*Mutagenesis
Plant Proteins/genetics/metabolism

@article {pmid30297850,
year = {2018},
author = {Serebrenik, YV and Shalem, O},
title = {CRISPR mutagenesis screening of mice.},
journal = {Nature cell biology},
volume = {20},
number = {11},
pages = {1235-1237},
doi = {10.1038/s41556-018-0224-y},
pmid = {30297850},
issn = {1476-4679},
mesh = {Amino Acids ; Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Germ Cells ; Mice ; Mutagenesis ; Neoplasm Proteins ; },
}

Amino Acids
Animals
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Germ Cells
Mice
Mutagenesis
Neoplasm Proteins

@article {pmid29472715,
year = {2018},
author = {Zhenilo, S and Deyev, I and Litvinova, E and Zhigalova, N and Kaplun, D and Sokolov, A and Mazur, A and Prokhortchouk, E},
title = {DeSUMOylation switches Kaiso from activator to repressor upon hyperosmotic stress.},
journal = {Cell death and differentiation},
volume = {25},
number = {11},
pages = {1938-1951},
pmid = {29472715},
issn = {1476-5403},
mesh = {Animals ; Apoptosis/drug effects ; Blood Pressure/drug effects ; CRISPR-Cas Systems/genetics ; Cell Line ; HEK293 Cells ; Humans ; Hypertension/pathology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mutagenesis, Site-Directed ; *Osmotic Pressure ; RNA Interference ; RNA, Small Interfering/metabolism ; Sodium Chloride/pharmacology ; Sumoylation/drug effects ; Transcription Factors/antagonists & inhibitors/genetics/*metabolism ; Transcription, Genetic ; Tripartite Motif Proteins/genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; },
abstract = {Kaiso is a member of the BTB/POZ zinc finger family, which is involved in cancer progression, cell cycle control, apoptosis, and WNT signaling. Depending on promoter context, it may function as either a transcriptional repressor or activator. Previous studies found that Kaiso might be SUMOylated due to heat shock, but the biological significance of Kaiso SUMOylation is unclear. Here, we find that K42 is the only amino acid within Kaiso that is modified with SUMO. Kaiso is monoSUMOylated at lysine 42 in cell lines of kidney origin under normal physiological conditions. SUMOylated Kaiso can activate transcription from exogenous methylated promoters, wherein the deSUMOylated form of the protein kept the ability to be a repressor. Rapid Kaiso deSUMOylation occurs in response to hyperosmotic stress and is reversible upon return to an isotonic environment. DeSUMOylation occurs within minutes in HEK293 cells treated with 100 mM NaCl and relaxes in 3 h even in a salt-containing medium. Genomic editing of Kaiso by conversion of K42 into R42 (K42R) in HEK293 cells that resulted in fully deSUMOylated endogenous protein led to misregulation of genes associated with ion transport, blood pressure, and the immune response. TRIM25 was significantly repressed in two K42R HEK293 clones. By a series of rescue experiments with K42R and KO HEK293 cells, we show that TRIM25 is a direct transcriptional target for Kaiso. In the absence of Kaiso, the level of TRIM25 is insensitive to hyperosmotic stress. Extending our observations to animal models, we show that in response to a high salt diet, Kaiso knockout mice are characterized by significantly higher blood pressure increases when compared to wild-type animals. Thus, we propose a novel biological role for Kaiso in the regulation of homeostasis.},
}

Animals
Apoptosis/drug effects
Blood Pressure/drug effects
CRISPR-Cas Systems/genetics
Cell Line
HEK293 Cells
Humans
Hypertension/pathology
Mice
Mice, Inbred C57BL
Mice, Knockout
Mutagenesis, Site-Directed
*Osmotic Pressure
RNA Interference
RNA, Small Interfering/metabolism
Sodium Chloride/pharmacology
Sumoylation/drug effects
Transcription Factors/antagonists & inhibitors/genetics/*metabolism
Transcription, Genetic
Tripartite Motif Proteins/genetics/metabolism
Ubiquitin-Protein Ligases/genetics/metabolism

@article {pmid31483929,
year = {2019},
author = {de Maagd, RA and Loonen, A and Chouaref, J and Pelé, A and Meijer-Dekens, F and Fransz, P and Bai, Y},
title = {CRISPR/Cas inactivation of RECQ4 increases homeologous crossovers in an interspecific tomato hybrid.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13248},
pmid = {31483929},
issn = {1467-7652},
abstract = {Crossover formation during meiosis in plants is required for proper chromosome segregation and is essential for crop breeding as it allows an (optimal) combination of traits by mixing parental alleles on each chromosome. Crossover formation commences with the production of a large number of DNA double-strand breaks, of which only a few result in crossovers. A small number of genes, which drive the resolution of DNA crossover intermediate structures towards non-crossovers, have been identified in Arabidopisis thaliana. In order to explore the potential of modification of these genes in interspecific hybrids between crops and their wild relatives towards increased production of crossovers, we have used CRISPR/Cas9-mutagenesis in an interspecific tomato hybrid to knock out RecQ4. A biallelic recq4 mutant was obtained in the F1 hybrid of Solanum lycopersicum and S. pimpinellifolium. Compared to the wild type F1 hybrid, the F1 recq4 mutant was shown to have a significant increase in crossovers: a 1.53-fold increase when directly observing ring bivalents in male meiocytes microscopically and a 1.8-fold extension of the genetic map when measured by analysing SNP markers in the progeny (F2) plants. This is one of the first demonstrations of increasing crossover frequency in interspecific hybrids by manipulating genes in crossover intermediate resolution pathways and the first to do so by directed mutagenesis. This article is protected by copyright. All rights reserved.},
}

@article {pmid31482512,
year = {2019},
author = {Dumeau, CE and Monfort, A and Kissling, L and Swarts, DC and Jinek, M and Wutz, A},
title = {Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1.},
journal = {Transgenic research},
volume = {},
number = {},
pages = {},
doi = {10.1007/s11248-019-00168-9},
pmid = {31482512},
issn = {1573-9368},
support = {31003A_152814/1//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; 31003A_175643/1//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; },
abstract = {CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using expression vectors, mRNA, or directly as ribonucleoprotein (RNP) complexes by different delivery methods. Whereas microinjection techniques are well established, more recently developed electroporation methods simplify RNP delivery but can provide less consistent efficiency. Previously, we have designed Cas12a-crRNA pairs to introduce large genomic deletions in the Ubn1, Ubn2, and Rbm12 genes in mouse embryonic stem cells (ESC). Here, we have optimized the conditions for electroporation of the same Cas12a RNP pairs into mouse zygotes. Using our protocol, large genomic deletions can be generated efficiently by electroporation of zygotes with or without an intact zona pellucida. Electroporation of as few as ten zygotes is sufficient to obtain a gene deletion in mice suggesting potential applicability of this method for species with limited availability of zygotes.},
}

@article {pmid31475782,
year = {2019},
author = {Wen, Z and Lu, M and Ledesma-Amaro, R and Li, Q and Jin, M and Yang, S},
title = {TargeTron Technology Applicable in Solventogenic Clostridia: Revisiting 12 years' Advances.},
journal = {Biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1002/biot.201900284},
pmid = {31475782},
issn = {1860-7314},
abstract = {Clostridium has great potential in industrial application and medical research. But low DNA repair capacity and plasmids transformation efficiency severely delayed development and application of genetic tools based on homologous recombination (HR). TargeTron is a gene editing technique dependent on the mobility of group II introns, rather than homologous recombination, which made it very suitable for gene disruption of Clostridium. The application of TargeTron technology in Clostridium was academically reported in 2007 and this tool has been introduced in various clostridia as it is easy to operate, time-saving, and reliable. TargeTron has made great progress in solventogenic Clostridium in the aspects of acetone-butanol-ethanol (ABE) fermentation pathway modification, important functional genes identification, and xylose metabolic pathway analysis & reconstruction. In the review, we revisited 12 years' advances of TargeTron technology applicable in solventogenic Clostridium, including its principle, technical characteristics, application and efforts to expand its capabilities, or to avoid potential drawbacks. Some other technologies as putative competitors or collaborators are also discussed. We believe that TargeTron combined with CRISPR/Cas-assisted gene/base editing and gene-expression regulation system will make a better future for clostridial genetic modification. This article is protected by copyright. All rights reserved.},
}

@article {pmid31474367,
year = {2019},
author = {Stanley, SY and Borges, AL and Chen, KH and Swaney, DL and Krogan, NJ and Bondy-Denomy, J and Davidson, AR},
title = {Anti-CRISPR-Associated Proteins Are Crucial Repressors of Anti-CRISPR Transcription.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2019.07.046},
pmid = {31474367},
issn = {1097-4172},
abstract = {Phages express anti-CRISPR (Acr) proteins to inhibit CRISPR-Cas systems that would otherwise destroy their genomes. Most acr genes are located adjacent to anti-CRISPR-associated (aca) genes, which encode proteins with a helix-turn-helix DNA-binding motif. The conservation of aca genes has served as a signpost for the identification of acr genes, but the function of the proteins encoded by these genes has not been investigated. Here we reveal that an acr-associated promoter drives high levels of acr transcription immediately after phage DNA injection and that Aca proteins subsequently repress this transcription. Without Aca activity, this strong transcription is lethal to a phage. Our results demonstrate how sufficient levels of Acr proteins accumulate early in the infection process to inhibit existing CRISPR-Cas complexes in the host cell. They also imply that the conserved role of Aca proteins is to mitigate the deleterious effects of strong constitutive transcription from acr promoters.},
}

@article {pmid31472270,
year = {2019},
author = {Ziegler, H and Nellen, W},
title = {CRISPR-Cas experiments for schools and the public.},
journal = {Methods (San Diego, Calif.)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymeth.2019.08.009},
pmid = {31472270},
issn = {1095-9130},
abstract = {The "gene scissors" CRISPR-Cas currently revolutionize the field of molecular biology with an enormous impact on society due to the broad application potentials in biomedicine, biotechnology and agriculture. We have developed simple CRISPR-Cas experiments that can serve to introduce pupils, students and non-scientists alike to the fascinating power of targeted gene editing. The experimental course is divided into two parts. In part 1, we target plasmid borne lacZ to convert blue E. coli to white E. coli. In part 2, we analyse the CRISPR-Cas9 mediated double strand breaks in the lacZ gene by a) colony PCR, b) colony cracking gel or c) restriction digest of the plasmids. Experimental work is embedded in short theoretical lecture parts that provide background of CRISPR-Cas and a step-by-step tutorial for the practical work. Though the experiment is robust, inexpensive and simple it should be noted that guidance by an expert instructor is required. Based on our experience, a full day lab course has a positive influence on the participants' attitude towards research in general. This is true for high school students as well as non-scientists (age groups 16 to 70 years).},
}

@article {pmid31469541,
year = {2019},
author = {Ju, E and Li, T and da Silva, SR and Gao, SJ},
title = {Gold Nanoclusters-Mediated Efficient Delivery of Cas9 Protein through pH-Induced Assembly-Disassembly for Inactivation of Virus Oncogenes.},
journal = {ACS applied materials & interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.9b12335},
pmid = {31469541},
issn = {1944-8252},
abstract = {The CRISPR/Cas gene editing system has been successfully applied to combating bacteria, cancer, virus, and genetic disorders. While viral vectors have been used for the delivery of the CRISPR/Cas9 system, the time required for insert cloning, and virus packaging and standardization hinders its efficient use. Additionally, the high molecular weight of the Cas9 endonuclease makes it not easy for packing in the vehicles. Herein, we report the self-assembly of gold nanoclusters (AuNCs) with SpCas9 protein (SpCas9-AuNCs) at the physiological condition and efficient delivery of SpCas9 into the cell nucleus. This assembly process is highly dependent on pH. SpCas9-AuNCs is stable at a higher pH but is disassembled at a lower pH. Significantly, this assembly-disassembly process facilitates the delivery of SpCas9 into cells and cell nucleus, where the SpCas9 exerts its cleavage function. As a proof-of-concept, the assembled SpCas9-AuNCs nanoparticles are successfully used for efficient knockout of E6 oncogene, restoring the function of tumor suppressive protein p53 and inducing apoptosis in cervical cancer cells with little effect on normal human cells. The SpCas9-AuNCs is useful for sgRNA functional validation, sgRNA library screening, and genomic manipulation.},
}

@article {pmid31467167,
year = {2019},
author = {Peng, R and Li, Z and Xu, Y and He, S and Peng, Q and Wu, LA and Wu, Y and Qi, J and Wang, P and Shi, Y and Gao, GF},
title = {Structural insight into multistage inhibition of CRISPR-Cas12a by AcrVA4.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {},
number = {},
pages = {},
doi = {10.1073/pnas.1909400116},
pmid = {31467167},
issn = {1091-6490},
abstract = {Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Lachnospiraceae bacterium Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.},
}

@article {pmid31462973,
year = {2019},
author = {Zhang, J and Chen, L and Zhang, J and Wang, Y},
title = {Drug Inducible CRISPR/Cas Systems.},
journal = {Computational and structural biotechnology journal},
volume = {17},
number = {},
pages = {1171-1177},
doi = {10.1016/j.csbj.2019.07.015},
pmid = {31462973},
issn = {2001-0370},
abstract = {Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have been employed as a powerful versatile technology for programmable gene editing, transcriptional modulation, epigenetic modulation, and genome labeling, etc. Yet better control of their activity is important to accomplish greater precision and to reduce undesired outcomes such as off-target events. The use of small molecules to control CRISPR/Cas activity represents a promising direction. Here, we provide an updated review on multiple drug inducible CRISPR/Cas systems and discuss their distinct properties. We arbitrarily divided the emerging drug inducible CRISPR/Cas systems into two categories based on whether at transcription or protein level does chemical control occurs. The first category includes Tet-On/Off system and Cre-dependent system. The second category includes chemically induced proximity systems, intein splicing system, 4-Hydroxytamoxifen-Estrogen Receptor based nuclear localization systems, allosterically regulated Cas9 system, and destabilizing domain mediated protein degradation systems. Finally, the advantages and limitations of each system were summarized.},
}

@article {pmid31461619,
year = {2019},
author = {Shao, N and Han, X and Song, Y and Zhang, P and Qin, L},
title = {CRISPR-Cas12a Coupled with Platinum Nano-Reporter for Visual Quantification of SNVs on a Volumetric Bar-Chart Chip.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.9b02925},
pmid = {31461619},
issn = {1520-6882},
abstract = {Methods that can detect and quantify single nucleotide variations (SNVs)/single nucleotide polymorphisms (SNPs) are greatly needed in bioanalysis measuring gene mutations and polymorphisms. Herein, a visual and instrument-free SNV quantification platform is developed. Platinum nanoparticles tethered to magnetic beads by single-stranded DNAs are designed as quantitative readout reporters for CRISPR-Cas12a nucleic acid detection system. The integration of platinum nano-reporter and the CRISPR-Cas system with a volumetric bar chart chip realizes volumetric quantification of nucleic acids. This platform enables quantification of multiple cancer mutations in pure DNA samples and mock cell-free DNA samples in serum, with allelic fractions as low as 0.01%. This platform could have great potential in quantification of SNVs/SNPs as well as other types of nucleic acid targets at the point of care.},
}

@article {pmid31451697,
year = {2019},
author = {Liu, Y and Wan, X and Wang, B},
title = {Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3693},
doi = {10.1038/s41467-019-11479-0},
pmid = {31451697},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/S018875/1//RCUK | Medical Research Council (MRC)/ ; BB/N007212/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; 202078/Z/16/Z//Wellcome Trust (Wellcome)/ ; RPG-2015-445//Leverhulme Trust/ ; },
abstract = {Transcriptional regulation by nuclease-deficient CRISPR/Cas is a popular and valuable tool for routine control of gene expression. CRISPR interference in bacteria can be reliably achieved with high efficiencies. Yet, options for CRISPR activation (CRISPRa) remained limited in flexibility and activity because they relied on σ70 promoters. Here we report a eukaryote-like bacterial CRISPRa system based on σ54-dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ54-dependent promoters with biotechnology relevance in non-model bacteria. It also supports orthogonal gene regulation on multiple levels. Combining our CRISPRa with dxCas9 further expands flexibility in DNA targeting, and boosts dynamic ranges into regimes that enable construction of cascaded CRISPRa circuits. Application-wise, we construct a reusable scanning platform for readily optimizing metabolic pathways without library reconstructions. This eukaryote-like CRISPRa system is therefore a powerful and versatile synthetic biology tool for diverse research and industrial applications.},
}

@article {pmid31440766,
year = {2019},
author = {Wallace, E and Howard, L and Liu, M and O'Brien, T and Ward, D and Shen, S and Prendiville, T},
title = {Long QT Syndrome: Genetics and Future Perspective.},
journal = {Pediatric cardiology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00246-019-02151-x},
pmid = {31440766},
issn = {1432-1971},
abstract = {Long QT syndrome (LQTS) is an inherited primary arrhythmia syndrome that may present with malignant arrhythmia and, rarely, risk of sudden death. The clinical symptoms include palpitations, syncope, and anoxic seizures secondary to ventricular arrhythmia, classically torsade de pointes. This predisposition to malignant arrhythmia is from a cardiac ion channelopathy that results in delayed repolarization of the cardiomyocyte action potential. The QT interval on the surface electrocardiogram is a summation of the individual cellular ventricular action potential durations, and hence is a surrogate marker of the abnormal cellular membrane repolarization. Severely affected phenotypes administered current standard of care therapies may not be fully protected from the occurrence of cardiac arrhythmias. There are 17 different subtypes of LQTS associated with monogenic mutations of 15 autosomal dominant genes. It is now possible to model the various LQTS phenotypes through the generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes. RNA interference can silence or suppress the expression of mutant genes. Thus, RNA interference can be a potential therapeutic intervention that may be employed in LQTS to knock out mutant mRNAs which code for the defective proteins. CRISPR/Cas9 is a genome editing technology that offers great potential in elucidating gene function and a potential therapeutic strategy for monogenic disease. Further studies are required to determine whether CRISPR/Cas9 can be employed as an efficacious and safe rescue of the LQTS phenotype. Current progress has raised opportunities to generate in vitro human cardiomyocyte models for drug screening and to explore gene therapy through genome editing.},
}

@article {pmid31439808,
year = {2019},
author = {Lee, J and Mou, H and Ibraheim, R and Liang, SQ and Liu, P and Xue, W and Sontheimer, EJ},
title = {Tissue-restricted Genome Editing in vivo Specified by MicroRNA-repressible Anti-CRISPR Proteins.},
journal = {RNA (New York, N.Y.)},
volume = {},
number = {},
pages = {},
doi = {10.1261/rna.071704.119},
pmid = {31439808},
issn = {1469-9001},
abstract = {CRISPR-Cas systems are bacterial adaptive immune pathways that have revolutionized biotechnology and biomedical applications. Despite the potential for human therapeutic development, there are many hurdles that must be overcome before its use in clinical settings. Some clinical safety concerns arise from editing activity in unintended cell types or tissues upon in vivo delivery [e.g. by adeno-associated virus (AAV) vectors]. Although tissue-specific promoters and serotypes with tissue tropisms can be used, suitably compact promoters are not always available for desired cell types, and AAV tissue tropism specificities are not absolute. To reinforce tissue-specific editing, we exploited anti-CRISPR proteins (Acrs) that have evolved as natural countermeasures against CRISPR immunity. To inhibit Cas9 in all ancillary tissues without compromising editing in the target tissue, we established a flexible platform in which an Acr transgene is repressed by endogenous, tissue-specific microRNAs (miRNAs). We demonstrate that miRNAs regulate the expression of an Acr transgene bearing miRNA-binding sites in its 3' UTR and control subsequent genome editing outcomes in a cell-type specific manner. We also show that the strategy is applicable to multiple Cas9 orthologs and their respective anti-CRISPRs. Furthermore, we validate this approach in vivo by demonstrating that AAV9 delivery of Nme2Cas9, along with an AcrIIC3Nme construct that is targeted for repression by liver-specific miR-122, allows editing in the liver while repressing editing in an unintended tissue (heart muscle) in adult mice. This strategy provides safeguards against off-tissue genome editing by confining Cas9 activity to selected cell types.},
}

@article {pmid31439663,
year = {2019},
author = {Meyer, MB and Lee, SM and Carlson, AH and Benkusky, NA and Kaufmann, M and Jones, G and Pike, JW},
title = {A chromatin-based mechanism controls differential regulation of the cytochrome P450 gene Cyp24a1 in renal and non-renal tissues.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA119.010173},
pmid = {31439663},
issn = {1083-351X},
abstract = {Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and CYP24A1 function maintain physiological levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the kidney. Renal Cyp27b1 and Cyp24a1 expression levels are transcriptionally regulated in a highly reciprocal manner by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3 In contrast, Cyp24a1 regulation in non-renal target cells (NRTCs) is limited to induction by 1,25(OH)2D3 Herein, we used ChIP-Seq analyses of mouse tissues to identify regulatory regions within the Cyp24a1 gene locus. We found an extended region downstream of Cyp24a1 containing a cluster of sites, termed C24-DS1, binding PTH-sensitive cAMP-responsive element-binding protein (CREB) and a cluster termed C24-DS2 binding the vitamin D receptor (VDR). VDR-occupied sites were present in both the kidney and in NRTCs, but pCREB sites were occupied only in the kidney. We deleted each segment in the mouse and observed that although the overt phenotypes of both cluster deletions were unremarkable, RNA analysis in the C24-DS1-deleted strain revealed a loss of basal renal Cyp24a1 expression, total resistance to FGF23 and PTH regulation, and secondary suppression of renal Cyp27b1; 1,25(OH)2D3 induction remained unaffected in all tissues. In contrast, loss of the VDR cluster in the C24-DS2-deleted strain did not affect 1,25(OH)2D3 induction of renal Cyp24a1 expression, yet reduced but did not eliminate Cyp24a1 responses in NRTCs. We conclude that a chromatin-based mechanism differentially regulates Cyp24a1 in the kidney and NRTCs and is essential for the specific functions of Cyp24a1 in these two tissue types.},
}

@article {pmid31433621,
year = {2019},
author = {Park, S and Beal, PA},
title = {Off-Target Editing by CRISPR-Guided DNA Base Editors.},
journal = {Biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.biochem.9b00573},
pmid = {31433621},
issn = {1520-4995},
abstract = {Base editing is a genome editing strategy that induces specific single-nucleotide changes within genomic DNA. Two major DNA base editors, cytosine base editors and adenine base editors, that consist of a Cas9 protein linked to a deaminase enzyme that catalyzes targeted base conversion directed by a single-guide RNA have been developed. This strategy has been used widely for precise genome editing because, unlike CRISPR-Cas nuclease-based genome editing systems, this strategy does not create double-strand DNA breaks that often result in high levels of undesirable indels. However, recent papers have reported that DNA base editors can cause substantial off-target editing in both genomic DNA and RNA. The off-target editing described in these studies is primarily independent of guide RNA and arises from the promiscuous reactivity of the deaminase enzymes used in DNA base editors. In this Perspective, we discuss the development of DNA base editors, the guide RNA-independent off-target activity reported in recent studies, and strategies that improve the selectivity of DNA base editors.},
}

@article {pmid31433273,
year = {2019},
author = {Kalinina, NO and Khromov, A and Love, AJ and Taliansky, M},
title = {CRISPR applications in plant virology: virus resistance and beyond.},
journal = {Phytopathology},
volume = {},
number = {},
pages = {},
doi = {10.1094/PHYTO-07-19-0267-IA},
pmid = {31433273},
issn = {0031-949X},
abstract = {CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) is a prokaryotic adaptive immune system which has been reprogrammed into a precise, simple and efficient gene targeting technology. This emerging technology is revolutionizing various areas of life sciences, medicine, biotechnology and has raised significant interest among plant biologists, both in basic science and in plant protection and breeding. In this review, we describe the basic principles of CRISPR/Cas systems, and how they can be deployed to model plants and crops for the control, monitoring and study of the mechanistic aspects of plant virus infections. We discuss how Cas endonucleases can be used to engineer plant virus resistance by directly targeting viral DNA or RNA, as well as how they can inactivate host susceptibility genes. Additionally, other applications of CRISPR/Cas in plant virology such as virus diagnostics and imaging are reviewed. The review also provides a systemic comparison between CRISPR/Cas technology and RNA interference approaches, the latter of which has also been used for development of virus resistant plants. Finally, we outline challenges to be solved before CRISPR/Cas can produce virus-resistant crop plants which can be marketed.},
}

@article {pmid31429483,
year = {2019},
author = {Wu, Q and Wang, B and Zhou, C and Lin, P and Qin, S and Gao, P and Wang, Z and Xia, Z and Wu, M},
title = {Bacterial Type I CRISPR-Cas systems influence inflammasome activation in mammalian host by promoting autophagy.},
journal = {Immunology},
volume = {},
number = {},
pages = {},
doi = {10.1111/imm.13108},
pmid = {31429483},
issn = {1365-2567},
abstract = {The CRISPR-Cas systems in prokaryotes function at defending against foreign DNAs, providing adaptive immunity to maintain homeostasis. CRISPR-Cas may also influence immune regulation ability in mammalian cells through alterations of pathogenic extent and nature. Recent research has implied that Type I CRISPR-Cas systems of Pseudomonas aeruginosa UCBPP-PA14 strain impede the recognition by toll-like receptor 4, and decreasing pro-inflammatory responses both in vitro and in vivo. However, the molecular mechanism by which CRISPR-Cas systems affect host immunity is largely undemonstrated. Here, we explored whether CRISPR-Cas systems can influence autophagy to alter the activation of inflammasome. Using the wild-type (WT) PA14 and total CRISPR-Cas region deletion (∆TCR) mutant strain, we elucidated the role and underlying mechanism of Type I CRISPR-Cas systems in bacterial infection, and showed that CRISPR-Cas systems impacted the release of mitochondrial DNA and induction of autophagy. CRISPR-Cas deficiency led to the increase of mitochondrial DNA release, decrease in autophagy, increase of inflammasome activation and ultimately elevation of pro-inflammatory response. Our findings illustrate a new important mechanism by which Type I CRISPR-Cas systems control their virulence potency to evade the host defense. This article is protected by copyright. All rights reserved.},
}

@article {pmid31428783,
year = {2019},
author = {Birkholz, N and Fagerlund, RD and Smith, LM and Jackson, SA and Fineran, PC},
title = {The autoregulator Aca2 mediates anti-CRISPR repression.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkz721},
pmid = {31428783},
issn = {1362-4962},
abstract = {CRISPR-Cas systems are widespread bacterial adaptive defence mechanisms that provide protection against bacteriophages. In response, phages have evolved anti-CRISPR proteins that inactivate CRISPR-Cas systems of their hosts, enabling successful infection. Anti-CRISPR genes are frequently found in operons with genes encoding putative transcriptional regulators. The role, if any, of these anti-CRISPR-associated (aca) genes in anti-CRISPR regulation is unclear. Here, we show that Aca2, encoded by the Pectobacterium carotovorum temperate phage ZF40, is an autoregulator that represses the anti-CRISPR-aca2 operon. Aca2 is a helix-turn-helix domain protein that forms a homodimer and interacts with two inverted repeats in the anti-CRISPR promoter. The inverted repeats are similar in sequence but differ in their Aca2 affinity, and we propose that they have evolved to fine-tune, and downregulate, anti-CRISPR production at different stages of the phage life cycle. Specific, high-affinity binding of Aca2 to the first inverted repeat blocks the promoter and induces DNA bending. The second inverted repeat only contributes to repression at high Aca2 concentrations in vivo, and no DNA binding was detectable in vitro. Our investigation reveals the mechanism by which an Aca protein regulates expression of its associated anti-CRISPR.},
}

@article {pmid31427601,
year = {2019},
author = {Lin, P and Pu, Q and Wu, Q and Zhou, C and Wang, B and Schettler, J and Wang, Z and Qin, S and Gao, P and Li, R and Li, G and Cheng, Z and Lan, L and Jiang, J and Wu, M},
title = {High-throughput screen reveals sRNAs regulating crRNA biogenesis by targeting CRISPR leader to repress Rho termination.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3728},
doi = {10.1038/s41467-019-11695-8},
pmid = {31427601},
issn = {2041-1723},
support = {R01AI109317-01A1//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; AI101973-01//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R15 AI101973/AI/NIAID NIH HHS/United States ; R01 AI138203/AI/NIAID NIH HHS/United States ; R01AI0138203-01//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 AI109317/AI/NIAID NIH HHS/United States ; },
abstract = {Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.},
}

@article {pmid31426522,
year = {2019},
author = {Shelake, RM and Pramanik, D and Kim, JY},
title = {Exploration of Plant-Microbe Interactions for Sustainable Agriculture in CRISPR Era.},
journal = {Microorganisms},
volume = {7},
number = {8},
pages = {},
doi = {10.3390/microorganisms7080269},
pmid = {31426522},
issn = {2076-2607},
support = {NRF 2017R1A4A1015515//National Research Foundation of Korea/ ; SSAC, Grant PJ01322601//Rural Development Administration (RDA), Republic of Korea/ ; },
abstract = {Plants and microbes are co-evolved and interact with each other in nature. Plant-associated microbes, often referred to as plant microbiota, are an integral part of plant life. Depending on the health effects on hosts, plant-microbe (PM) interactions are either beneficial or harmful. The role of microbiota in plant growth promotion (PGP) and protection against various stresses is well known. Recently, our knowledge of community composition of plant microbiome and significant driving factors have significantly improved. So, the use of plant microbiome is a reliable approach for a next green revolution and to meet the global food demand in sustainable and eco-friendly agriculture. An application of the multifaceted PM interactions needs the use of novel tools to know critical genetic and molecular aspects. Recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated genome editing (GE) tools are of great interest to explore PM interactions. A systematic understanding of the PM interactions will enable the application of GE tools to enhance the capacity of microbes or plants for agronomic trait improvement. This review focuses on applying GE techniques in plants or associated microbiota for discovering the fundamentals of the PM interactions, disease resistance, PGP activity, and future implications in agriculture.},
}

@article {pmid31416510,
year = {2019},
author = {Ma, F and Shi, CC and Liang, PP and Li, ST and Gu, X and Xiao, X and Hao, H},
title = {[Construction of a mouse model of cblC type methylmalonic acidemia with W203X mutation based on the CRISPR/Cas9 technology].},
journal = {Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics},
volume = {21},
number = {8},
pages = {824-829},
pmid = {31416510},
issn = {1008-8830},
mesh = {Amino Acid Metabolism, Inborn Errors ; Animals ; *CRISPR-Cas Systems ; Carrier Proteins ; Heterozygote ; Mice ; Mutation ; },
abstract = {OBJECTIVE: To construct a W203X-mutant mouse model of cblC type methylmalonic acidemia based on the CRISPR/Cas9 technology.

METHODS: At first, BLAST was used to compare the conservative nature of the cblC gene and protein sequences in humans and mice, and then, the CRISPR/Cas9 technology was used for microinjection of mouse fertilized eggs to obtain heterozygous F1 mice. Hybridization was performed for these mice to obtain homozygous W203X-mutant mice. The blood level of the metabolite propionyl carnitine (C3) was measured for homozygous mutant mice, heterozygous littermates, and wild-type mice.

RESULTS: The gene and protein sequences of MMACHC, the pathogenic gene for cblC type methylmalonic acidemia, were highly conserved in humans and mice. The homozygous W203X-mutant mice were successfully obtained by the CRISPR/Cas9 technology, and there was a significant increase in C3 in these mice at 24 hours after birth (P<0.001).

CONCLUSIONS: A W203X-mutant mouse model of cblC type methylmalonic acidemia is successfully constructed by the CRISPR/Cas9 technology.},
}

Amino Acid Metabolism, Inborn Errors
Animals
*CRISPR-Cas Systems
Carrier Proteins
Heterozygote
Mice
Mutation

@article {pmid31416467,
year = {2019},
author = {Keough, KC and Lyalina, S and Olvera, MP and Whalen, S and Conklin, BR and Pollard, KS},
title = {AlleleAnalyzer: a tool for personalized and allele-specific sgRNA design.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {167},
doi = {10.1186/s13059-019-1783-3},
pmid = {31416467},
issn = {1474-760X},
support = {UM1HL098179/NH/NIH HHS/United States ; P01HL089707/NH/NIH HHS/United States ; R01MH109907/NH/NIH HHS/United States ; U01HL100406/NH/NIH HHS/United States ; P01HL089707/NH/NIH HHS/United States ; R01HL130533/NH/NIH HHS/United States ; 1R01EY028249/NH/NIH HHS/United States ; },
abstract = {The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing by even a single base pair. Target sites might carry genetic variations that are not distinguishable by sgRNA designing tools based on one reference genome. AlleleAnalyzer is an open-source software that incorporates single-nucleotide variants and short insertions and deletions to design sgRNAs for precisely editing 1 or multiple haplotypes of a sequenced genome, currently supporting 11 Cas proteins. It also leverages patterns of shared genetic variation to optimize sgRNA design for different human populations. AlleleAnalyzer is available at https://github.com/keoughkath/AlleleAnalyzer .},
}

@article {pmid31411686,
year = {2019},
author = {Tang, Z and Chen, S and Chen, A and He, B and Zhou, Y and Chai, G and Guo, F and Huang, J},
title = {CasPDB: an integrated and annotated database for Cas proteins from bacteria and archaea.},
journal = {Database : the journal of biological databases and curation},
volume = {2019},
number = {},
pages = {},
doi = {10.1093/database/baz093},
pmid = {31411686},
issn = {1758-0463},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) constitute CRISPR-Cas systems, which are antiphage immune systems present in numerous bacterial and most archaeal species. In recent years, CRISPR-Cas systems have been developed into reliable and powerful genome editing tools. Nevertheless, finding similar or better tools from bacteria or archaea remains crucial. This requires the exploration of different CRISPR systems, identification and characterization new Cas proteins. Archives tailored for Cas proteins are urgently needed and necessitate the prediction and grouping of Cas proteins into an information center with all available experimental evidence. Here, we constructed Cas Protein Data Bank (CasPDB), an integrated and annotated online database for Cas proteins from bacteria and archaea. The CasPDB database contains 287 reviewed Cas proteins, 257 745 putative Cas proteins and 3593 Cas operons from 32 023 bacteria species and 1802 archaea species. The database can be freely browsed and searched. The CasPDB web interface also represents all the 3593 putative Cas operons and its components. Among these operons, 328 are members of the type II CRISPR-Cas system.},
}

@article {pmid31409685,
year = {2019},
author = {Hullahalli, K and Rodrigues, M and Nguyen, UT and Palmer, K},
title = {Erratum for Hullahalli et al., "An Attenuated CRISPR-Cas System in Enterococcus faecalis Permits DNA Acquisition".},
journal = {mBio},
volume = {10},
number = {4},
pages = {},
doi = {10.1128/mBio.01775-19},
pmid = {31409685},
issn = {2150-7511},
}

@article {pmid31409682,
year = {2019},
author = {Gloag, ES and Marshall, CW and Snyder, D and Lewin, GR and Harris, JS and Santos-Lopez, A and Chaney, SB and Whiteley, M and Cooper, VS and Wozniak, DJ},
title = {Pseudomonas aeruginosa Interstrain Dynamics and Selection of Hyperbiofilm Mutants during a Chronic Infection.},
journal = {mBio},
volume = {10},
number = {4},
pages = {},
doi = {10.1128/mBio.01698-19},
pmid = {31409682},
issn = {2150-7511},
support = {U01 AI124302/AI/NIAID NIH HHS/United States ; R01 AI134895/AI/NIAID NIH HHS/United States ; R01 GM110444/GM/NIGMS NIH HHS/United States ; R01 AI097511/AI/NIAID NIH HHS/United States ; R01 AI077628/AI/NIAID NIH HHS/United States ; },
abstract = {Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse Pseudomonas aeruginosa infections. Wounds were infected with a mixed community of six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the wsp pathway, conferring hyperbiofilm phenotypes. Several of the wsp mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes.IMPORTANCE Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of Pseudomonas aeruginosa in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection, P. aeruginosa experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes.},
}

@article {pmid31407519,
year = {2019},
author = {Li, QV and Rosen, BP and Huangfu, D},
title = {Decoding pluripotency: Genetic screens to interrogate the acquisition, maintenance, and exit of pluripotency.},
journal = {Wiley interdisciplinary reviews. Systems biology and medicine},
volume = {},
number = {},
pages = {e1464},
doi = {10.1002/wsbm.1464},
pmid = {31407519},
issn = {1939-005X},
support = {R01DK096239/DK/NIDDK NIH HHS/United States ; T32GM008539//NIH T32 Training Grant/ ; P30 CA008748/CA/NCI NIH HHS/United States ; 1-19-IBS-125//American Diabetes Association/ ; 3-SRA-2017-364-S-B//Juvenile Diabetes Research Foundation International/ ; #2016-004//Tri-Institutional Stem Cell Initiative/ ; 2016-032//Tri-Institutional Stem Cell Initiative/ ; C32593GG//New York State Stem Cell Science/ ; C029156//New York State Stem Cell Science/ ; C029567//New York State Stem Cell Science/ ; },
abstract = {Pluripotent stem cells have the ability to unlimitedly self-renew and differentiate to any somatic cell lineage. A number of systems biology approaches have been used to define this pluripotent state. Complementary to systems level characterization, genetic screens offer a unique avenue to functionally interrogate the pluripotent state and identify the key players in pluripotency acquisition and maintenance, exit of pluripotency, and lineage differentiation. Here we review how genetic screens have helped us decode pluripotency regulation. We will summarize results from RNA interference (RNAi) based screens, discuss recent advances in CRISPR/Cas-based genetic perturbation methods, and how these advances have made it possible to more comprehensively interrogate pluripotency and differentiation through genetic screens. Such investigations will not only provide a better understanding of this unique developmental state, but may enhance our ability to use pluripotent stem cells as an experimental model to study human development and disease progression. Functional interrogation of pluripotency also provides a valuable roadmap for utilizing genetic perturbation to gain systems level understanding of additional cellular states, from later stages of development to pathological disease states. This article is categorized under: Developmental Biology > Stem Cell Biology and Regeneration Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates.},
}

@article {pmid31403072,
year = {2019},
author = {Hanewich-Hollatz, MH and Chen, Z and Hochrein, LM and Huang, J and Pierce, NA},
title = {Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology.},
journal = {ACS central science},
volume = {5},
number = {7},
pages = {1241-1249},
doi = {10.1021/acscentsci.9b00340},
pmid = {31403072},
issn = {2374-7943},
abstract = {A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF "terminator switch" mechanism, ≈15-fold for an ON → OFF "splinted switch" mechanism, and ≈3-fold for an OFF → ON "toehold switch" mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.},
}

@article {pmid31401729,
year = {2019},
author = {Hsu, CT and Cheng, YJ and Yuan, YH and Hung, WF and Cheng, QW and Wu, FH and Lee, LY and Gelvin, SB and Lin, CS},
title = {Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco.},
journal = {Plant molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s11103-019-00907-w},
pmid = {31401729},
issn = {1573-5028},
support = {105-2313-B-001 -007 -MY3//Ministry of Science and Technology, Taiwan/ ; AS-KPQ-107-ITAR-10//Innovative Translational Agricultural Research Administrative Office/ ; },
abstract = {KEY MESSAGE: Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants. Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.},
}

@article {pmid31400605,
year = {2019},
author = {Liu, X and Li, G and Zhou, X and Qiao, Y and Wang, R and Tang, S and Liu, J and Wang, L and Huang, X},
title = {Improving Editing Efficiency for the Sequences with NGH PAM Using xCas9-Derived Base Editors.},
journal = {Molecular therapy. Nucleic acids},
volume = {17},
number = {},
pages = {626-635},
doi = {10.1016/j.omtn.2019.06.024},
pmid = {31400605},
issn = {2162-2531},
abstract = {The development of CRISPR/Cas9-mediated base editors (BEs) provided a versatile tool for precise genome editing. The recently developed xCas9-derived base editors (xBEs) that recognize the NG PAM substantially expand the targeting scope in the genome, while their editing efficiency needs to be improved. Here, we described an improved version of xBEs by fusing the BPNLS and Gam to the N terminus of xBEs (BPNLS-Gam-xBE3 and BPNLS-xABE), and this version of base editor displayed higher targeting efficiency for the majority of detected sites. By using this improved version of xBEs, we successfully created and corrected pathogenic mutations at genomic sites with the NGN protospacer-adjacent motif in human cells. Lastly, we used BPNLS-Gam-xBE3 to model pathogenic mutations in discarded human tripronuclear (3PN) zygotes, and no obvious off-targets and indels were detected. Taken together, the data in our study offer an efficient tool for precise genome editing and, thus, an enriched base editing toolkit.},
}

@article {pmid31399410,
year = {2019},
author = {Maikova, A and Kreis, V and Boutserin, A and Severinov, K and Soutourina, O},
title = {Using endogenous CRISPR-Cas system for genome editing in the human pathogen Clostridium difficile.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1128/AEM.01416-19},
pmid = {31399410},
issn = {1098-5336},
abstract = {Human enteropathogen Clostridium difficile constitutes a key public health issue in industrialized countries. Many aspects of C. difficile pathophysiology and adaptation inside the host remain poorly understood. We have recently reported that this bacterium possesses an active CRISPR-Cas system of subtype I-B for defence against phages and other mobile genetic elements that could contribute to its success during infection. In this paper, we demonstrate that redirecting this endogenous CRISPR-Cas system towards autoimmunity allows efficient genome editing in C. difficile We provide detailed description of this newly developed approach and show, as a proof of principle, its efficient application for deletion of a specific gene in reference 630Δerm and in epidemic R20291 C. difficile strains. The new method expands the arsenal of the currently limiting set of gene engineering tools available for investigation of C. difficile and may serve as the basis for new strategies to control C. difficile infections.ImportanceClostridium difficile represents today a real danger for human and animal health. It is the leading cause of diarrhoea associated with healthcare in adults in industrialized countries. The incidence of these infections continues to increase and this trend is accentuated by the general aging of the population. Many questions remain unanswered on the mechanisms contributing to C. difficile success inside the host. The set of genetic tools available for this pathogen is limited and new developments are badly needed. C. difficile has developed efficient defence systems that are directed against foreign DNA and could contribute to its survival in phage-rich gut communities. We show how one of such defence systems, named CRISPR-Cas, can be hijacked for C. difficile genome editing. Our results also show a great potential of CRISPR-Cas system for development of new therapeutic strategies against C. difficile infections.},
}

@article {pmid31399023,
year = {2019},
author = {Kruse, T and Ratnadevi, CM and Erikstad, HA and Birkeland, NK},
title = {Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph "Candidatus Methylacidiphilum kamchatkense" strain Kam1 and comparison with its closest relatives.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {642},
doi = {10.1186/s12864-019-5995-4},
pmid = {31399023},
issn = {1471-2164},
support = {261923//Norges Forskningsråd/ ; },
abstract = {BACKGROUND: The candidate genus "Methylacidiphilum" comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the "Ca. Methylacidiphilum" and the sister genus "Ca. Methylacidimicrobium" represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.

RESULTS: Here we present the closed genome of "Ca. Methylacidiphilum kamchatkense" strain Kam1 and a comparison with the genomes of its two closest relatives "Ca. Methylacidiphilum fumariolicum" strain SolV and "Ca. Methylacidiphilum infernorum" strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of "Ca. Methylacidiphilum" is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between "Ca. M. kamchatkense" strain Kam1 and strains SolV and V4 is ≤95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements.

CONCLUSIONS: Comparing three closed genomes of "Ca. Methylacidiphilum" spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.},
}

@article {pmid31397669,
year = {2019},
author = {Knott, GJ and Cress, BF and Liu, JJ and Thornton, BW and Lew, RJ and Al-Shayeb, B and Rosenberg, DJ and Hammel, M and Adler, BA and Lobba, MJ and Xu, M and Arkin, AP and Fellmann, C and Doudna, JA},
title = {Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
doi = {10.7554/eLife.49110},
pmid = {31397669},
issn = {2050-084X},
support = {HR0011-17-2-0043//Defense Advanced Research Projects Agency/ ; MCB-1244557//National Science Foundation/ ; P50GM082250/NH/NIH HHS/United States ; DGE 1752814//National Science Foundation/ ; RM1HG009490/NH/NIH HHS/United States ; R00GM118909/GM/NIGMS NIH HHS/United States ; Graduate Research Fellowship,DGE 1752814//National Science Foundation/ ; P50GM082250/NH/NIH HHS/United States ; R00GM118909/GM/NIGMS NIH HHS/United States ; RM1HG009490/NH/NIH HHS/United States ; R00 GM118909/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.},
}

@article {pmid31392984,
year = {2019},
author = {Kim, JG and Garrett, S and Wei, Y and Graveley, BR and Terns, MP},
title = {CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkz677},
pmid = {31392984},
issn = {1362-4962},
abstract = {CRISPR-Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3' hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to coordinate directional and site-specific spacer integration into the CRISPR locus to ensure precise duplication of the repeat required for CRISPR immunity.},
}

@article {pmid31392501,
year = {2019},
author = {Ai, L and Guo, W and Chen, W and Teng, Y and Bai, L},
title = {The gal80 Deletion by CRISPR-Cas9 in Engineered Saccharomyces cerevisiae Produces Artemisinic Acid Without Galactose Induction.},
journal = {Current microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00284-019-01752-2},
pmid = {31392501},
issn = {1432-0991},
support = {2017ZX09101002-003-003//The Drug Innovation Major Project/ ; 31870059//National Natural Science Foundation of China/ ; 2018ZX09711001-007-003//Drug Innovation Major Project/ ; 2018YFA0902000//National Key Research and Development Program of China/ ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has emerged as the dominating tool for genome engineering, while also changes the speed and efficiency of metabolic engineering in conventional and non-conventional yeasts. Among these CRISPR-Cas systems, CRISPR-Cas9 technology has usually been applied for removing unfavorable target genes. Here, we used CRISPR-Cas9 technology to delete the gal80 gene in uracil-deficient strain and had successfully remolded the engineered Saccharomyces cerevisiae that can produce artemisinic acid without galactose induction. An L9(34) orthogonal test was adopted to investigate the effects of different factors on artemisinic acid production. Fermentation medium III with sucrose as carbon sources, 1% inoculum level, and 84-h culture time were identified as the optimal fermentation conditions. Under this condition, the maximum artemisinic acid production by engineered S. cerevisiae 1211-2 was 740 mg/L in shake-flask cultivation level. This study provided an effective approach to reform metabolic pathway of artemisinic acid-producing strain. The engineered S. cerevisiae 1211-2 may be applied to artemisinic acid production by industrial fermentation in the future.},
}

@article {pmid31392299,
year = {2019},
author = {Zhang, F and Song, G and Tian, Y},
title = {Anti-CRISPRs: The natural inhibitors for CRISPR-Cas systems.},
journal = {Animal models and experimental medicine},
volume = {2},
number = {2},
pages = {69-75},
doi = {10.1002/ame2.12069},
pmid = {31392299},
issn = {2576-2095},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated protein) systems serve as the adaptive immune system by which prokaryotes defend themselves against phages. It has also been developed into a series of powerful gene-editing tools. As the natural inhibitors of CRISPR-Cas systems, anti-CRISPRs (Acrs) can be used as the "off-switch" for CRISPR-Cas systems to limit the off-target effects caused by Cas9. Since the discovery of CRISPR-Cas systems, much research has focused on the identification, mechanisms and applications of Acrs. In light of the rapid development and scientific significance of this field, this review summarizes the history and research status of Acrs, and considers future applications.},
}

@article {pmid31391532,
year = {2019},
author = {Martens, KJA and van Beljouw, SPB and van der Els, S and Vink, JNA and Baas, S and Vogelaar, GA and Brouns, SJJ and van Baarlen, P and Kleerebezem, M and Hohlbein, J},
title = {Visualisation of dCas9 target search in vivo using an open-microscopy framework.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3552},
doi = {10.1038/s41467-019-11514-0},
pmid = {31391532},
issn = {2041-1723},
abstract = {CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.},
}

@article {pmid31391371,
year = {2019},
author = {Yamauchi, T},
title = {[Exploration of novel therapeutic targets in acute myeloid leukemia via genome-wide CRISPR screening].},
journal = {[Rinsho ketsueki] The Japanese journal of clinical hematology},
volume = {60},
number = {7},
pages = {810-817},
doi = {10.11406/rinketsu.60.810},
pmid = {31391371},
issn = {0485-1439},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Leukemia, Myeloid, Acute/*genetics ; Mice ; *RNA Splicing ; *RNA Stability ; RNA, Small Interfering ; },
abstract = {Acute myeloid leukemia (AML) remains a devasting disease. Progress has been made to define molecular mechanisms underlying disease pathogenesis due, in part, to the near-complete understanding of AML genome. Nonetheless, functional studies are necessary to assess the significance of AML-associated mutations and devise urgently needed therapies. Genome-wide knockout screening, employing CRISPR-Cas9 genome editing, is a powerful tool in functional genomics. In this study, genome-wide CRISPR screening was performed using mouse leukemia cell lines developed in our Center, followed by in vivo screening. Among 20,611 genes, 130 AML essential genes were identified, including clinically actionable candidates. It was shown that mRNA decapping enzyme scavenger (DCPS), an enzyme implicated in mRNA decay pathway, is essential for AML survival. ShRNA-mediated gene knockdown and DCPS inhibitor (RG3039) were employed to validate findings. RG3039 induced cell-cycle arrest and apoptosis in vitro. Furthermore, mass spectrometry analysis revealed an association between DCPS and RNA metabolic pathways, and RNA-Seq showed that RG3039 treatment induced aberrant mRNA splicing in AML cells. Importantly, RG3039 exhibited anti-leukemia effects in PDX models. These findings identify DCPS as a novel therapeutic target for AML, shedding new light on the nuclear RNA metabolic pathway in leukemogenesis.},
}

Animals
*CRISPR-Cas Systems
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Leukemia, Myeloid, Acute/*genetics
Mice
*RNA Splicing
*RNA Stability
RNA, Small Interfering

@article {pmid31389773,
year = {2019},
author = {Young, CS and Pyle, AD and Spencer, MJ},
title = {CRISPR for Neuromuscular Disorders: Gene Editing and Beyond.},
journal = {Physiology (Bethesda, Md.)},
volume = {34},
number = {5},
pages = {341-353},
doi = {10.1152/physiol.00012.2019},
pmid = {31389773},
issn = {1548-9221},
abstract = {This is a review describing advances in CRISPR/Cas-mediated therapies for neuromuscular disorders (NMDs). We explore both CRISPR-mediated editing and dead Cas approaches as potential therapeutic strategies for multiple NMDs. Last, therapeutic considerations, including delivery and off-target effects, are also discussed.},
}

@article {pmid31389128,
year = {2019},
author = {Kim, Y and Lee, SJ and Yoon, HJ and Kim, NK and Lee, BJ and Suh, JY},
title = {Anti-CRISPR AcrIIC3 discriminates between Cas9 orthologs via targeting the variable surface of the HNH nuclease domain.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.15037},
pmid = {31389128},
issn = {1742-4658},
support = {500-20170161//Creative-Pioneering Researchers Program through Seoul National University/ ; NRF-2016R1C1B2014609//National Research Foundation/ ; PJ013181//Cooperative Research Program for Agriculture Science & Technology Development, Rural Development Administration/ ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems constitute the adaptive immunity of bacteria and archaea, degrading nucleic acids of invading phages and plasmids. In response, phages employ anti-CRISPR (Acr) proteins as a counterdefense mechanism to neutralize the host immunity. AcrIIC3 directly inhibits target DNA cleavage of type II-C Cas9 of Neisseria meningitidis. Here, we show that AcrIIC3 interacts with the HNH nuclease domain of N. meningitidis Cas9 to inhibit its nuclease activity in an allosteric manner. The crystal structure of the AcrIIC3-HNH complex reveals that AcrIIC3 binds opposite the active site on the HNH nuclease domain. AcrIIC3 employs a unique interface for HNH, allowing it to discriminate between Cas9 orthologs, which contrasts with the broad spectrum of Cas9 inhibition by AcrIIC1. Interface residues of HNH provide key electrostatic and hydrophobic interactions that determine the host specificity of AcrIIC3. Mutations that replace HNH interfaces of N. meningitidis Cas9 with those of Geobacillus stearothermophilus Cas9 or Campylobacter jejuni Cas9 significantly attenuate AcrIIC3 binding, illustrating that the divergent interaction surface confers the host specificity of AcrIIC3. Our study demonstrates that the variable sequences of binding interface can define the target specificity of Acr proteins, suggesting potential applications in Cas9 control for gene editing.},
}

@article {pmid31385197,
year = {2019},
author = {Hajizadeh Dastjerdi, A and Newman, A and Burgio, G},
title = {The Expanding Class 2 CRISPR Toolbox: Diversity, Applicability, and Targeting Drawbacks.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1007/s40259-019-00369-y},
pmid = {31385197},
issn = {1179-190X},
abstract = {The class 2 clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, one of the prokaryotic adaptive immune systems, has sparked a lot of attention for its use as a gene editing tool. Currently, type II, V, and VI effector modules of this class have been characterized and extensively tested for nucleic acid editing, imaging, and disease diagnostics. Due to the unique composition of their nuclease catalytic center, the effector modules substantially vary in their function and possible biotechnology applications. In this review, we discuss the structural and functional diversity in class 2 CRISPR effectors, and debate their suitability for nucleic acid targeting and their shortcomings as gene editing tools.},
}

@article {pmid31381206,
year = {2019},
author = {Wolter, F and Puchta, H},
title = {In planta gene targeting can be enhanced by the use of CRISPR/Cas12a.},
journal = {The Plant journal : for cell and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/tpj.14488},
pmid = {31381206},
issn = {1365-313X},
abstract = {The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a DSB and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell specific expression of the potent but less broadly applicable SaCas9 nuclease. We now tested whether we can improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20 to 80 fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for GT and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower NHEJ induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns. This article is protected by copyright. All rights reserved.},
}

@article {pmid31378358,
year = {2019},
author = {Guest, M and Goodchild, JA and Bristow, JA and Flemming, AJ},
title = {RDL A301S alone does not confer high levels of resistance to cyclodiene organochlorine or phenyl pyrazole insecticides in Plutella xylostella.},
journal = {Pesticide biochemistry and physiology},
volume = {158},
number = {},
pages = {32-39},
doi = {10.1016/j.pestbp.2019.04.005},
pmid = {31378358},
issn = {1095-9939},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dieldrin/pharmacology ; Endosulfan/pharmacology ; Insecticide Resistance/genetics ; Insecticides/*pharmacology ; Moths/*drug effects/genetics ; Mutation/genetics ; Pyrazoles/pharmacology ; Receptors, GABA-A/genetics ; },
abstract = {Mutations in the GABA-gated chloride channel are associated with resistance to cyclodiene organochlorine and phenyl pyrazole insecticides. The best characterised of these is A301S, which was initially identified in a Dieldrin resistant strain of Drosophila melanogaster. The orthologous mutation has been found in a variety of different crop pests including the diamond back moth Plutella xylostella. However, the contribution of this mutation to resistance in this species remains unclear. We have used the CRISPR/Cas9 system in order to edit Plutella xylostella PxGABARalpha1 to Serine at the 301 orthologous position (282 in PxGABARalpha1) in an insecticide sensitive strain isolated from Vero Beach (VB) USA. In this edited line, no high level of resistance is conferred to Dieldrin, Endosulfan or Fipronil, rather only a subtle shift in sensitivity which could not confer commercially important resistance. We conclude that the high level of commercial resistance to cyclodiene organochlorine and phenyl pyrazole insecticides observed in some field isolates of Plutella xylostella cannot arise from A282S in PxGABARalpha1 alone.},
}

Animals
CRISPR-Cas Systems/genetics
Dieldrin/pharmacology
Endosulfan/pharmacology
Insecticide Resistance/genetics
Insecticides/*pharmacology
Moths/*drug effects/genetics
Mutation/genetics
Pyrazoles/pharmacology
Receptors, GABA-A/genetics

@article {pmid31373227,
year = {2019},
author = {Li, F and Hung, SSC and Mohd Khalid, MKN and Wang, JH and Chrysostomou, V and Wong, VHY and Singh, V and Wing, K and Tu, L and Bender, JA and Pebay, A and King, AE and Cook, AL and Wong, RCB and Bui, BV and Hewitt, AW and Liu, GS},
title = {Utility of self-destructing CRISPR/Cas constructs for targeted gene editing in the retina.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2019.021},
pmid = {31373227},
issn = {1557-7422},
abstract = {Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing "kamikaze" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were initially designed to target Streptococcus pyogenes Cas9 (SpCas9) and after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein, YFP), in the presence of mCherry. Both constructs were packaged into AAV2 vectors and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks the expression of SpCas9 and the efficacy of YFP gene disruption was quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated-YFP/SpCas9 targeting CRISPR/Cas compared to those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (~85.5% reduction compared to LacZ/SpCas9 targeting CRISPR/Cas) in the transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive "kamikaze" CRISPR/Cas system can be used as a robust tool for genome editing in the retina, without compromising on-target efficiency.},
}

@article {pmid31372754,
year = {2019},
author = {Fofanov, MV and Morozova, VV and Kozlova, YN and Tikunov, AY and Babkin, IV and Poletaeva, YE and Ryabchikova, EI and Tikunova, NV},
title = {Raoultella bacteriophage RP180, a new member of the genus Kagunavirus, subfamily Guernseyvirinae.},
journal = {Archives of virology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00705-019-04349-z},
pmid = {31372754},
issn = {1432-8798},
support = {18-29-08015//Russian Foundation of Basic Research/ ; # АААА-А17-117020210027-9//Russian State funded budget project/ ; },
abstract = {A novel lytic Raoultella phage, RP180, was isolated and characterized. The RP180 genome has 44,851 base pairs and contains 65 putative genes, 35 of them encoding proteins whose functions were predicted based on sequence similarity to known proteins. The RP180 genome possesses a gene synteny typical of members of the subfamily Guernseyvirinae. Phylogenetic analysis of the RP180 genome and similar phage genomes revealed that phage RP180 is the first member of the genus Kagunavirus, subfamily Guernseyvirinae, that is specific for Raoultella sp. The genome of RP180 encodes a putative protein with similarity to CRISPR-like Cas4 nucleases, which belong to the pfam12705/PDDEXK_1 family. Cas4-like proteins of this family have been shown to interfere with the bacterial host type II-C CRISPR-Cas system.},
}

@article {pmid31371352,
year = {2019},
author = {Engreitz, J and Abudayyeh, O and Gootenberg, J and Zhang, F},
title = {CRISPR Tools for Systematic Studies of RNA Regulation.},
journal = {Cold Spring Harbor perspectives in biology},
volume = {11},
number = {8},
pages = {},
doi = {10.1101/cshperspect.a035386},
pmid = {31371352},
issn = {1943-0264},
abstract = {SUMMARYRNA molecules perform diverse functions in mammalian cells, including transferring genetic information from DNA to protein and playing diverse regulatory roles through interactions with other cellular components. Here, we discuss how clustered regularly interspaced short palindromic repeat (CRISPR)-based technologies for directed perturbations of DNA and RNA are revealing new insights into RNA regulation. First, we review the fundamentals of CRISPR-Cas enzymes and functional genomics tools that leverage these systems. Second, we explore how these new perturbation technologies are transforming the study of regulation of and by RNA, focusing on the functions of DNA regulatory elements and long noncoding RNAs (lncRNAs). Third, we highlight an emerging class of RNA-targeting CRISPR-Cas enzymes that have the potential to catalyze studies of RNA biology by providing tools to directly perturb or measure RNA modifications and functions. Together, these tools enable systematic studies of RNA function and regulation in mammalian cells.},
}

@article {pmid31370935,
year = {2019},
author = {Hanswillemenke, A and Stafforst, T},
title = {Protocols for the generation of caged guideRNAs for light-triggered RNA-targeting with SNAP-ADARs.},
journal = {Methods in enzymology},
volume = {624},
number = {},
pages = {47-68},
doi = {10.1016/bs.mie.2019.06.004},
pmid = {31370935},
issn = {1557-7988},
abstract = {The SNAP-tag technology offers a convenient way to assemble guideRNA-protein conjugates for transcript-specific RNA editing in vitro, in cell culture and in vivo. In contrast to other methods, including CRISPR/Cas-based, the SNAP-tag is small, well expressed and of human origin. Furthermore, the SNAP-ADAR approach enables the ready inclusion of photo control by caging/decaging of the benzylguanine moiety required for the conjugation reaction with the SNAP-tag. Beyond site-directed RNA editing, the method has high potential for various applications in the field of RNA targeting. However, the generation of the required guideRNAs includes some basic chemistry. Here, we provide step-by-step protocols for (a) conduction of photo controlled RNA editing reaction, (b) the generation of photo activatable guideRNAs, and (c) the synthesis of the caged benzylguanine moiety. With this we hope to foster a broader application of these attractive methods to researchers with less experience in chemistry.},
}

@article {pmid31370789,
year = {2019},
author = {Jouanin, A and Schaart, JG and Boyd, LA and Cockram, J and Leigh, FJ and Bates, R and Wallington, EJ and Visser, RGF and Smulders, MJM},
title = {Outlook for coeliac disease patients: towards bread wheat with hypoimmunogenic gluten by gene editing of α- and γ-gliadin gene families.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {333},
doi = {10.1186/s12870-019-1889-5},
pmid = {31370789},
issn = {1471-2229},
support = {FP7-PEOPLE-2013_ITN-607178//FP7 People: Marie-Curie Actions/ ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Electrophoresis, Polyacrylamide Gel ; Gene Editing/*methods ; Genes, Plant/*genetics ; Gliadin/*genetics ; Glutens/*genetics/immunology ; Plant Breeding/methods ; Plants, Genetically Modified ; Sequence Alignment ; Triticum/*genetics ; },
abstract = {BACKGROUND: Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1-2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes.

RESULTS: In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods.

CONCLUSIONS: The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods.},
}

CRISPR-Associated Protein 9
CRISPR-Cas Systems
Electrophoresis, Polyacrylamide Gel
Gene Editing/*methods
Genes, Plant/*genetics
Gliadin/*genetics
Glutens/*genetics/immunology
Plant Breeding/methods
Plants, Genetically Modified
Sequence Alignment
Triticum/*genetics

@article {pmid31362003,
year = {2019},
author = {Karl, M and Sommer, C and Gabriel, CH and Hecklau, K and Venzke, M and Hennig, AF and Radbruch, A and Selbach, M and Baumgrass, R},
title = {Recruitment of Histone Methyltransferase Ehmt1 to Foxp3 TSDR Counteracts Differentiation of Induced Regulatory T Cells.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jmb.2019.07.031},
pmid = {31362003},
issn = {1089-8638},
abstract = {Differentiation toward CD4+ regulatory T (Treg) cells is essentially dependent on an epigenetic program at Treg signature genes, which involves remodeling of the Treg-specific demethylated regions (TSDRs). In particular, the epigenetic status of the conserved non-coding sequence 2 of Foxp3 (Foxp3 TSDR) determines expression stability of the master transcription factor and thus Treg lineage identity. However, the molecular mechanisms controlling the epigenetic remodeling at TSDRs in Treg and conventional T cells are largely unknown. Using a combined approach of DNA pull-down and mass spectrometric analysis, we report a novel regulatory mechanism in which transcription factor Wiz recruits the histone methyltransferase Ehmt1 to Foxp3 TSDR. We show that both Wiz and Ehmt1 are crucial for shaping the region with the repressive histone modification H3K9me2 in conventional T cells. Consistently, knocking out either Ehmt1 or Wiz by CRISPR/Cas resulted in the loss of H3K9me2 and enhanced Foxp3 expression during iTreg differentiation. Moreover, the essential role of the Wiz-Ehmt1 interaction as observed at several TSDRs indicates a global function of Ehmt1 in the Treg differentiation program.},
}

@article {pmid31358538,
year = {2019},
author = {Zhang, Y and Zhou, L and Bandyopadhyay, D and Sharma, K and Allen, AJ and Kmieciak, M and Grant, S},
title = {The Covalent CDK7 Inhibitor THZ1 Potently Induces Apoptosis in Multiple Myeloma Cells In Vitro and In Vivo.},
journal = {Clinical cancer research : an official journal of the American Association for Cancer Research},
volume = {},
number = {},
pages = {},
doi = {10.1158/1078-0432.CCR-18-3788},
pmid = {31358538},
issn = {1078-0432},
abstract = {Purpose: The goal of this study was to characterize the activity of the covalent CDK7 inhibitor THZ1 in multiple myeloma models.Experimental Design: Multiple myeloma lines were exposed to varying THZ1 concentrations alone or with carfilzomib or ABT-199, after which apoptosis was monitored by flow cytometry, protein expression by Western blot analysis, mRNA by RT-PCR. Analogous studies were performed in cells ectopically expressing c-MYC, MCL-1, or BCL-XL, or CRISPER-Cas CDK7 sgRNA knockout. Primary multiple myeloma cells were exposed to THZ1 ± carfilzomib or ABT-199. In vivo effects of THZ1 were examined in a systemic U266 xenograft model.Results: THZ1 markedly diminished multiple myeloma cell proliferation and survival despite bortezomib or stromal cell resistance in association with G2-M arrest, inactivation of CTD RNA Pol II, dephosphorylation of CDKs 7 as well as 1, 2, and 9, and MCL-1, BCL-xL, and c-MYC mRNA or protein downregulation. Ectopic MCL-1, c-MYC, or BCL-XL expression significantly protected cells from THZ1 lethality. Both THZ1 and CRISPR-Cas CDK7 knockout sharply diminished multiple myeloma cell proliferation and significantly increased carfilzomib and ABT-199 lethality. Parallel effects and interactions were observed in primary CD138+ (N = 22) or primitive multiple myeloma cells (CD138-/CD19+/CD20+/CD27+; N = 16). THZ1 administration [10 mg/kg i.p. twice daily (BID), 5 days/week] significantly improved survival in a systemic multiple myeloma xenograft model with minimal toxicity and induced similar events observed in vitro, for example, MCL-1 and c-MYC downregulation.Conclusions: THZ1 potently reduces multiple myeloma cell proliferation through transcriptional downregulation of MCL-1, BCL-XL, and c-MYC in vitro and in vivo It warrants further attention as a therapeutic agent in multiple myeloma.},
}

@article {pmid31358055,
year = {2019},
author = {Caron, J and Pène, V and Tolosa, L and Villaret, M and Luce, E and Fourrier, A and Heslan, JM and Saheb, S and Bruckert, E and Gómez-Lechón, MJ and Nguyen, TH and Rosenberg, AR and Weber, A and Dubart-Kupperschmitt, A},
title = {Low-density lipoprotein receptor-deficient hepatocytes differentiated from induced pluripotent stem cells allow familial hypercholesterolemia modeling, CRISPR/Cas-mediated genetic correction, and productive hepatitis C virus infection.},
journal = {Stem cell research & therapy},
volume = {10},
number = {1},
pages = {221},
doi = {10.1186/s13287-019-1342-6},
pmid = {31358055},
issn = {1757-6512},
support = {FP7-HEALTH.2011.1.4-2-278152 "InnovaLiv"//FP7 Health/ ; FP7-HEALTH.2011.1.4-2-278152 "InnovaLiv"//FP7 Health/ ; FP7-HEALTH.2011.1.4-2-278152 "InnovaLiv"//FP7 Health/ ; FP7-HEALTH.2011.1.4-2-278152 "InnovaLiv"//FP7 Health/ ; ANR-2010-RFCS-004 "Liv-iPS"//Agence Nationale de la Recherche/ ; ANR-2010-RFCS-004 "Liv-iPS".//Agence Nationale de la Recherche/ ; ANR-14-CE16-0026//Agence Nationale de la Recherche/ ; ANR-10-IBHU-005//Agence Nationale de la Recherche/ ; N/A//Ministère de l'Enseignement Supérieur et de la Recherche/ ; FDT20160435349//Fondation pour la Recherche Médicale (FR)/ ; N/A//DHU Hepatinov (FR)/ ; N/A//DHU Hepatinov (FR)/ ; Miguel Servet I Contract (CP16/00097)//Instituto de Salud Carlos III/ ; DIM Biothérapies//Conseil Régional, Île-de-France/ ; N/A//Société Francaise d'Hématologie (FR)/ ; N/A//Région Pays de La Loire - Nantes Métropole (FR)/ ; },
abstract = {BACKGROUND: Familial hypercholesterolemia type IIA (FH) is due to mutations in the low-density lipoprotein receptor (LDLR) resulting in elevated levels of low-density lipoprotein cholesterol (LDL-c) in plasma and in premature cardiovascular diseases. As hepatocytes are the only cells capable of metabolizing cholesterol, they are therefore the target cells for cell/gene therapy approaches in the treatment of lipid metabolism disorders. Furthermore, the LDLR has been reported to be involved in hepatitis C virus (HCV) entry into hepatocytes; however, its role in the virus infection cycle is still disputed.

METHODS: We generated induced pluripotent stem cells (iPSCs) from a homozygous LDLR-null FH-patient (FH-iPSCs). We constructed a correction cassette bearing LDLR cDNA under the control of human hepatic apolipoprotein A2 promoter that targets the adeno-associated virus integration site AAVS1. We differentiated both FH-iPSCs and corrected FH-iPSCs (corr-FH-iPSCs) into hepatocytes to study statin-mediated regulation of genes involved in cholesterol metabolism. Upon HCV particle inoculation, viral replication and production were quantified in these cells.

RESULTS: We showed that FH-iPSCs displayed the disease phenotype. Using homologous recombination mediated by the CRISPR/Cas9 system, FH-iPSCs were genetically corrected by the targeted integration of a correction cassette at the AAVS1 locus. Both FH-iPSCs and corr-FH-iPSCs were then differentiated into functional polarized hepatocytes using a stepwise differentiation approach (FH-iHeps and corr-FH-iHeps). The correct insertion and expression of the correction cassette resulted in restoration of LDLR expression and function (LDL-c uptake) in corr-FH-iHeps. We next demonstrated that pravastatin treatment increased the expression of genes involved in cholesterol metabolism in both cell models. Moreover, LDLR expression and function were also enhanced in corr-FH-iHeps after pravastatin treatment. Finally, we demonstrated that both FH-iHeps and corr-FH-iHeps were as permissive to viral infection as primary human hepatocytes but that virus production in FH-iHeps was significantly decreased compared to corr-FH-iHeps, suggesting a role of the LDLR in HCV morphogenesis.

CONCLUSIONS: Our work provides the first LDLR-null FH cell model and its corrected counterpart to study the regulation of cholesterol metabolism and host determinants of HCV life cycle, and a platform to screen drugs for treating dyslipidemia and HCV infection.},
}