@article {pmid36351608,
year = {2022},
author = {Kiga, K},
title = {[RNA functions in bacterial infections and its application to antimicrobial therapy].},
journal = {Nihon saikingaku zasshi. Japanese journal of bacteriology},
volume = {77},
number = {3},
pages = {139-144},
doi = {10.3412/jsb.77.139},
pmid = {36351608},
issn = {1882-4110},
abstract = {In the concept of central dogma (RNA is transcribed from DNA to produce proteins), RNA was thought to be merely an intermediary for genetic information to synthesize proteins from DNA. Since the discovery of RNA interference in 2000, research on RNA has progressed remarkably, especially in mammals. On the other hand, the role of RNA in bacterial infections was largely unknown. At that time, we started research on RNA and bacterial infection and revealed that miR-210, a small RNA in the gastric epithelial cells, is involved in gastric diseases caused by Helicobacter pylori in-fection. Furthermore, we have successfully developed sequence-specific antimicrobials by loading CRISPR-Cas13, an RNA-targeting CRISPR-Cas, on bacteriophage. The constructed antimicrobials were effective against at least Escherichia coli and Staphylococcus aureus. In this paper, we would like to introduce the importance of RNA in bacteriology.},
}

@article {pmid36350608,
year = {2022},
author = {Fan, J and Shi, L and Liu, Q and Zhu, Z and Wang, F and Song, R and Su, J and Zhou, D and Chen, X and Li, K and Xue, L and Sun, L and Mao, F},
title = {Annotation and evaluation of base editing outcomes in multiple cell types using CRISPRbase.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkac967},
pmid = {36350608},
issn = {1362-4962},
support = {32170656//National Natural Science Foundation of China/ ; Z211100002121039//Beijing Nova Program/ ; PKU2022LCXQ043//Clinical Medicine Plus X - Young Scholars Project, Peking University/ ; BYSYYZD2021001//Peking University Third Hospital/ ; XT202218//the Project of Collaborative Innovation Center of GDAAS/ ; },
abstract = {CRISPR-Cas base editing (BE) system is a powerful tool to expand the scope and efficiency of genome editing with single-nucleotide resolution. The editing efficiency, product purity, and off-target effect differ among various BE systems. Herein, we developed CRISPRbase (http://crisprbase.maolab.org), by integrating 1 252 935 records of base editing outcomes in more than 50 cell types from 17 species. CRISPRbase helps to evaluate the putative editing precision of different BE systems by integrating multiple annotations, functional predictions and a blasting system for single-guide RNA sequences. We systematically assessed the editing window, editing efficiency and product purity of various BE systems. Intensive efforts were focused on increasing the editing efficiency and product purity of base editors since the byproduct could be detrimental in certain applications. Remarkably, more than half of cancer-related off-target mutations were non-synonymous and extremely damaging to protein functions in most common tumor types. Luckily, most of these cancer-related mutations were passenger mutations (4840/5703, 84.87%) rather than cancer driver mutations (863/5703, 15.13%), indicating a weak effect of off-target mutations on carcinogenesis. In summary, CRISPRbase is a powerful and convenient tool to study the outcomes of different base editors and help researchers choose appropriate BE designs for functional studies.},
}

@article {pmid36347819,
year = {2022},
author = {Dai, P and Hu, D},
title = {The making of hypervirulent Klebsiella pneumoniae.},
journal = {Journal of clinical laboratory analysis},
volume = {},
number = {},
pages = {e24743},
doi = {10.1002/jcla.24743},
pmid = {36347819},
issn = {1098-2825},
support = {2023KY1326//Zhejiang Medical and Health Research Program/ ; },
abstract = {Klebsiella pneumoniae is a notorious bacterium in clinical practice. Virulence, carbapenem-resistance and their convergence among K. pneumoniae are extensively discussed in this article. Hypervirulent K. pneumoniae (HvKP) has spread from the Asian Pacific Rim to the world, inducing various invasive infections, such as pyogenic liver abscess, endophthalmitis, and meningitis. Furthermore, HvKP has acquired more and more drug resistance. Among multidrug-resistant HvKP, hypervirulent carbapenem-resistant K. pneumoniae (Hv-CRKP), and carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) are both devastating for their extreme drug resistance and virulence. The hypervirulence of HvKP is primarily attributed to hypercapsule, macromolecular exopolysaccharides, or excessive siderophores, although it has many other factors, for example, lipopolysaccharides, fimbriae, and porins. In contrast with classical determination of HvKP, that is, animal lethality test, molecular determination could be an optional and practical method after improvement. HvKP, including Hv-CRKP and CR-HvKP, has been progressing. R-M and CRISPR-Cas systems may play pivotal roles in such evolutions. Hv-CRKP and CR-HvKP, in particular the former, should be of severe concern due to their being more and more prevalent.},
}

@article {pmid36344749,
year = {2022},
author = {Chen, PJ and Liu, DR},
title = {Prime editing for precise and highly versatile genome manipulation.},
journal = {Nature reviews. Genetics},
volume = {},
number = {},
pages = {},
pmid = {36344749},
issn = {1471-0064},
abstract = {Programmable gene-editing tools have transformed the life sciences and have shown potential for the treatment of genetic disease. Among the CRISPR-Cas technologies that can currently make targeted DNA changes in mammalian cells, prime editors offer an unusual combination of versatility, specificity and precision. Prime editors do not require double-strand DNA breaks and can make virtually any substitution, small insertion and small deletion within the DNA of living cells. Prime editing minimally requires a programmable nickase fused to a polymerase enzyme, and an extended guide RNA that both specifies the target site and templates the desired genome edit. In this Review, we summarize prime editing strategies to generate programmed genomic changes, highlight their limitations and recent developments that circumvent some of these bottlenecks, and discuss applications and future directions.},
}

@article {pmid36344504,
year = {2022},
author = {Kato, K and Okazaki, S and Kannan, S and Altae-Tran, H and Esra Demircioglu, F and Isayama, Y and Ishikawa, J and Fukuda, M and Macrae, RK and Nishizawa, T and Makarova, KS and Koonin, EV and Zhang, F and Nishimasu, H},
title = {Structure of the IscB-ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6719},
pmid = {36344504},
issn = {2041-1723},
support = {21H05281//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
abstract = {Transposon-encoded IscB family proteins are RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely ancestors of the RNA-guided nuclease Cas9 in the type II CRISPR-Cas adaptive immune system. IscB associates with its cognate ωRNA to form a ribonucleoprotein complex that cleaves double-stranded DNA targets complementary to an ωRNA guide segment. Although IscB shares the RuvC and HNH endonuclease domains with Cas9, it is much smaller than Cas9, mainly due to the lack of the α-helical nucleic-acid recognition lobe. Here, we report the cryo-electron microscopy structure of an IscB protein from the human gut metagenome (OgeuIscB) in complex with its cognate ωRNA and a target DNA, at 2.6-Å resolution. This high-resolution structure reveals the detailed architecture of the IscB-ωRNA ribonucleoprotein complex, and shows how the small IscB protein assembles with the ωRNA and mediates RNA-guided DNA cleavage. The large ωRNA scaffold structurally and functionally compensates for the recognition lobe of Cas9, and participates in the recognition of the guide RNA-target DNA heteroduplex. These findings provide insights into the mechanism of the programmable DNA cleavage by the IscB-ωRNA complex and the evolution of the type II CRISPR-Cas9 effector complexes.},
}

@article {pmid36333335,
year = {2022},
author = {Schmiderer, L and Yudovich, D and Oburoglu, L and Hjort, M and Larsson, J},
title = {Site-specific CRISPR-based mitochondrial DNA manipulation is limited by gRNA import.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {18687},
pmid = {36333335},
issn = {2045-2322},
mesh = {Humans ; *RNA, Guide/genetics/metabolism ; *DNA, Mitochondrial/genetics ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Mitochondria/genetics/metabolism ; },
abstract = {Achieving CRISPR Cas9-based manipulation of mitochondrial DNA (mtDNA) has been a long-standing goal and would be of great relevance for disease modeling and for clinical applications. In this project, we aimed to deliver Cas9 into the mitochondria of human cells and analyzed Cas9-induced mtDNA cleavage and measured the resulting mtDNA depletion with multiplexed qPCR. In initial experiments, we found that measuring subtle effects on mtDNA copy numbers is challenging because of high biological variability, and detected no significant Cas9-caused mtDNA degradation. To overcome the challenge of being able to detect Cas9 activity on mtDNA, we delivered cytosine base editor Cas9-BE3 to mitochondria and measured its effect (C → T mutations) on mtDNA. Unlike regular Cas9-cutting, this leaves a permanent mark on mtDNA that can be detected with amplicon sequencing, even if the efficiency is low. We detected low levels of C → T mutations in cells that were exposed to mitochondrially targeted Cas9-BE3, but, surprisingly, these occurred regardless of whether a guide RNA (gRNA) specific to the targeted site, or non-targeting gRNA was used. This unspecific off-target activity shows that Cas9-BE3 can technically edit mtDNA, but also strongly indicates that gRNA import to mitochondria was not successful. Going forward mitochondria-targeted Cas9 base editors will be a useful tool for validating successful gRNA delivery to mitochondria without the ambiguity of approaches that rely on quantifying mtDNA copy numbers.},
}

Humans
*RNA, Guide/genetics/metabolism
*DNA, Mitochondrial/genetics
Gene Editing/methods
CRISPR-Cas Systems/genetics
Mitochondria/genetics/metabolism

@article {pmid36305833,
year = {2022},
author = {Shangguan, Q and Graham, S and Sundaramoorthy, R and White, MF},
title = {Structure and mechanism of the type I-G CRISPR effector.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11214-11228},
pmid = {36305833},
issn = {1362-4962},
support = {202008060345//China Scholarship Council/ ; //University of St Andrews/ ; BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/S021647/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *CRISPR-Associated Proteins/metabolism ; Cryoelectron Microscopy ; CRISPR-Cas Systems/genetics ; DNA/genetics/metabolism ; },
abstract = {Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3' end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.},
}

Humans
*CRISPR-Associated Proteins/metabolism
Cryoelectron Microscopy
CRISPR-Cas Systems/genetics
DNA/genetics/metabolism

@article {pmid36272409,
year = {2022},
author = {Shi, YJ and Duan, M and Ding, JM and Wang, FQ and Bi, LL and Zhang, CX and Zhang, YZ and Duan, JY and Huang, AH and Lei, XL and Yin, H and Zhang, Y},
title = {DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9.},
journal = {Molecular cell},
volume = {82},
number = {21},
pages = {4160-4175.e6},
doi = {10.1016/j.molcel.2022.09.032},
pmid = {36272409},
issn = {1097-4164},
mesh = {Animals ; *CRISPR-Cas Systems ; *Escherichia coli/genetics/metabolism ; Gene Editing ; DNA/genetics ; Plasmids ; Mammals/metabolism ; },
abstract = {CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology. AtCas9 has a relaxed PAM of N4CNNN and N4RNNA (R = A/G) and is able to bind but not cleave targets with mutated PAMs. When PAM-mutated DNA was in underwound topology, AtCas9 exhibited enhanced binding affinity and high cleavage activity. Mechanistically, AtCas9 has a unique loop motif, which docked into the DNA major groove, and this interaction can be regulated by DNA topology. More importantly, AtCas9 showed near-PAMless editing of supercoiled plasmid in E. coli. In mammalian cells, AtCas9 exhibited broad PAM preference to edit plasmid with up to 72% efficiency and effective base editing at four endogenous loci, representing a potentially powerful tool for near-PAMless editing.},
}

Animals
*CRISPR-Cas Systems
*Escherichia coli/genetics/metabolism
Gene Editing
DNA/genetics
Plasmids
Mammals/metabolism

@article {pmid36263801,
year = {2022},
author = {Liu, X and Cui, S and Qi, Q and Lei, H and Zhang, Y and Shen, W and Fu, F and Tian, T and Zhou, X},
title = {G-quadruplex-guided RNA engineering to modulate CRISPR-based genomic regulation.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11387-11400},
pmid = {36263801},
issn = {1362-4962},
support = {22177089//National Natural Science Foundation of China/ ; 2019CFA064//Hubei Natural Science Foundation for Distinguished Young Scholars/ ; 2042019kf0189//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Humans ; *RNA, Guide/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genomics ; RNA/genetics ; },
abstract = {It is important to develop small moelcule-based methods to modulate gene editing and expression in human cells. The roles of the G-quadruplex (G4) in biological systems have been widely studied. Here, G4-guided RNA engineering is performed to generate guide RNA with G4-forming units (G4-gRNA). We further demonstrate that chemical targeting of G4-gRNAs holds promise as a general approach for modulating gene editing and expression in human cells. The rich structural diversity of RNAs offers a reservoir of targets for small molecules to bind, thus creating the potential to modulate RNA biology.},
}

Humans
*RNA, Guide/genetics/metabolism
*CRISPR-Cas Systems/genetics
Gene Editing/methods
Genomics
RNA/genetics

@article {pmid36252775,
year = {2022},
author = {Radtke, L and Majchrzak-Celińska, A and Awortwe, C and Vater, I and Nagel, I and Sebens, S and Cascorbi, I and Kaehler, M},
title = {CRISPR/Cas9-induced knockout reveals the role of ABCB1 in the response to temozolomide, carmustine and lomustine in glioblastoma multiforme.},
journal = {Pharmacological research},
volume = {185},
number = {},
pages = {106510},
doi = {10.1016/j.phrs.2022.106510},
pmid = {36252775},
issn = {1096-1186},
mesh = {Humans ; Temozolomide/pharmacology/therapeutic use ; *Glioblastoma/drug therapy/genetics/pathology ; Carmustine/pharmacology/therapeutic use ; ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism ; Lomustine/therapeutic use/pharmacology ; CRISPR-Cas Systems ; ATP-Binding Cassette Transporters/metabolism ; Neoplasm Proteins/metabolism ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism ; },
abstract = {Glioblastoma multiforme (GBM) is the most common malignant brain tumor with limited therapeutic options. Besides surgery, chemotherapy using temozolomide, carmustine or lomustine is the main pillar of therapy. However, therapy success is limited and prognosis still is very poor. One restraining factor is drug resistance caused by drug transporters of the ATP-binding cassette family, e.g. ABCB1 and ABCG2, located at the blood-brain barrier and on tumor cells. The active efflux of xenobiotics including drugs, e.g. temozolomide, leads to low intracellular drug concentrations and subsequently insufficient anti-tumor effects. Nevertheless, the role of efflux transporters in GBM is controversially discussed. In the present study, we analyzed the role of ABCB1 and ABCG2 in GBM cells showing that ABCB1, but marginally ABCG2, is relevant. Applying a CRISPR/Cas9-derived ABCB1 knockout, the response to temozolomide was significantly augmented demonstrated by decreased cell number (p < 0.001) and proliferation rate (p = 0.04), while apoptosis was increased (p = 0.04). For carmustine, a decrease of cells in G1-phase was detected pointing to cell cycle arrest in the ABCB1 knockout (p = 0.006). For lomustine, however, loss of ABCB1 did not alter the response to the treatment. Overall, this study shows that ABCB1 is involved in the active transport of temozolomide out of the tumor cells diminishing the response to temozolomide. Interestingly, loss of ABCB1 also affected the response to the lipophilic drug carmustine. These findings show that ABCB1 is not only relevant at the blood-brain barrier, but also in the tumor cells diminishing success of chemotherapy.},
}

Humans
Temozolomide/pharmacology/therapeutic use
*Glioblastoma/drug therapy/genetics/pathology
Carmustine/pharmacology/therapeutic use
ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism
Lomustine/therapeutic use/pharmacology
CRISPR-Cas Systems
ATP-Binding Cassette Transporters/metabolism
Neoplasm Proteins/metabolism
Cell Line, Tumor
Drug Resistance, Neoplasm
ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism

@article {pmid36243978,
year = {2022},
author = {Wu, J and Zou, Z and Liu, Y and Liu, X and Zhangding, Z and Xu, M and Hu, J},
title = {CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11128-11137},
pmid = {36243978},
issn = {1362-4962},
support = {NK2022010101//Ministry of Agriculture and Rural Affairs of China/ ; 32122018//NSFC/ ; Z201100005320011//Beijing Municipal Science and Technology Commission/ ; //Center for Life Sciences/ ; //Boehringer Ingelheim-Peking University Postdoctoral Program/ ; },
mesh = {Animals ; Mice ; *T-Lymphocytes ; *Gene Editing ; CRISPR-Cas Systems/genetics ; DNA, Viral ; Receptors, Antigen, T-Cell/genetics ; },
abstract = {CRISPR/Cas9 has been adapted to disrupt endogenous genes in adoptive T-lymphocyte therapy to prevent graft-versus-host disease. However, genome editing also generates prevalent deleterious structural variations (SVs), including chromosomal translocations and large deletions, raising safety concerns about reinfused T cells. Here, we dynamically monitored the progression of SVs in a mouse model of T-cell receptor (TCR)-transgenic T-cell adoptive transfer, mimicking TCR T therapeutics. Remarkably, CRISPR/Cas9-induced SVs persist and undergo clonal expansion in vivo after three weeks or even two months, evidenced by high enrichment and low junctional diversity of identified SVs post infusion. Specifically, we detected 128 expanded translocations, with 20 615 as the highest number of amplicons. The identified SVs are stochastically selected among different individuals and show an inconspicuous locus preference. Similar to SVs, viral DNA integrations are routinely detected in edited T cells and also undergo clonal expansion. The persistent SVs and viral DNA integrations in the infused T cells may constantly threaten genome integrity, drawing immediate attention to the safety of CRISPR/Cas9-engineered T cells mediated immunotherapy.},
}

Animals
Mice
*T-Lymphocytes
*Gene Editing
CRISPR-Cas Systems/genetics
DNA, Viral
Receptors, Antigen, T-Cell/genetics

@article {pmid36228744,
year = {2022},
author = {Abe, T and Horisawa, Y and Kikuchi, O and Ozawa-Umeta, H and Kishimoto, A and Katsuura, Y and Imaizumi, A and Hashimoto, T and Shirakawa, K and Takaori-Kondo, A and Yusa, K and Asakura, T and Kakeya, H and Kanai, M},
title = {Pharmacologic characterization of TBP1901, a prodrug form of aglycone curcumin, and CRISPR-Cas9 screen for therapeutic targets of aglycone curcumin.},
journal = {European journal of pharmacology},
volume = {935},
number = {},
pages = {175321},
doi = {10.1016/j.ejphar.2022.175321},
pmid = {36228744},
issn = {1879-0712},
mesh = {Mice ; Animals ; *Curcumin/pharmacology ; *Prodrugs/pharmacology/therapeutic use ; Glucuronides/metabolism/pharmacology/therapeutic use ; Reactive Oxygen Species/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; NF-kappa B/metabolism ; Glucuronidase/metabolism ; },
abstract = {Curcumin (aglycone curcumin) has antitumor properties in a variety of malignancies via the alteration of multiple cancer-related biological pathways; however, its clinical application has been hampered due to its poor bioavailability. To overcome this limitation, we have developed a synthesized curcumin β-D-glucuronide sodium salt (TBP1901), a prodrug form of aglycone curcumin. In this study, we aimed to clarify the pharmacologic characteristics of TBP1901. In β-glucuronidase (GUSB)-proficient mice, both curcumin β-D-glucuronide and its active metabolite, aglycone curcumin, were detected in the blood after TBP1901 injection, whereas only curcumin β-D-glucuronide was detected in GUSB-impaired mice, suggesting that GUSB plays a pivotal role in the conversion of TBP1901 into aglycone curcumin in vivo. TBP1901 itself had minimal antitumor effects in vitro, whereas it demonstrated significant antitumor effects in vivo. Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screen disclosed the genes associated with NF-κB signaling pathway and mitochondria were among the highest hit. In vitro, aglycone curcumin inhibited NF-kappa B signaling pathways whereas it caused production of reactive oxygen species (ROS). ROS scavenger, N-acetyl-L-cysteine, partially reversed antitumor effects of aglycone curcumin. In summary, TBP1901 can exert antitumor effects as a prodrug of aglycone curcumin through GUSB-dependent activation.},
}

Mice
Animals
*Curcumin/pharmacology
*Prodrugs/pharmacology/therapeutic use
Glucuronides/metabolism/pharmacology/therapeutic use
Reactive Oxygen Species/metabolism
CRISPR-Cas Systems/genetics
Cell Line, Tumor
NF-kappa B/metabolism
Glucuronidase/metabolism

@article {pmid36215034,
year = {2022},
author = {Patterson, A and White, A and Waymire, E and Fleck, S and Golden, S and Wilkinson, RA and Wiedenheft, B and Bothner, B},
title = {Anti-CRISPR proteins function through thermodynamic tuning and allosteric regulation of CRISPR RNA-guided surveillance complex.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11243-11254},
pmid = {36215034},
issn = {1362-4962},
support = {//M.J. Murdock Charitable Trust/ ; //Office of the Vice President for Research, Economic Development and Graduate Education/ ; R35GM134867/GF/NIH HHS/United States ; //Amgen/ ; //Montana State University Agricultural Experimental Station/ ; P20GM103474/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/metabolism ; Allosteric Regulation ; Models, Molecular ; Proteins/genetics ; Thermodynamics ; RNA ; CRISPR-Cas Systems ; },
abstract = {CRISPR RNA-guided detection and degradation of foreign DNA is a dynamic process. Viruses can interfere with this cellular defense by expressing small proteins called anti-CRISPRs. While structural models of anti-CRISPRs bound to their target complex provide static snapshots that inform mechanism, the dynamics and thermodynamics of these interactions are often overlooked. Here, we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) and differential scanning fluorimetry (DSF) experiments to determine how anti-CRISPR binding impacts the conformational landscape of the type IF CRISPR RNA guided surveillance complex (Csy) upon binding of two different anti-CRISPR proteins (AcrIF9 and AcrIF2). The results demonstrate that AcrIF2 binding relies on enthalpic stabilization, whereas AcrIF9 uses an entropy driven reaction to bind the CRISPR RNA-guided surveillance complex. Collectively, this work reveals the thermodynamic basis and mechanistic versatility of anti-CRISPR-mediated immune suppression. More broadly, this work presents a striking example of how allosteric effectors are employed to regulate nucleoprotein complexes.},
}

*CRISPR-Associated Proteins/metabolism
Allosteric Regulation
Models, Molecular
Proteins/genetics
Thermodynamics
RNA
CRISPR-Cas Systems

@article {pmid36191879,
year = {2022},
author = {Dong, M and Liu, J and Liu, C and Wang, H and Sun, W and Liu, B},
title = {CRISPR/CAS9: A promising approach for the research and treatment of cardiovascular diseases.},
journal = {Pharmacological research},
volume = {185},
number = {},
pages = {106480},
doi = {10.1016/j.phrs.2022.106480},
pmid = {36191879},
issn = {1096-1186},
mesh = {Humans ; *CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; Gene Editing/methods ; Genetic Engineering/methods ; },
abstract = {The development of gene-editing technology has been one of the biggest advances in biomedicine over the past two decades. Not only can it be used as a research tool to build a variety of disease models for the exploration of disease pathogenesis at the genetic level, it can also be used for prevention and treatment. This is done by intervening with the expression of target genes and carrying out precise molecular targeted therapy for diseases. The simple and flexible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing technology overcomes the limitations of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). For this reason, it has rapidly become a preferred method for gene editing. As a new gene intervention method, CRISPR/Cas9 has been widely used in the clinical treatment of tumours and rare diseases; however, its application in the field of cardiovascular diseases is currently limited. This article reviews the application of the CRISPR/Cas9 editing technology in cardiovascular disease research and treatment, and discusses the limitations and prospects of this technology.},
}

Humans
*CRISPR-Cas Systems
*Cardiovascular Diseases/genetics/therapy
Gene Editing/methods
Genetic Engineering/methods

@article {pmid36127475,
year = {2022},
author = {Armat, M and Vaz, PK and Browning, GF and Noormohammadi, AH and Hartley, CA and Devlin, JM},
title = {Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques.},
journal = {Virus genes},
volume = {58},
number = {6},
pages = {540-549},
pmid = {36127475},
issn = {1572-994X},
support = {DP180101417//Australian Resuscitation Council/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; Australia ; *Herpesvirus 1, Gallid/genetics ; Chickens ; Glycoproteins/genetics ; Green Fluorescent Proteins/genetics ; Homologous Recombination ; *Herpesviridae Infections ; *Poultry Diseases ; },
abstract = {In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.},
}

Animals
CRISPR-Cas Systems
Australia
*Herpesvirus 1, Gallid/genetics
Chickens
Glycoproteins/genetics
Green Fluorescent Proteins/genetics
Homologous Recombination
*Herpesviridae Infections
*Poultry Diseases

@article {pmid36063049,
year = {2022},
author = {Sottolano, CJ and Revaitis, NT and Geneva, AJ and Yakoby, N},
title = {Nebulous without white: annotated long-read genome assembly and CRISPR/Cas9 genome engineering in Drosophila nebulosa.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {11},
pages = {},
pmid = {36063049},
issn = {2160-1836},
support = {2R15GM101597-02//National Institute of General Medical Sciences of the National Institutes of Health/ ; IOS-1926802//National Science Foundation/ ; },
mesh = {Animals ; Male ; *CRISPR-Cas Systems ; *Gene Editing ; Drosophila/genetics ; Drosophila melanogaster/genetics ; Animals, Genetically Modified ; },
abstract = {The diversity among Drosophila species presents an opportunity to study the molecular mechanisms underlying the evolution of biological phenomena. A challenge to investigating these species is that, unlike the plethora of molecular and genetics tools available for D. melanogaster research, many other species do not have sequenced genomes; a requirement for employing these tools. Selecting transgenic flies through white (w) complementation has been commonly practiced in numerous Drosophila species. While tolerated, the disruption of w is associated with impaired vision, among other effects in D. melanogaster. The D. nebulosa fly has a unique mating behavior which requires vision, and is thus unable to successfully mate in dark conditions. Here, we hypothesized that the disruption of w will impede mating success. As a first step, using PacBio long-read sequencing, we assembled a high-quality annotated genome of D. nebulosa. Using these data, we employed CRISPR/Cas9 to successfully disrupt the w gene. As expected, D. nebulosa males null for w did not court females, unlike several other mutant strains of Drosophila species whose w gene has been disrupted. In the absence of mating, no females became homozygous null for w. We conclude that gene disruption via CRISPR/Cas9 genome engineering is a successful tool in D. nebulosa, and that the w gene is necessary for mating. Thus, an alternative selectable marker unrelated to vision is desirable.},
}

Animals
Male
*CRISPR-Cas Systems
*Gene Editing
Drosophila/genetics
Drosophila melanogaster/genetics
Animals, Genetically Modified

@article {pmid35929067,
year = {2022},
author = {Gao, K and Zhang, X and Zhang, Z and Wu, X and Guo, Y and Fu, P and Sun, A and Peng, J and Zheng, J and Yu, P and Wang, T and Ye, Q and Jiang, J and Wang, H and Lin, CP and Gao, G},
title = {Transcription-coupled donor DNA expression increases homologous recombination for efficient genome editing.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {e109},
pmid = {35929067},
issn = {1362-4962},
support = {32070846//National Natural Science Foundation of China/ ; 2019M661652//National Postdoctoral Fellowship/ ; 19JC1413600//Science, and Technology Commission of Shanghai Municipality/ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Homologous Recombination/genetics ; DNA Breaks, Double-Stranded ; DNA/genetics ; Mammals/genetics ; },
abstract = {Genomes can be edited by homologous recombination stimulated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated peptide 9]-induced DNA double-strand breaks. However, this approach is inefficient for inserting or deleting long fragments in mammalian cells. Here, we describe a simple genome-editing method, termed transcription-coupled Cas9-mediated editing (TEd), that can achieve higher efficiencies than canonical Cas9-mediated editing (CEd) in deleting genomic fragments, inserting/replacing large DNA fragments and introducing point mutations into mammalian cell lines. We also found that the transcription on DNA templates is crucial for the promotion of homology-directed repair, and that tethering transcripts from TEd donors to targeted sites further improves editing efficiency. The superior efficiency of TEd for the insertion and deletion of long DNA fragments expands the applications of CRISPR for editing mammalian genomes.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Homologous Recombination/genetics
DNA Breaks, Double-Stranded
DNA/genetics
Mammals/genetics

@article {pmid36343261,
year = {2022},
author = {Nethery, MA and Hidalgo-Cantabrana, C and Roberts, A and Barrangou, R},
title = {CRISPR-based engineering of phages for in situ bacterial base editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {46},
pages = {e2206744119},
doi = {10.1073/pnas.2206744119},
pmid = {36343261},
issn = {1091-6490},
support = {DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; },
abstract = {Investigation of microbial gene function is essential to the elucidation of ecological roles and complex genetic interactions that take place in microbial communities. While microbiome studies have increased in prevalence, the lack of viable in situ editing strategies impedes experimental progress, rendering genetic knowledge and manipulation of microbial communities largely inaccessible. Here, we demonstrate the utility of phage-delivered CRISPR-Cas payloads to perform targeted genetic manipulation within a community context, deploying a fabricated ecosystem (EcoFAB) as an analog for the soil microbiome. First, we detail the engineering of two classical phages for community editing using recombination to replace nonessential genes through Cas9-based selection. We show efficient engineering of T7, then demonstrate the expression of antibiotic resistance and fluorescent genes from an engineered λ prophage within an Escherichia coli host. Next, we modify λ to express an APOBEC-1-based cytosine base editor (CBE), which we leverage to perform C-to-T point mutations guided by a modified Cas9 containing only a single active nucleolytic domain (nCas9). We strategically introduce these base substitutions to create premature stop codons in-frame, inactivating both chromosomal (lacZ) and plasmid-encoded genes (mCherry and ampicillin resistance) without perturbation of the surrounding genomic regions. Furthermore, using a multigenera synthetic soil community, we employ phage-assisted base editing to induce host-specific phenotypic alterations in a community context both in vitro and within the EcoFAB, observing editing efficiencies from 10 to 28% across the bacterial population. The concurrent use of a synthetic microbial community, soil matrix, and EcoFAB device provides a controlled and reproducible model to more closely approximate in situ editing of the soil microbiome.},
}

@article {pmid36342558,
year = {2022},
author = {Yasmeen, A and Shakoor, S and Azam, S and Bakhsh, A and Shahid, N and Latif, A and Shahid, AA and Husnain, T and Rao, AQ},
title = {CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes.},
journal = {Planta},
volume = {256},
number = {6},
pages = {107},
pmid = {36342558},
issn = {1432-2048},
abstract = {MAIN CONCLUSION: VInv gene editing in potato using CRISPR/Cas9 resulted in knockdown of expression and a lower VInv enzymatic activity resulting in a decrease in post-harvest cold-storage sugars formation and sweetening in potatoes. CRISPR-Cas9-mediated knockdown of vacuolar invertase (VInv) gene was carried out using two sgRNAs in local cultivar of potato plants. The transformation efficiency of potatoes was found to be 11.7%. The primary transformants were screened through PCR, Sanger sequencing, digital PCR, and ELISA. The overall editing efficacy was determined to be 25.6% as per TIDE analysis. The amplicon sequencing data showed maximum indel frequency for potato plant T12 (14.3%) resulting in 6.2% gene knockout and 6% frame shift. While for plant B4, the maximum indel frequency of 2.0% was found which resulted in 4.4% knockout and 4% frameshift as analyzed by Geneious. The qRT-PCR data revealed that mRNA expression of VInv gene was reduced 90-99-fold in edited potato plants when compared to the non-edited control potato plant. Following cold storage, chips analysis of potatoes proved B4 and T12 as best lines. Reducing sugars' analysis by titration method determined fivefold reduction in percentage of reducing sugars in tubers of B4 transgenic lines as compared to the control. Physiologically genome-edited potatoes behaved like their conventional counterpart. This is first successful report of knockdown of potato VInv gene in Pakistan that addressed cold-induced sweetening resulting in minimum accumulation of reducing sugars in genome edited tubers.},
}

@article {pmid36342156,
year = {2022},
author = {Yu, L and Zhang, Y and Marchisio, MA},
title = {Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {188},
pages = {},
doi = {10.3791/64539},
pmid = {36342156},
issn = {1940-087X},
abstract = {Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to β-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.},
}

@article {pmid36341480,
year = {2022},
author = {Paßreiter, A and Naumann, N and Thomas, A and Grogna, N and Delahaut, P and Thevis, M},
title = {How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d2an01318e},
pmid = {36341480},
issn = {1364-5528},
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool kit constitutes one of today's most frequently used gene editing techniques. Editing of virtually any DNA sequence can be realised, due to the quickly progressing research into different Cas effectors and their ever-expanding range of targets. Moreover, the simplicity and cost-effectiveness of those CRISPR tools can, unfortunately, also facilitate the illicit utilisation of CRISPR/Cas in order to achieve performance enhancements amongst athletes. Consequently, there is an urgent need for the direct detection of illegally applied CRISPR/Cas methods in doping control samples, for which a promising strategy is presented herein employing Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) for targeted nucleic acid detection. An analytical method was developed that enables the detection of sgRNA associated with Cas9 from Streptococcus pyogenes (SpCas9) in serum samples by means of reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection via SHERLOCK in combination with a complementary gel-based screening procedure in order to uncover doping attempts with lipid mediated CRISPR ribonucleoprotein (RNP) complexes. Initial qualitative method characterisation confirmed the specificity of both procedures and established a detection sensitivity of 10 nM uncomplexed target sequence and 100 pM sgRNA in the form of RNP complexes. Furthermore, a proof-of-concept in vivo adimistration study simulating a hypothetical gene doping scenario employing a mouse model revealed a detection window of 8 h after intravenous injection, supporting the principal applicability of the test strategy to authentic doping control samples in the future.},
}

@article {pmid36340792,
year = {2022},
author = {Altay, HY and Ozdemir, F and Afghah, F and Kilinc, Z and Ahmadian, M and Tschopp, M and Agca, C},
title = {Gene regulatory and gene editing tools and their applications for retinal diseases and neuroprotection: From proof-of-concept to clinical trial.},
journal = {Frontiers in neuroscience},
volume = {16},
number = {},
pages = {924917},
doi = {10.3389/fnins.2022.924917},
pmid = {36340792},
issn = {1662-4548},
abstract = {Gene editing and gene regulatory fields are continuously developing new and safer tools that move beyond the initial CRISPR/Cas9 technology. As more advanced applications are emerging, it becomes crucial to understand and establish more complex gene regulatory and editing tools for efficient gene therapy applications. Ophthalmology is one of the leading fields in gene therapy applications with more than 90 clinical trials and numerous proof-of-concept studies. The majority of clinical trials are gene replacement therapies that are ideal for monogenic diseases. Despite Luxturna's clinical success, there are still several limitations to gene replacement therapies including the size of the target gene, the choice of the promoter as well as the pathogenic alleles. Therefore, further attempts to employ novel gene regulatory and gene editing applications are crucial to targeting retinal diseases that have not been possible with the existing approaches. CRISPR-Cas9 technology opened up the door for corrective gene therapies with its gene editing properties. Advancements in CRISPR-Cas9-associated tools including base modifiers and prime editing already improved the efficiency and safety profile of base editing approaches. While base editing is a highly promising effort, gene regulatory approaches that do not interfere with genomic changes are also becoming available as safer alternatives. Antisense oligonucleotides are one of the most commonly used approaches for correcting splicing defects or eliminating mutant mRNA. More complex gene regulatory methodologies like artificial transcription factors are also another developing field that allows targeting haploinsufficiency conditions, functionally equivalent genes, and multiplex gene regulation. In this review, we summarized the novel gene editing and gene regulatory technologies and highlighted recent translational progress, potential applications, and limitations with a focus on retinal diseases.},
}

@article {pmid36338968,
year = {2022},
author = {Garrood, WT and Cuber, P and Willis, K and Bernardini, F and Page, NM and Haghighat-Khah, RE},
title = {Driving down malaria transmission with engineered gene drives.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {891218},
doi = {10.3389/fgene.2022.891218},
pmid = {36338968},
issn = {1664-8021},
abstract = {The last century has witnessed the introduction, establishment and expansion of mosquito-borne diseases into diverse new geographic ranges. Malaria is transmitted by female Anopheles mosquitoes. Despite making great strides over the past few decades in reducing the burden of malaria, transmission is now on the rise again, in part owing to the emergence of mosquito resistance to insecticides, antimalarial drug resistance and, more recently, the challenges of the COVID-19 pandemic, which resulted in the reduced implementation efficiency of various control programs. The utility of genetically engineered gene drive mosquitoes as tools to decrease the burden of malaria by controlling the disease-transmitting mosquitoes is being evaluated. To date, there has been remarkable progress in the development of CRISPR/Cas9-based homing endonuclease designs in malaria mosquitoes due to successful proof-of-principle and multigenerational experiments. In this review, we examine the lessons learnt from the development of current CRISPR/Cas9-based homing endonuclease gene drives, providing a framework for the development of gene drive systems for the targeted control of wild malaria-transmitting mosquito populations that overcome challenges such as with evolving drive-resistance. We also discuss the additional substantial works required to progress the development of gene drive systems from scientific discovery to further study and subsequent field application in endemic settings.},
}

@article {pmid36338309,
year = {2022},
author = {Liu, L and Xu, Z and Awayda, K and Dollery, SJ and Bao, M and Fan, J and Cormier, D and O'Connell, M and Tobin, GJ and Du, K},
title = {Gold Nanoparticle-Labeled CRISPR-Cas13a Assay for the Sensitive Solid-State Nanopore Molecular Counting.},
journal = {Advanced materials technologies},
volume = {7},
number = {3},
pages = {},
doi = {10.1002/admt.202101550},
pmid = {36338309},
issn = {2365-709X},
abstract = {A gold nanoparticle (AuNP) labeled CRISPR-Cas13a nucleic acid assay has been developed for sensitive solid-state nanopore sensing. Instead of directly detecting the translocation of RNA through a nanopore, our system utilizes non-covalent conjugates of AuNPs and RNA targets. Upon CRISPR activation, the AuNPs are liberated from the RNA, isolated, and passed through a nanopore sensor. Detection of the AuNPs can be observed as increasing ionic current in the chip. Each AuNP that is detected is enumerated as an event, leading to quantitative of molecular targets. Leveraging the high signal-to-noise ratio enabled by the AuNPs, a detection limit of 50 fM before front-end target amplification is achieved using SARS-CoV-2 RNA segments as a Cas13 target. Furthermore, a dynamic range of six orders of magnitude is demonstrated for quantitative RNA sensing. This simplified AuNP-based CRISPR assay is performed at the physiological temperature without relying on thermal cyclers. In addition, the nanopore reader is similar in size to a smartphone, making the assay system suitable for rapid and portable nucleic acid biomarker detection in either low-resource settings or hospitals.},
}

@article {pmid36326816,
year = {2022},
author = {Schubert, OT and Bloom, JS and Sadhu, MJ and Kruglyak, L},
title = {Genome-wide base editor screen identifies regulators of protein abundance in yeast.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
doi = {10.7554/eLife.79525},
pmid = {36326816},
issn = {2050-084X},
support = {R01 GM102308/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *RNA, Guide/genetics ; Genetic Testing ; CRISPR-Cas Systems ; },
abstract = {Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network.},
}

*Saccharomyces cerevisiae/genetics/metabolism
*RNA, Guide/genetics
Genetic Testing
CRISPR-Cas Systems

@article {pmid36162731,
year = {2022},
author = {Ghosh, D and Nilavar, NM and Raghavan, SC},
title = {A novel KU70-mutant human leukemic cell line generated using CRISPR-Cas9 shows increased sensitivity to DSB inducing agents and reduced NHEJ activity.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1866},
number = {12},
pages = {130246},
doi = {10.1016/j.bbagen.2022.130246},
pmid = {36162731},
issn = {1872-8006},
mesh = {Humans ; Mice ; Animals ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; DNA-Binding Proteins/genetics/metabolism ; DNA/metabolism ; Cell Line ; },
abstract = {KU70 (XRCC6 gene in humans) is one of the proteins in the KU70-KU80 heterodimer which is the first component recruited to broken DNA ends during DNA double-strand break repair through nonhomologous end joining (NHEJ). Previous studies have shown that Ku70 deficient mouse cells are defective in NHEJ and V(D)J recombination. In contrast, heterozygous KU70 mutant human cell lines did not show any significant change in cell viability and sensitivity towards ionizing radiation. In this study, we used CRISPR-Cas9 technique to generate a KU70 mutant (heterozygous) human pre-B leukemic cell line (N6-KU70-2-DG). We observed that the N6-KU70-2-DG cells showed a prominent reduction in the expression of both KU70 mRNA and protein. The mutant cells showed reduced cell viability, increased sensitivity to DSB inducing agents such as ionizing radiation (IR) and etoposide, and increased number of unrepaired DSBs after exposure to IR. In addition, the mutant cells showed a reduction in the NHEJ activity and increased rate of microhomology mediated joining (MMEJ) activity. KU70 mutant cells also revealed enhanced level of senescence markers following irradiation. Thus, we report a novel KU70-mutant leukemic cell line (heterozygous) with reduced NHEJ, which is sensitive to DNA damaging agents, unlike the previously reported other KU heterozygous mutant cell lines.},
}

Humans
Mice
Animals
*DNA Breaks, Double-Stranded
*CRISPR-Cas Systems
DNA-Binding Proteins/genetics/metabolism
DNA/metabolism
Cell Line

@article {pmid36334915,
year = {2022},
author = {Subramaniam, S and Smith, GR},
title = {RecBCD enzyme and Chi recombination hotspots as determinants of self vs. non-self: Myths and mechanisms.},
journal = {Advances in genetics},
volume = {109},
number = {},
pages = {1-37},
doi = {10.1016/bs.adgen.2022.06.001},
pmid = {36334915},
issn = {0065-2660},
abstract = {Bacteria face a challenge when DNA enters their cells by transformation, mating, or phage infection. Should they treat this DNA as an invasive foreigner and destroy it, or consider it one of their own and potentially benefit from incorporating new genes or alleles to gain useful functions? It is frequently stated that the short nucleotide sequence Chi (5' GCTGGTGG 3'), a hotspot of homologous genetic recombination recognized by Escherichia coli's RecBCD helicase-nuclease, allows E. coli to distinguish its DNA (self) from any other DNA (non-self) and to destroy non-self DNA, and that Chi is "over-represented" in the E. coli genome. We show here that these latter statements (dogmas) are not supported by available evidence. We note Chi's wide-spread occurrence and activity in distantly related bacterial species and phages. We illustrate multiple, highly non-random features of the genomes of E. coli and coliphage P1 that account for Chi's high frequency and genomic position, leading us to propose that P1 selects for Chi's enhancement of recombination, whereas E. coli selects for the preferred codons in Chi. We discuss other, previously described mechanisms for self vs. non-self determination involving RecBCD and for RecBCD's destruction of DNA that cannot recombine, whether foreign or domestic, with or without Chi.},
}

@article {pmid36334630,
year = {2022},
author = {Beyers, WC and Detry, AM and Di Pietro, SM},
title = {OCA7 is a melanosome membrane protein that defines pigmentation by regulating early stages of melanosome biogenesis.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {102669},
doi = {10.1016/j.jbc.2022.102669},
pmid = {36334630},
issn = {1083-351X},
abstract = {Mutations in C10orf11 (OCA7) cause OculoCutaneous Albinism, a disorder that presents with hypopigmentation in skin, eyes and hair. The OCA7 pathophysiology is unknown and there is virtually no information on the OCA7 protein and its cellular function. Here, we discover that OCA7 localizes to the limiting membrane of melanosomes, the specialized pigment cell organelles where melanin is synthesized. We demonstrate that OCA7 is recruited through interaction with a canonical effector binding surface of melanosome proteins Rab32 and Rab38. Using newly generated OCA7-KO MNT1 cells, we show OCA7 regulates overall melanin levels in a melanocyte autonomous manner by controlling melanosome maturation. Importantly, we found OCA7 regulates premelanosome protein (PMEL) processing, impacting fibrillation and the striations that define transition from melanosome Stage I to Stage II. Furthermore, the melanosome lumen of OCA7-KO cells displays lower pH than control cells. Together, our results reveal OCA7 regulates pigmentation through two well established determinants of melanosome biogenesis and function, PMEL processing and organelle pH.},
}

@article {pmid36323848,
year = {2022},
author = {Chen, F and Lian, M and Ma, B and Gou, S and Luo, X and Yang, K and Shi, H and Xie, J and Ge, W and Ouyang, Z and Lai, C and Li, N and Zhang, Q and Jin, Q and Liang, Y and Chen, T and Wang, J and Zhao, X and Li, L and Yu, M and Ye, Y and Wang, K and Wu, H and Lai, L},
title = {Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {1163},
pmid = {36323848},
issn = {2399-3642},
support = {32100410//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Cytosine ; Adenine ; Point Mutation ; },
abstract = {Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.},
}

*Gene Editing
*CRISPR-Cas Systems
Cytosine
Adenine
Point Mutation

@article {pmid36323688,
year = {2022},
author = {Hoberecht, L and Perampalam, P and Lun, A and Fortin, JP},
title = {A comprehensive Bioconductor ecosystem for the design of CRISPR guide RNAs across nucleases and technologies.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6568},
pmid = {36323688},
issn = {2041-1723},
mesh = {*RNA, Guide/genetics ; *CRISPR-Cas Systems/genetics ; Ecosystem ; Endonucleases/genetics/metabolism ; Deoxyribonucleases/genetics ; },
abstract = {The success of CRISPR-mediated gene perturbation studies is highly dependent on the quality of gRNAs, and several tools have been developed to enable optimal gRNA design. However, these tools are not all adaptable to the latest CRISPR modalities or nucleases, nor do they offer comprehensive annotation methods for advanced CRISPR applications. Here, we present a new ecosystem of R packages, called crisprVerse, that enables efficient gRNA design and annotation for a multitude of CRISPR technologies. This includes CRISPR knockout (CRISPRko), CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), CRISPR base editing (CRISPRbe) and CRISPR knockdown (CRISPRkd). The core package, crisprDesign, offers a user-friendly and unified interface to add off-target annotations, rich gene and SNP annotations, and on- and off-target activity scores. These functionalities are enabled for any RNA- or DNA-targeting nucleases, including Cas9, Cas12, and Cas13. The crisprVerse ecosystem is open-source and deployed through the Bioconductor project (https://github.com/crisprVerse).},
}

*RNA, Guide/genetics
*CRISPR-Cas Systems/genetics
Ecosystem
Endonucleases/genetics/metabolism
Deoxyribonucleases/genetics

@article {pmid36322747,
year = {2022},
author = {Lee, J and Kim, DH and Karolak, MC and Shin, S and Lee, K},
title = {Generation of genome-edited chicken and duck lines by adenovirus-mediated in vivo genome editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {45},
pages = {e2214344119},
doi = {10.1073/pnas.2214344119},
pmid = {36322747},
issn = {1091-6490},
support = {2020-67015-31537//USDA | National Institute of Food and Agriculture (NIFA)/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Chickens/genetics ; Ducks/genetics ; CRISPR-Cas Systems ; Adenoviridae/genetics ; Germ Cells ; },
abstract = {Conventional avian genome editing is mediated by isolation, culture, and genome editing of primordial germ cells (PGCs); screening and propagating the genome-edited PGCs; and transplantation of the PGCs into recipient embryos. The PGC-mediated procedures, however, are technically difficult, and therefore, the conventional method has previously been utilized only in chickens. Here, we generated germline mosaic founder chicken and duck lines without the PGC-mediated procedures by injecting an adenovirus containing the CRISPR-Cas9 system into avian blastoderms. Genome-edited chicken and duck offspring produced from the founders carried different insertion or deletion mutations without mutations in the potential off-target sites. Our data demonstrate successful applications of the adenovirus-mediated method for production of genome-edited chicken and duck lines.},
}

Animals
*Gene Editing/methods
*Chickens/genetics
Ducks/genetics
CRISPR-Cas Systems
Adenoviridae/genetics
Germ Cells

@article {pmid36255392,
year = {2022},
author = {Xu, X and Liu, C and Wang, S and Mäkilä, E and Wang, J and Koivisto, O and Zhou, J and Rosenholm, JM and Shu, Y and Zhang, H},
title = {Microfluidic-assisted biomineralization of CRISPR/Cas9 in near-infrared responsive metal-organic frameworks for programmable gene-editing.},
journal = {Nanoscale},
volume = {14},
number = {42},
pages = {15832-15844},
doi = {10.1039/d2nr04095f},
pmid = {36255392},
issn = {2040-3372},
mesh = {*Gene Editing ; CRISPR-Cas Systems ; *Metal-Organic Frameworks/metabolism ; Microfluidics ; Biomineralization ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Ribonucleoprotein (RNP) based CRISPR/Cas9 gene-editing system shows great potential in biomedical applications. However, due to the large size, charged surface and high biological sensitivity of RNP, its efficient delivery with precise control remains highly challenging. Herein, a microfluidic-assisted metal-organic framework (MOF) based biomineralization strategy is designed and utilized for the efficient delivery and remote regulation of CRISPR/Cas9 RNP gene editing. The strategy is realized by biomimetic growing of thermo-responsive EuMOFs onto photothermal template Prussian blue (PB). The RNP is loaded during MOFs crystallization in microfluidic channels. By adjusting different microfluidic parameters, well-defined and comparable RNP encapsulated nanocarrier (PB@RNP-EuMOFs) are obtained with high loading efficiency (60%), remarkable RNP protection and NIR-stimulated release capacity. Upon laser exposure, the nanocarrier induces effective endosomal escape (4 h) and precise gene knockout of green fluorescent protein by 40% over 2 days. Moreover, the gene-editing activity can be programmed by tuning exposure times (42% for three times and 47% for four times), proving more controllable and inducible editing modality compared to control group without laser irradiation. This novel microfluidic-assisted MOFs biomineralization strategy thus offers an attractive route to optimize delivery systems and reduce off-target side effects by NIR-triggered remote control of CRISPR/Cas9 RNP, improving the potential for its highly efficient and precise therapeutic application.},
}

*Gene Editing
CRISPR-Cas Systems
*Metal-Organic Frameworks/metabolism
Microfluidics
Biomineralization
Ribonucleoproteins/genetics/metabolism

@article {pmid36180232,
year = {2022},
author = {Geng, K and Merino, LG and Wedemann, L and Martens, A and Sobota, M and Sanchez, YP and Søndergaard, JN and White, RJ and Kutter, C},
title = {Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells.},
journal = {Genome research},
volume = {32},
number = {10},
pages = {1876-1891},
doi = {10.1101/gr.276901.122},
pmid = {36180232},
issn = {1549-5469},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nanopore Sequencing ; Genomics ; RNA, Guide/genetics ; DNA/genetics ; Alleles ; },
abstract = {The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.},
}

Humans
*CRISPR-Cas Systems
*Nanopore Sequencing
Genomics
RNA, Guide/genetics
DNA/genetics
Alleles

@article {pmid36063051,
year = {2022},
author = {Dvir, E and Shohat, S and Flint, J and Shifman, S},
title = {Identification of genetic mechanisms for tissue-specific genetic effects based on CRISPR screens.},
journal = {Genetics},
volume = {222},
number = {3},
pages = {},
doi = {10.1093/genetics/iyac134},
pmid = {36063051},
issn = {1943-2631},
support = {466/21//Israel Science Foundation/ ; 2612/18//Israel Science Foundation Broad Institute Joint Program/ ; },
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Cell Line ; *Neoplasms/genetics ; Gene Amplification ; },
abstract = {A major challenge in genetic studies of complex diseases is to determine how the action of risk genes is restricted to a tissue or cell type. Here, we investigate tissue specificity of gene action using CRISPR screens from 786 cancer cell lines originating from 24 tissues. We find that the expression pattern of the gene across tissues explains only a minority of cases of tissue-specificity (9%), while gene amplification and the expression levels of paralogs account for 39.5% and 15.5%, respectively. In addition, the transfer of small molecules to mutant cells explains tissue-specific gene action in blood. The tissue-specific genes we found are not specific just for human cancer cell lines: we found that the tissue-specific genes are intolerant to functional mutations in the human population and are associated with human diseases more than genes that are essential across all cell types. Our findings offer important insights into genetic mechanisms for tissue specificity of human diseases.},
}

Humans
*Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Cell Line
*Neoplasms/genetics
Gene Amplification

@article {pmid35941224,
year = {2022},
author = {Doman, JL and Sousa, AA and Randolph, PB and Chen, PJ and Liu, DR},
title = {Designing and executing prime editing experiments in mammalian cells.},
journal = {Nature protocols},
volume = {17},
number = {11},
pages = {2431-2468},
pmid = {35941224},
issn = {1750-2799},
support = {R01EB031172//U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; R35GM118062//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; Liu investigatorship//Howard Hughes Medical Institute (HHMI)/ ; Liu grant//Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation)/ ; U01AI142756//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; R01 EB031172/EB/NIBIB NIH HHS/United States ; RM1HG009490//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; DNA Breaks, Double-Stranded ; DNA Repair ; DNA/genetics ; RNA, Guide/genetics ; Mammals/genetics ; },
abstract = {Prime editing (PE) is a precision gene editing technology that enables the programmable installation of substitutions, insertions and deletions in cells and animals without requiring double-strand DNA breaks (DSBs). The mechanism of PE makes it less dependent on cellular replication and endogenous DNA repair than homology-directed repair-based approaches, and its ability to precisely install edits without creating DSBs minimizes indels and other undesired outcomes. The capabilities of PE have also expanded since its original publication. Enhanced PE systems, PE4 and PE5, manipulate DNA repair pathways to increase PE efficiency and reduce indels. Other advances that improve PE efficiency include engineered pegRNAs (epegRNAs), which include a structured RNA motif to stabilize and protect pegRNA 3' ends, and the PEmax architecture, which improves editor expression and nuclear localization. New applications such as twin PE (twinPE) can precisely insert or delete hundreds of base pairs of DNA and can be used in tandem with recombinases to achieve gene-sized (>5 kb) insertions and inversions. Achieving optimal PE requires careful experimental design, and the large number of parameters that influence PE outcomes can be daunting. This protocol describes current best practices for conducting PE and twinPE experiments and describes the design and optimization of pegRNAs. We also offer guidelines for how to select the proper PE system (PE1 to PE5 and twinPE) for a given application. Finally, we provide detailed instructions on how to perform PE in mammalian cells. Compared with other procedures for editing human cells, PE offers greater precision and versatility, and can be completed within 2-4 weeks.},
}

Animals
Humans
*CRISPR-Cas Systems
*Gene Editing/methods
DNA Breaks, Double-Stranded
DNA Repair
DNA/genetics
RNA, Guide/genetics
Mammals/genetics

@article {pmid36327561,
year = {2022},
author = {Guk, K and Yi, S and Kim, H and Bae, Y and Yong, D and Kim, S and Lee, KS and Lim, EK and Kang, T and Jung, J},
title = {Hybrid CRISPR/Cas protein for one-pot detection of DNA and RNA.},
journal = {Biosensors & bioelectronics},
volume = {219},
number = {},
pages = {114819},
doi = {10.1016/j.bios.2022.114819},
pmid = {36327561},
issn = {1873-4235},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as next-generation molecular diagnostics. In CRISPR-based diagnostics, Cas12 and Cas13 proteins have been widely employed to detect DNA and RNA, respectively. Herein, we developed a novel hybrid Cas protein capable of detecting universal nucleic acids (DNA and RNA). The CRISPR/hybrid Cas system simultaneously recognizes both DNA and RNA, enabling the dual detection of pathogenic viruses in a single tube. Using wild-type (WT) and N501Y mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as detection models, we successfully detected both virus strains with a detection limit of 10 viral copies per reaction without cross-reactivity. Furthermore, it is demonstrated the detection of WT SARS-CoV-2 and N501Y mutant variants in clinical samples by using the CRISPR/hybrid Cas system. The hybrid Cas protein is expected to be utilized in a molecular diagnostic method for infectious diseases, tissue and liquid biopsies, and other nucleic acid biomarkers.},
}

@article {pmid36327313,
year = {2022},
author = {Qin, P and Chen, P and Deng, N and Tan, L and Yin, BC and Ye, BC},
title = {Switching the Activity of CRISPR/Cas12a Using an Allosteric Inhibitory Aptamer for Biosensing.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.2c04315},
pmid = {36327313},
issn = {1520-6882},
abstract = {The current CRISPR/Cas12a-based diagnostic techniques focus on designing the crRNA or substrate DNA elements to indirectly switch the trans-cleavage activity of Cas12a responsive to target information. Here, we propose the use of an allosteric DNA probe to directly regulate the trans-cleavage activity of Cas12a and present a method for sensing different types of analytes. An allosteric inhibitor probe is rationally designed to couple the target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system and enables binding to a specific target to induce the change of conformation, which leads to the loss of its inhibitory function on Cas12a. As a result, the structure-switchable probe can regulate the degree of activity of Cas12a depending on the dose of target. Scalability of our strategy can be achieved by simply replacing the loop domain with different target recognition sequences. The proposed method was validated by detecting adenosine triphosphate and let-7a, giving the detection limits of 490 nM and 26 pM, respectively, and showing an excellent specificity. We believe that this work exploits a viable approach to use the inhibitory aptamer of Cas12a as a regulatory element for biosensing purposes, enriching the arsenal of CRISPR/Cas12a-based methods for molecular diagnostics and spurring further development and application of aptamers of the CRISPR/Cas system.},
}

@article {pmid36326904,
year = {2022},
author = {Maharajan, T and Krishna, TPA and Rakkammal, K and Ceasar, SA and Ramesh, M},
title = {Application of CRISPR/Cas system in cereal improvement for biotic and abiotic stress tolerance.},
journal = {Planta},
volume = {256},
number = {6},
pages = {106},
pmid = {36326904},
issn = {1432-2048},
support = {Under Seed Money for Faculty Minor Research//Rajagiri College of Social Sciences/ ; },
abstract = {MAIN CONCLUSION: Application of the recently developed CRISPR/Cas tools might help enhance cereals' growth and yield under biotic and abiotic stresses. Cereals are the most important food crops for human life and an essential source of nutrients for people in developed and developing countries. The growth and yield of all major cereals are affected by both biotic and abiotic stresses. To date, molecular breeding and functional genomic studies have contributed to the understanding and improving cereals' growth and yield under biotic and abiotic stresses. Clustered, regularly inter-spaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been predicted to play a major role in precision plant breeding and developing non-transgenic cereals that can tolerate adverse effects of climate change. Variants of next-generation CRISPR/Cas tools, such as prime editor, base editor, CRISPR activator and repressor, chromatin imager, Cas12a, and Cas12b, are currently used in various fields, including plant science. However, few studies have been reported on applying the CRISPR/Cas system to understand the mechanism of biotic and abiotic stress tolerance in cereals. Rice is the only plant used frequently for such studies. Genes responsible for biotic and abiotic stress tolerance have not yet been studied by CRISPR/Cas system in other major cereals (sorghum, barley, maize and small millets). Examining the role of genes that respond to biotic and abiotic stresses using the CRISPR/Cas system may help enhance cereals' growth and yield under biotic and abiotic stresses. It will help to develop new and improved cultivars with biotic- and abiotic-tolerant traits for better yields to strengthen food security. This review provides information for cereal researchers on the current status of the CRISPR/Cas system for improving biotic and abiotic stress tolerance in cereals.},
}

@article {pmid36324900,
year = {2022},
author = {Rittiner, J and Cumaran, M and Malhotra, S and Kantor, B},
title = {Therapeutic modulation of gene expression in the disease state: Treatment strategies and approaches for the development of next-generation of the epigenetic drugs.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {1035543},
doi = {10.3389/fbioe.2022.1035543},
pmid = {36324900},
issn = {2296-4185},
abstract = {Epigenetic dysregulation is an important determinant of many pathological conditions and diseases. Designer molecules that can specifically target endogenous DNA sequences provide a means to therapeutically modulate gene function. The prokaryote-derived CRISPR/Cas editing systems have transformed our ability to manipulate the expression program of genes through specific DNA and RNA targeting in living cells and tissues. The simplicity, utility, and robustness of this technology have revolutionized epigenome editing for research and translational medicine. Initial success has inspired efforts to discover new systems for targeting and manipulating nucleic acids on the epigenetic level. The evolution of nuclease-inactive and RNA-targeting Cas proteins fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions opened up an unprecedented level of possibilities for the development of "next-generation" gene therapy therapeutics. The rational design and construction of different types of designer molecules paired with viral-mediated gene-to-cell transfers, specifically using lentiviral vectors (LVs) and adeno-associated vectors (AAVs) are reviewed in this paper. Furthermore, we explore and discuss the potential of these molecules as therapeutic modulators of endogenous gene function, focusing on modulation by stable gene modification and by regulation of gene transcription. Notwithstanding the speedy progress of CRISPR/Cas-based gene therapy products, multiple challenges outlined by undesirable off-target effects, oncogenicity and other virus-induced toxicities could derail the successful translation of these new modalities. Here, we review how CRISPR/Cas-based gene therapy is translated from research-grade technological system to therapeutic modality, paying particular attention to the therapeutic flow from engineering sophisticated genome and epigenome-editing transgenes to delivery vehicles throughout efficient and safe manufacturing and administration of the gene therapy regimens. In addition, the potential solutions to some of the obstacles facing successful CRISPR/Cas utility in the clinical research are discussed in this review. We believe, that circumventing these challenges will be essential for advancing CRISPR/Cas-based tools towards clinical use in gene and cell therapies.},
}

@article {pmid35708734,
year = {2022},
author = {Wilson, J and Loizou, JI},
title = {Exploring the genetic space of the DNA damage response for cancer therapy through CRISPR-based screens.},
journal = {Molecular oncology},
volume = {16},
number = {21},
pages = {3778-3791},
doi = {10.1002/1878-0261.13272},
pmid = {35708734},
issn = {1878-0261},
support = {//Austrian Academy of Sciences/ ; ERC Synergy Grant DDREAMM 855741/ERC_/European Research Council/International ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/genetics ; Mutation ; Genetic Testing ; DNA Damage ; },
abstract = {The concepts of synthetic lethality and viability have emerged as powerful approaches to identify vulnerabilities and resistances within the DNA damage response for the treatment of cancer. Historically, interactions between two genes have had a longstanding presence in genetics and have been identified through forward genetic screens that rely on the molecular basis of the characterized phenotypes, typically caused by mutations in single genes. While such complex genetic interactions between genes have been studied extensively in model organisms, they have only recently been prioritized as therapeutic strategies due to technological advancements in genetic screens. Here, we discuss synthetic lethal and viable interactions within the DNA damage response and present how CRISPR-based genetic screens and chemical compounds have allowed for the systematic identification and targeting of such interactions for the treatment of cancer.},
}

Humans
*CRISPR-Cas Systems
*Neoplasms/genetics
Mutation
Genetic Testing
DNA Damage

@article {pmid36323073,
year = {2022},
author = {Lu, Q and Chen, R and Du, S and Chen, C and Pan, Y and Luan, X and Yang, J and Zeng, F and He, B and Han, X and Song, Y},
title = {Activation of the cGAS-STING pathway combined with CRISPR-Cas9 gene editing triggering long-term immunotherapy.},
journal = {Biomaterials},
volume = {291},
number = {},
pages = {121871},
doi = {10.1016/j.biomaterials.2022.121871},
pmid = {36323073},
issn = {1878-5905},
abstract = {Effective activation of cGAS-STING pathway combined with immune checkpoint blockade (ICB) within the immunosuppressive tumor microenvironment to induce stronger immune responsiveness yet remains challenging. CRISPR-Cas9 gene editing technology, which offers the benefits of permanence and irreversibility, could recognize the target genome sequence with sgRNA (Guide RNA) and guide the Cas9 protease to knock down the target gene. Herein, a nanoplatform (HMnMPH) for dual activation of cGAS-STING pathway in combination with CRISPR-Cas9 gene editing to silence programmed death ligand 1 (PD-L1) to trigger long-term immunotherapy was reported. The HMnMPH consists of hollow manganese dioxide (HMn) loaded with STING agonist (MSA-2) and CRISPR-Cas9/sg-PD-L1 plasmid with further modification of hyaluronic acid (HA). In acidic and GSH overexpressed tumor environment, HMnPMH was degraded to release large amounts of Mn ions and STING agonists, strongly and persistently activating the cGAS-STING pathway to promote the release of type I interferon and pro-inflammatory factors. Meanwhile, the released CRISPR-Cas9 plasmid could knockdown the PD-L1 immune checkpoint and restart immunosuppressive T cells to differentiate into cytotoxic T lymphocytes significantly, which reduced the activity of primary and distal tumors and demonstrated a long-term immune memory effect on distal tumors.},
}

@article {pmid36321657,
year = {2022},
author = {Zhang, B and Lin, J and Perčulija, V and Li, Y and Lu, Q and Chen, J and Ouyang, S},
title = {Structural insights into target DNA recognition and cleavage by the CRISPR-Cas12c1 system.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkac987},
pmid = {36321657},
issn = {1362-4962},
support = {82225028//National Nature Science Foundation of China/ ; 2021YFC2301403//National Key Research and Development Program of China/ ; 2020L3008//Special Funds of the Central Government Guiding Local Science and Technology Development/ ; Z0210509//High-level personnel introduction grant of Fujian Normal University/ ; },
abstract = {Cas12c is the recently characterized dual RNA-guided DNase effector of type V-C CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems. Due to minimal requirements for a protospacer adjacent motif (PAM), Cas12c is an attractive candidate for genome editing. Here we report the crystal structure of Cas12c1 in complex with single guide RNA (sgRNA) and target double-stranded DNA (dsDNA) containing the 5'-TG-3' PAM. Supported by biochemical and mutation assays, this study reveals distinct structural features of Cas12c1 and the associated sgRNA, as well as the molecular basis for PAM recognition, target dsDNA unwinding, heteroduplex formation and recognition, and cleavage of non-target and target DNA strands. Cas12c1 recognizes the PAM through a mechanism that is interdependent on sequence identity and Cas12c1-induced conformational distortion of the PAM region. Another special feature of Cas12c1 is the cleavage of both non-target and target DNA strands at a single, uniform site with indistinguishable cleavage capacity and order. Location of the sgRNA seed region and minimal length of target DNA required for triggering Cas12c1 DNase activity were also determined. Our findings provide valuable information for developing the CRISPR-Cas12c1 system into an efficient, high-fidelity genome editing tool.},
}

@article {pmid36320584,
year = {2022},
author = {Zhao, X and Na, N and Ouyang, J},
title = {CRISPR/Cas9-based coronal nanostructures for targeted mitochondria single molecule imaging.},
journal = {Chemical science},
volume = {13},
number = {38},
pages = {11433-11441},
doi = {10.1039/d2sc03329a},
pmid = {36320584},
issn = {2041-6520},
abstract = {The biological state at the subcellular level is highly relevant to many diseases, and the monitoring of organelles such as mitochondria is crucial based on this. However, most DNA and protein based nanoprobes used for the detection of mitochondrial RNAs (mitomiRs) lack spatial selectivity, which leads to inefficiencies in probe delivery and signal turn-on. Herein, we constructed a novel DNA nanoprobe named protein delivery nano-corona (PDNC) to improve the delivery efficiency of Cas protein, for spatially selective imaging of mitomiRs in living cells switched on by a CRISPR/Cas system. Combined with a single-molecule counting method, this strategy enables highly sensitive detection of low-abundance mitomiR. Therefore, the strategy in this work opens up new opportunities for cell identification, early clinical diagnosis, and research in biological behaviour at the subcellular level.},
}

@article {pmid36320315,
year = {2022},
author = {Lohia, A and Sahel, DK and Salman, M and Singh, V and Mariappan, I and Mittal, A and Chitkara, D},
title = {Delivery strategies for CRISPR/Cas genome editing tool for retinal dystrophies: challenges and opportunities.},
journal = {Asian journal of pharmaceutical sciences},
volume = {17},
number = {2},
pages = {153-176},
doi = {10.1016/j.ajps.2022.02.001},
pmid = {36320315},
issn = {2221-285X},
abstract = {CRISPR/Cas, an adaptive immune system in bacteria, has been adopted as an efficient and precise tool for site-specific gene editing with potential therapeutic opportunities. It has been explored for a variety of applications, including gene modulation, epigenome editing, diagnosis, mRNA editing, etc. It has found applications in retinal dystrophic conditions including progressive cone and cone-rod dystrophies, congenital stationary night blindness, X-linked juvenile retinoschisis, retinitis pigmentosa, age-related macular degeneration, leber's congenital amaurosis, etc. Most of the therapies for retinal dystrophic conditions work by regressing symptoms instead of reversing the gene mutations. CRISPR/Cas9 through indel could impart beneficial effects in the reversal of gene mutations in dystrophic conditions. Recent research has also consolidated on the approaches of using CRISPR systems for retinal dystrophies but their delivery to the posterior part of the eye is a major concern due to high molecular weight, negative charge, and in vivo stability of CRISPR components. Recently, non-viral vectors have gained interest due to their potential in tissue-specific nucleic acid (miRNA/siRNA/CRISPR) delivery. This review highlights the opportunities of retinal dystrophies management using CRISPR/Cas nanomedicine.},
}

@article {pmid36317180,
year = {2022},
author = {Medishetti, R and Balamurugan, K and Yadavalli, K and Rani, R and Sevilimedu, A and Challa, AK and Parsa, K and Chatti, K},
title = {CRISPR-Cas9-induced gene knockout in zebrafish.},
journal = {STAR protocols},
volume = {3},
number = {4},
pages = {101779},
pmid = {36317180},
issn = {2666-1667},
mesh = {Animals ; Humans ; Gene Knockout Techniques ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Germ Cells ; },
abstract = {The application of CRISPR has greatly facilitated genotype-phenotype studies of human disease models. In this protocol, we describe CRISPR-Cas9-induced gene knockout in zebrafish, utilizing purified Cas9 protein and in vitro-transcribed sgRNA. This protocol targets the PHLPP1 gene in an Indian wild-caught strain, but is broadly applicable. Major factors influencing protocol success include zebrafish health and fecundity, sgRNA efficiency and specificity, germline transmission, and mutant viability. For complete details on the use and execution of this protocol, please refer to Balamurugan et al. (2022).},
}

Animals
Humans
Gene Knockout Techniques
*Zebrafish/genetics
*CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics
Germ Cells

@article {pmid36305591,
year = {2022},
author = {Vos, PD and Filipovska, A and Rackham, O},
title = {Frankenstein Cas9: engineering improved gene editing systems.},
journal = {Biochemical Society transactions},
volume = {50},
number = {5},
pages = {1505-1516},
doi = {10.1042/BST20220873},
pmid = {36305591},
issn = {1470-8752},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; RNA, Guide/genetics/metabolism ; Endonucleases/genetics/metabolism ; },
abstract = {The discovery of CRISPR-Cas9 and its widespread use has revolutionised and propelled research in biological sciences. Although the ability to target Cas9's nuclease activity to specific sites via an easily designed guide RNA (gRNA) has made it an adaptable gene editing system, it has many characteristics that could be improved for use in biotechnology. Cas9 exhibits significant off-target activity and low on-target nuclease activity in certain contexts. Scientists have undertaken ambitious protein engineering campaigns to bypass these limitations, producing several promising variants of Cas9. Cas9 variants with improved and alternative activities provide exciting new tools to expand the scope and fidelity of future CRISPR applications.},
}

*Gene Editing
*CRISPR-Cas Systems
RNA, Guide/genetics/metabolism
Endonucleases/genetics/metabolism

@article {pmid36318504,
year = {2022},
author = {Gong, S and Wang, X and Zhou, P and Pan, W and Li, N and Tang, B},
title = {AND Logic-Gate-Based CRISPR/Cas12a Biosensing Platform for the Sensitive Colorimetric Detection of Dual miRNAs.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.2c03666},
pmid = {36318504},
issn = {1520-6882},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) system has been widely explored for the detection of disease-related nucleic acids. Nevertheless, the simultaneous detection of multiple nucleic acids within one assay using the CRISPR-Cas system is still challenging. In this study, we develop an AND logic-gate-based CRISPR-Cas12a biosensing platform to achieve the sensitive colorimetric detection of dual miRNAs. Specifically, the DNA probe was designed to recognize the binary input of miRNAs and to output trigger DNA, which activated the CRISPR-Cas12a system to cut single-stranded DNA (ssDNA). The ssDNA on magnetic beads (MBs) was cleaved by the activated CRISPR-Cas12a, causing the separation of glucose oxidase (GOx) from MB and the subsequent generation of a colorimetric signal. The color change induced by 1 pM of target miRNAs can be directly distinguished by the naked eye and the instrumental limit of detection reaches 36.4 fM. The overexpressed miR-205 and miR-944 in the real human serum can be detected, allowing us to differentiate between lung cancer patients and healthy people. Furthermore, the developed strategy achieves simultaneous detection of dual miRNAs using CRISPR-Cas12a with one kind of crRNA, avoiding sophisticated nucleic acid amplifications and the use of bulky instruments. The current method can broaden the CRISPR-Cas12a-based applications for multiple biomarkers detection and precise disease diagnosis.},
}

@article {pmid36317989,
year = {2022},
author = {Blicharska, D and Szućko-Kociuba, I and Filip, E and Skuza, L},
title = {CRISPR/Cas as the intelligent immune system of bacteria and archea.},
journal = {Postepy biochemii},
volume = {68},
number = {3},
pages = {235-245},
doi = {10.18388/pb.2021_453},
pmid = {36317989},
issn = {0032-5422},
abstract = {The invention of CRISPR is considered to be one of the most breakthrough discoveries in recent years in the history of biology, biotechnology, medicine, as well as the pharmaceutical and agricultural industries. The methods developed using CRISPR create new, previously unattainable possibilities that can significantly improve the quality of life. From the invention of this intelligent immune system to the present day, much research has been done using the CRISPR/Cas systems. The result of these studies was the development of a modern tool for genetic manipulation, which allows for the introduction of many modifications within the DNA, which may contribute to the silencing of the expression of given genes or their overexpression through e.g. mutations or deletions. The paper describes the application of the method for genetic manipulation with the use of the second class system – CRISPR/Cas9 and the advantages of this method and its advantage over the previously used genetic engineering tools, as well as its limitations and disadvantages, which significantly limit the possibility of its application. The potential use of the method was also presented as well as the research carried out with the use of CRSPR/Cas9.},
}

@article {pmid36317129,
year = {2022},
author = {Robinson, EL and Port, JD},
title = {Utilization and Potential of RNA-Based Therapies in Cardiovascular Disease.},
journal = {JACC. Basic to translational science},
volume = {7},
number = {9},
pages = {956-969},
doi = {10.1016/j.jacbts.2022.02.003},
pmid = {36317129},
issn = {2452-302X},
abstract = {Cardiovascular disease (CVD) remains the largest cause of mortality worldwide. The development of new effective therapeutics is a major unmet need. The current review focuses broadly on the concept of nucleic acid (NA)-based therapies, considering the use of various forms of NAs, including mRNAs, miRNAs, siRNA, and guide RNAs, the latter specifically for the purpose of CRISPR-Cas directed gene editing. We describe the current state-of-the-art of RNA target discovery and development, the status of RNA therapeutics in the context of CVD, and some of the challenges and hurdles to be overcome.},
}

@article {pmid36316452,
year = {2022},
author = {Guan, J and Oromí-Bosch, A and Mendoza, SD and Karambelkar, S and Berry, JD and Bondy-Denomy, J},
title = {Bacteriophage genome engineering with CRISPR-Cas13a.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {36316452},
issn = {2058-5276},
support = {R01GM127489//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01GM127489//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01GM127489//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
abstract = {Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous 'phage nucleus' structure that protects from DNA-targeting CRISPR-Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the ФKZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR-Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function.},
}

@article {pmid36309986,
year = {2022},
author = {Huang, S and Xue, Y and Zhou, C and Ma, Y},
title = {An efficient CRISPR/Cas9-based genome editing system for alkaliphilic Bacillus sp. N16-5 and application in engineering xylose utilization for D-lactic acid production.},
journal = {Microbial biotechnology},
volume = {15},
number = {11},
pages = {2730-2743},
doi = {10.1111/1751-7915.14131},
pmid = {36309986},
issn = {1751-7915},
support = {2020YFA0906800//National Key R&D Program of China/ ; 31870789//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing ; CRISPR-Cas Systems ; Xylose ; *Bacillus/genetics ; Lactic Acid ; },
abstract = {Alkaliphiles are considered more suitable chassis than traditional neutrophiles due to their excellent resistance to microbial contamination. Alkaliphilic Bacillus sp. N16-5, an industrially interesting strain with great potential for the production of lactic acid and alkaline polysaccharide hydrolases, can only be engineered genetically by the laborious and time-consuming homologous recombination. In this study, we reported the successful development of a CRISPR/Cas9-based genome editing system with high efficiency for single-gene deletion, large gene fragment deletion and exogenous DNA chromosomal insertion. Moreover, based on a catalytically dead variant of Cas9 (dCas9), we also developed a CRISPRi system to efficiently regulate gene expression. Finally, this efficient genome editing system was successfully applied to engineer the xylose metabolic pathway for the efficient bioproduction of D-lactic acid. Compared with the wild-type Bacillus sp. N16-5, the final engineered strain with XylR deletion and AraE overexpression achieved 34.3% and 27.7% increases in xylose consumption and D-lactic acid production respectively. To our knowledge, this is the first report on the development and application of CRISPR/Cas9-based genome editing system in alkaliphilic Bacillus, and this study will significantly facilitate functional genomic studies and genome manipulation in alkaliphilic Bacillus, laying a foundation for the development of more robust microbial chassis.},
}

*Gene Editing
CRISPR-Cas Systems
Xylose
*Bacillus/genetics
Lactic Acid

@article {pmid36309521,
year = {2022},
author = {Xu, Z and Chen, D and Li, T and Yan, J and Zhu, J and He, T and Hu, R and Li, Y and Yang, Y and Liu, M},
title = {Microfluidic space coding for multiplexed nucleic acid detection via CRISPR-Cas12a and recombinase polymerase amplification.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6480},
pmid = {36309521},
issn = {2041-1723},
support = {22174150//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Recombinases ; CRISPR-Cas Systems/genetics ; Microfluidics ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; Nucleotidyltransferases ; },
abstract = {Fast, inexpensive, and multiplexed detection of multiple nucleic acids is of great importance to human health, yet it still represents a significant challenge. Herein, we propose a nucleic acid testing platform, named MiCaR, which couples a microfluidic device with CRISPR-Cas12a and multiplex recombinase polymerase amplification. With only one fluorescence probe, MiCaR can simultaneously test up to 30 nucleic acid targets through microfluidic space coding. The detection limit achieves 0.26 attomole, and the multiplexed assay takes only 40 min. We demonstrate the utility of MiCaR by efficiently detecting the nine HPV subtypes targeted by the 9-valent HPV vaccine, showing a sensitivity of 97.8% and specificity of 98.1% in the testing of 100 patient samples at risk for HPV infection. Additionally, we also show the generalizability of our approach by successfully testing eight of the most clinically relevant respiratory viruses. We anticipate this effective, undecorated and versatile platform to be widely used in multiplexed nucleic acid detection.},
}

Humans
*Recombinases
CRISPR-Cas Systems/genetics
Microfluidics
Nucleic Acid Amplification Techniques
*Nucleic Acids
Nucleotidyltransferases

@article {pmid36309502,
year = {2022},
author = {Ciciani, M and Demozzi, M and Pedrazzoli, E and Visentin, E and Pezzè, L and Signorini, LF and Blanco-Miguez, A and Zolfo, M and Asnicar, F and Casini, A and Cereseto, A and Segata, N},
title = {Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6474},
pmid = {36309502},
issn = {2041-1723},
support = {825825//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; RNA, Guide/genetics ; Metagenome ; Gene Editing/methods ; Endonucleases/metabolism ; },
abstract = {The identification of the protospacer adjacent motif (PAM) sequences of Cas9 nucleases is crucial for their exploitation in genome editing. Here we develop a computational pipeline that was used to interrogate a massively expanded dataset of metagenome and virome assemblies for accurate and comprehensive PAM predictions. This procedure allows the identification and isolation of sequence-tailored Cas9 nucleases by using the target sequence as bait. As proof of concept, starting from the disease-causing mutation P23H in the RHO gene, we find, isolate and experimentally validate a Cas9 which uses the mutated sequence as PAM. Our PAM prediction pipeline will be instrumental to generate a Cas9 nuclease repertoire responding to any PAM requirement.},
}

*CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems/genetics
RNA, Guide/genetics
Metagenome
Gene Editing/methods
Endonucleases/metabolism

@article {pmid36307542,
year = {2022},
author = {Bäckström, A and Yudovich, D and Žemaitis, K and Nilsén Falck, L and Subramaniam, A and Larsson, J},
title = {Combinatorial gene targeting in primary human hematopoietic stem and progenitor cells.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {18169},
pmid = {36307542},
issn = {2045-2322},
support = {648894/ERC_/European Research Council/International ; },
mesh = {Humans ; Mice ; Animals ; *Hematopoietic Stem Cells/metabolism ; *Gene Editing ; Gene Targeting ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {The CRISPR/Cas9 system offers enormous versatility for functional genomics but many applications have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, to establish a paradigm for multiplexed gene editing in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs), we used co-delivery of lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The fluorescent markers allow for tracking of either single- or double-edited cells, and we could achieve robust double knockout of the cell surface molecules CD45 and CD44 with an efficiency of ~ 70%. As a functional proof of concept, we demonstrate that this system can be used to model gene dependencies for cell survival, by simultaneously targeting the cohesin genes STAG1 and STAG2. Moreover, we show combinatorial effects with potential synergy for HSPC expansion by targeting the Aryl Hydrocarbon Receptor (AHR) in conjunction with members of the CoREST complex. Taken together, our traceable multiplexed CRISPR/Cas9 system enables studies of genetic dependencies and cooperation in primary HSPCs, and has important implications for modelling polygenic diseases, as well as investigation of the underlying mechanisms of gene interactions.},
}

Humans
Mice
Animals
*Hematopoietic Stem Cells/metabolism
*Gene Editing
Gene Targeting
Cell Line
CRISPR-Cas Systems

@article {pmid36307508,
year = {2022},
author = {Westermann, L and Li, Y and Göcmen, B and Niedermoser, M and Rhein, K and Jahn, J and Cascante, I and Schöler, F and Moser, N and Neubauer, B and Hofherr, A and Behrens, YL and Göhring, G and Köttgen, A and Köttgen, M and Busch, T},
title = {Wildtype heterogeneity contributes to clonal variability in genome edited cells.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {18211},
pmid = {36307508},
issn = {2045-2322},
support = {NakSys//Else Kröner-Fresenius-Stiftung/ ; SFB1453//Deutsche Forschungsgemeinschaft/ ; TRR152//Deutsche Forschungsgemeinschaft/ ; CIBSS//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Reproducibility of Results ; *Gene Editing/methods ; Cell Line ; Cells, Cultured ; },
abstract = {Genome editing tools such as CRISPR/Cas9 enable the rapid and precise manipulation of genomes. CRISPR-based genome editing has greatly simplified the study of gene function in cell lines, but its widespread use has also highlighted challenges of reproducibility. Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype-phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts (477 up- and 306 downregulated) when comparing two populations of wild-type cells. Furthermore, we show a variety of cellular and biochemical alterations in different wild-type clones in a range that is commonly interpreted as biologically relevant in genome-edited cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability. We therefore propose to generate matched isogenic control cells prior to genome editing to increase reproducibility.},
}

*CRISPR-Cas Systems/genetics
Reproducibility of Results
*Gene Editing/methods
Cell Line
Cells, Cultured

@article {pmid36307477,
year = {2022},
author = {Bellinvia, E and García-González, J and Cifrová, P and Martinek, J and Sikorová, L and Havelková, L and Schwarzerová, K},
title = {CRISPR-Cas9 Arabidopsis mutants of genes for ARPC1 and ARPC3 subunits of ARP2/3 complex reveal differential roles of complex subunits.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {18205},
pmid = {36307477},
issn = {2045-2322},
support = {19-10845S//Ministerstvo Školství, Mládeže a Tělovýchovy/ ; CZ.1.05/4.1.00/16.0347//European Regional Development Fund/ ; LM2018129//Czech Bio-imaging/ ; e-INFRA LM2018140//e-Infrastruktura CZ/ ; },
mesh = {*Actin-Related Protein 2-3 Complex/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; Actins/metabolism ; CRISPR-Cas Systems ; Actin-Related Protein 2/genetics ; Actin-Related Protein 3/metabolism ; },
abstract = {Protein complex Arp2/3 has a conserved role in the nucleation of branched actin filaments. It is constituted of seven subunits, including actin-like subunits ARP2 and ARP3 plus five other subunits called Arp2/3 Complex Component 1 to 5, which are not related to actin. Knock-out plant mutants lacking individual plant ARP2/3 subunits have a typical phenotype of distorted trichomes, altered pavement cells shape and defects in cell adhesion. While knock-out mutant Arabidopsis plants for most ARP2/3 subunits have been characterized before, Arabidopsis plant mutants missing ARPC1 and ARPC3 subunits have not yet been described. Using CRISPR/Cas9, we generated knock-out mutants lacking ARPC1 and ARPC3 subunits. We confirmed that the loss of ARPC1 subunits results in the typical ARP2/3 mutant phenotype. However, the mutants lacking ARPC3 subunits resulted in plants with surprisingly different phenotypes. Our results suggest that plant ARP2/3 complex function in trichome shaping does not require ARPC3 subunit, while the fully assembled complex is necessary for the establishment of correct cell adhesion in the epidermis.},
}

*Actin-Related Protein 2-3 Complex/genetics/metabolism
*Arabidopsis/genetics/metabolism
Actins/metabolism
CRISPR-Cas Systems
Actin-Related Protein 2/genetics
Actin-Related Protein 3/metabolism

@article {pmid36307400,
year = {2022},
author = {Coelho, R and Tozzi, A and Disler, M and Lombardo, F and Fedier, A and López, MN and Freuler, F and Jacob, F and Heinzelmann-Schwarz, V},
title = {Overlapping gene dependencies for PARP inhibitors and carboplatin response identified by functional CRISPR-Cas9 screening in ovarian cancer.},
journal = {Cell death & disease},
volume = {13},
number = {10},
pages = {909},
pmid = {36307400},
issn = {2041-4889},
mesh = {Humans ; Female ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Carcinoma, Ovarian Epithelial/drug therapy/genetics ; Carboplatin/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Early Detection of Cancer ; Phthalazines/pharmacology/therapeutic use ; *Ovarian Neoplasms/drug therapy/genetics/pathology ; Genes, Overlapping ; },
abstract = {PARP inhibitors (PARPi) have revolutionized the therapeutic landscape of epithelial ovarian cancer (EOC) treatment with outstanding benefits in regard to progression-free survival, especially in patients either carrying BRCA1/2 mutations or harboring defects in the homologous recombination repair system. Yet, it remains uncertain which PARPi to apply and how to predict responders when platinum sensitivity is unknown. To shed light on the predictive power of genes previously suggested to be associated with PARPi response, we systematically reviewed the literature and identified 79 publications investigating a total of 93 genes. The top candidate genes were further tested using a comprehensive CRISPR-Cas9 mutagenesis screening in combination with olaparib treatment. Therefore, we generated six constitutive Cas9+ EOC cell lines and profiled 33 genes in a CRISPR-Cas9 cell competition assay using non-essential (AAVS1) and essential (RPA3 and PCNA) genes for cell fitness as negative and positive controls, respectively. We identified only ATM, MUS81, NBN, BRCA2, and RAD51B as predictive markers for olaparib response. As the major survival benefit of PARPi treatment was reported in platinum-sensitive tumors, we next assessed nine top candidate genes in combination with three PARPi and carboplatin. Interestingly, we observed similar dropout rates in a gene and compound independent manner, supporting the strong correlation of cancer cell response to compounds that rely on DNA repair for their effectiveness. In addition, we report on CDK12 as a common vulnerability for EOC cell survival and proliferation without altering the olaparib response, highlighting its potential as a therapeutic target in EOC.},
}

Humans
Female
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use
Carcinoma, Ovarian Epithelial/drug therapy/genetics
Carboplatin/pharmacology/therapeutic use
CRISPR-Cas Systems/genetics
Early Detection of Cancer
Phthalazines/pharmacology/therapeutic use
*Ovarian Neoplasms/drug therapy/genetics/pathology
Genes, Overlapping

@article {pmid36306089,
year = {2023},
author = {Kohm, K and Lutz, VT and Friedrich, I and Hertel, R},
title = {CRISPR-Cas9 Shaped Viral Metagenomes Associated with Bacillus subtilis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2555},
number = {},
pages = {205-212},
pmid = {36306089},
issn = {1940-6029},
mesh = {*Bacillus subtilis/genetics ; CRISPR-Cas Systems/genetics ; Metagenome ; *Bacteriophages ; },
abstract = {Phages are viruses of bacteria and have been known for over a century. They do not have a metabolism or protein synthesis machinery and rely on host cells for replication. The model organism Bacillus subtilis has served as a host strain for decades and enabled the isolation of many unique viral strains. However, many viral species representatives remained orphans as no, or only a few, related phages were ever re-isolated.The presented protocol describes how a CRISPR-Cas9 system with an artificial CRISPR-array can be set up and used to discriminate abundant and well-known B. subtilis phage from a host-based metagenome enrichment. The obtained viral suspension can be used for metagenome sequencing and isolating new viral strains.},
}

*Bacillus subtilis/genetics
CRISPR-Cas Systems/genetics
Metagenome
*Bacteriophages

@article {pmid36243247,
year = {2022},
author = {Valcárcel-Hernández, V and Guillén-Yunta, M and Bueno-Arribas, M and Montero-Pedrazuela, A and Grijota-Martínez, C and Markossian, S and García-Aldea, Á and Flamant, F and Bárez-López, S and Guadaño-Ferraz, A},
title = {A CRISPR/Cas9-engineered avatar mouse model of monocarboxylate transporter 8 deficiency displays distinct neurological alterations.},
journal = {Neurobiology of disease},
volume = {174},
number = {},
pages = {105896},
doi = {10.1016/j.nbd.2022.105896},
pmid = {36243247},
issn = {1095-953X},
mesh = {Animals ; Mice ; *Monocarboxylic Acid Transporters/genetics/metabolism ; *Symporters/genetics/metabolism ; CRISPR-Cas Systems ; Thyroid Hormones/metabolism ; Disease Models, Animal ; Mammals/metabolism ; },
abstract = {Inactivating mutations in the specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8) lead to an X-linked rare disease named MCT8 deficiency or Allan-Herndon-Dudley Syndrome. Patients exhibit a plethora of severe endocrine and neurological alterations, with no effective treatment for the neurological symptoms. An optimal mammalian model is essential to explore the pathological mechanisms and potential therapeutic approaches. Here we have generated by CRISPR/Cas9 an avatar mouse model for MCT8 deficiency with a point mutation found in two MCT8-deficient patients (P253L mice). We have predicted by in silico studies that this mutation alters the substrate binding pocket being the probable cause for impairing thyroid hormone transport. We have characterized the phenotype of MCT8-P253L mice and found endocrine alterations similar to those described in patients and in MCT8-deficient mice. Importantly, we detected brain hypothyroidism, structural and functional neurological alterations resembling the patient's neurological impairments. Thus, the P253L mouse provides a valuable model for studying the pathophysiology of MCT8 deficiency and in the future will allow to test therapeutic alternatives such as in vivo gene therapy and pharmacological chaperone therapy to improve the neurological impairments in MCT8 deficiency.},
}

Animals
Mice
*Monocarboxylic Acid Transporters/genetics/metabolism
*Symporters/genetics/metabolism
CRISPR-Cas Systems
Thyroid Hormones/metabolism
Disease Models, Animal
Mammals/metabolism

@article {pmid36174845,
year = {2022},
author = {Luo, C and Wang, Q and Guo, R and Zhang, J and Zhang, J and Zhang, R and Ma, X and Wang, P and Adam, FEA and Zeshan, B and Yang, Z and Zhou, Y and Wang, X},
title = {A novel Pseudorabies virus vaccine developed using HDR-CRISPR/Cas9 induces strong humoral and cellular immune response in mice.},
journal = {Virus research},
volume = {322},
number = {},
pages = {198937},
doi = {10.1016/j.virusres.2022.198937},
pmid = {36174845},
issn = {1872-7492},
mesh = {Swine ; Mice ; Animals ; Pseudorabies Vaccines/genetics ; *Herpesvirus 1, Suid ; Granulocyte-Macrophage Colony-Stimulating Factor/genetics ; CRISPR-Cas Systems ; *Swine Diseases ; Antibodies, Viral ; *Pseudorabies/prevention & control ; Immunity, Cellular ; Viral Envelope Proteins/genetics ; },
abstract = {Outbreaks of Pseudorabies (PR) by numerous highly virulent and antigenic variant Pseudorabies virus (PRV) strains have been causing severe economic losses to the pig industry in China since 2011. However, current commercial vaccines are often unable to induce thorough protective immunity. In this study, a TK/gI/gE deleted recombinant PRV expressing GM-CSF was developed by using the HDR-CRISPR/Cas9 system. Here, a four-sgRNA along with the Cas9D10A targeting system was utilized for TK/gI/gE gene deletion and GM-CSF insertion. Our study showed that the four-sgRNA targeting system appeared to have higher knock-in efficiency for PRVs editing. The replication of the recombinant PRVs were slightly lower than that of the parental strain, but they appeared to have similar properties in terms of growth curves and plaque morphology. The mice vaccinated with the recombinant PRV expressing GM-CSF via intramuscular injection showed no obvious clinical symptoms, milder pathological lesions, and were completely protected against wild-type PRV challenge. When compared to the triple gene-deleted PRV, the gB antibodies and neutralizing antibody titers were improved and the immunized mice appeared to have lower viral load and higher mRNA levels of IL-2, IL-4, IL-6, and IFN-γ in spleens. Our study offers a novel approach for recombinant PRV construction, and the triple gene-deleted PRV expressing GM-CSF could serve as a promising vaccine candidate for PR control.},
}

Swine
Mice
Animals
Pseudorabies Vaccines/genetics
*Herpesvirus 1, Suid
Granulocyte-Macrophage Colony-Stimulating Factor/genetics
CRISPR-Cas Systems
*Swine Diseases
Antibodies, Viral
*Pseudorabies/prevention & control
Immunity, Cellular
Viral Envelope Proteins/genetics

@article {pmid36036114,
year = {2022},
author = {Chen, Z and Zhu, J and Chen, Z and Du, M and Yao, R and Fu, W and Lei, A and Wang, J},
title = {High-throughput sequencing revealed low-efficacy genome editing using Cas9 RNPs electroporation and single-celled microinjection provided an alternative to deliver CRISPR reagents into Euglena gracilis.},
journal = {Plant biotechnology journal},
volume = {20},
number = {11},
pages = {2048-2050},
pmid = {36036114},
issn = {1467-7652},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Euglena gracilis ; Microinjections ; Indicators and Reagents ; Electroporation ; Ribonucleoproteins/genetics ; High-Throughput Nucleotide Sequencing ; RNA, Guide/genetics ; },
}

*Gene Editing
CRISPR-Cas Systems/genetics
*Euglena gracilis
Microinjections
Indicators and Reagents
Electroporation
Ribonucleoproteins/genetics
High-Throughput Nucleotide Sequencing
RNA, Guide/genetics

@article {pmid35916139,
year = {2022},
author = {Ishikawa, M and Nomura, T and Tamaki, S and Ozasa, K and Suzuki, T and Toyooka, K and Hirota, K and Yamada, K and Suzuki, K and Mochida, K},
title = {CRISPR/Cas9-mediated generation of non-motile mutants to improve the harvesting efficiency of mass-cultivated Euglena gracilis.},
journal = {Plant biotechnology journal},
volume = {20},
number = {11},
pages = {2042-2044},
pmid = {35916139},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; *Euglena gracilis/genetics ; Gene Editing ; Ribonucleoproteins/genetics ; },
}

*CRISPR-Cas Systems/genetics
*Euglena gracilis/genetics
Gene Editing
Ribonucleoproteins/genetics

@article {pmid35796629,
year = {2022},
author = {Herath, D and Voogd, C and Mayo-Smith, M and Yang, B and Allan, AC and Putterill, J and Varkonyi-Gasic, E},
title = {CRISPR-Cas9-mediated mutagenesis of kiwifruit BFT genes results in an evergrowing but not early flowering phenotype.},
journal = {Plant biotechnology journal},
volume = {20},
number = {11},
pages = {2064-2076},
pmid = {35796629},
issn = {1467-7652},
mesh = {*Actinidia/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; Flowers/genetics/metabolism ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Amino Acid Sequence ; Phenotype ; Mutagenesis ; Plants, Genetically Modified/genetics/metabolism ; },
abstract = {Phosphatidylethanolamine-binding protein (PEBP) genes regulate flowering and architecture in many plant species. Here, we study kiwifruit (Actinidia chinensis, Ac) PEBP genes with homology to BROTHER OF FT AND TFL1 (BFT). CRISPR-Cas9 was used to target AcBFT genes in wild-type and fast-flowering kiwifruit backgrounds. The editing construct was designed to preferentially target AcBFT2, whose expression is elevated in dormant buds. Acbft lines displayed an evergrowing phenotype and increased branching, while control plants established winter dormancy. The evergrowing phenotype, encompassing delayed budset and advanced budbreak after defoliation, was identified in multiple independent lines with edits in both alleles of AcBFT2. RNA-seq analyses conducted using buds from gene-edited and control lines indicated that Acbft evergrowing plants had a transcriptome similar to that of actively growing wild-type plants, rather than dormant controls. Mutations in both alleles of AcBFT2 did not promote flowering in wild-type or affect flowering time, morphology and fertility in fast-flowering transgenic kiwifruit. In summary, editing of AcBFT2 has the potential to reduce plant dormancy with no adverse effect on flowering, giving rise to cultivars better suited for a changing climate.},
}

*Actinidia/genetics/metabolism
Gene Expression Regulation, Plant/genetics
Flowers/genetics/metabolism
Plant Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Amino Acid Sequence
Phenotype
Mutagenesis
Plants, Genetically Modified/genetics/metabolism

@article {pmid36315306,
year = {2022},
author = {Singh, S and Chaudhary, R and Deshmukh, R and Tiwari, S},
title = {Opportunities and challenges with CRISPR-Cas mediated homologous recombination based precise editing in plants and animals.},
journal = {Plant molecular biology},
volume = {},
number = {},
pages = {},
pmid = {36315306},
issn = {1573-5028},
support = {BIRAC/TechTransfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; Core Grant//National Agri-Food Biotechnology Institute/ ; },
abstract = {KEY MESSAGE: We summarise recent advancements to achieve higher homologous recombination based gene targeting efficiency in different animals and plants. The genome editing has revolutionized the agriculture and human therapeutic sectors by its ability to create precise, stable and predictable mutations in the genome. It depends upon targeted double-strand breaks induction by the engineered endonucleases, which then gets repaired by highly conserved endogenous DNA repair mechanisms. The repairing could be done either through non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The HDR-based editing can be applied for precise gene targeting such as insertion of a new gene, gene replacement and altering of the regulatory sequence of a gene to control the existing protein expression. However, HDR-mediated editing is considered challenging because of lower efficiency in higher eukaryotes, thus, preventing its widespread application. This article reviews the recent progress of HDR-mediated editing and discusses novel strategies such as cell cycle synchronization, modulation of DNA damage repair factors, engineering of Cas protein favoring HDR and CRISPR-Cas reagents delivery methods to improve efficiency for generating knock-in events in both plants and animals. Further, multiplexing of described methods may be promising towards achieving higher donor template-assisted homologous recombination efficiency at the target locus.},
}

@article {pmid36312941,
year = {2022},
author = {Adade, NE and Aniweh, Y and Mosi, L and Valvano, MA and Duodu, S and Ahator, SD},
title = {Comparative analysis of Vibrio cholerae isolates from Ghana reveals variations in genome architecture and adaptation of outbreak and environmental strains.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {998182},
doi = {10.3389/fmicb.2022.998182},
pmid = {36312941},
issn = {1664-302X},
abstract = {Recurrent epidemics of cholera denote robust adaptive mechanisms of Vibrio cholerae for ecological shifting and persistence despite variable stress conditions. Tracking the evolution of pathobiological traits requires comparative genomic studies of isolates from endemic areas. Here, we investigated the genetic differentiation among V. cholerae clinical and environmental isolates by highlighting the genomic divergence associated with gene decay, genome plasticity, and the acquisition of virulence and adaptive traits. The clinical isolates showed high phylogenetic relatedness due to a higher frequency of shared orthologs and fewer gene variants in contrast to the evolutionarily divergent environmental strains. Divergence of the environmental isolates is linked to extensive genomic rearrangements in regions containing mobile genetic elements resulting in numerous breakpoints, relocations, and insertions coupled with the loss of virulence determinants acf, zot, tcp, and ctx in the genomic islands. Also, four isolates possessed the CRISPR-Cas systems with spacers specific for Vibrio phages and plasmids. Genome synteny and homology analysis of the CRISPR-Cas systems suggest horizontal acquisition. The marked differences in the distribution of other phage and plasmid defense systems such as Zorya, DdmABC, DdmDE, and type-I Restriction Modification systems among the isolates indicated a higher propensity for plasmid or phage disseminated traits in the environmental isolates. Our results reveal that V. cholerae strains undergo extensive genomic rearrangements coupled with gene acquisition, reflecting their adaptation during ecological shifts and pathogenicity.},
}

@article {pmid36302757,
year = {2022},
author = {Li, M and Zhong, A and Wu, Y and Sidharta, M and Beaury, M and Zhao, X and Studer, L and Zhou, T},
title = {Transient inhibition of p53 enhances prime editing and cytosine base-editing efficiencies in human pluripotent stem cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6354},
pmid = {36302757},
issn = {2041-1723},
support = {P30CA008748//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; Tumor Suppressor Protein p53/genetics ; Cytosine ; Gene Editing/methods ; *Pluripotent Stem Cells/physiology ; *Induced Pluripotent Stem Cells ; CRISPR-Cas Systems ; },
abstract = {Precise gene editing in human pluripotent stem cells (hPSCs) holds great promise for studying and potentially treating human diseases. Both prime editing and base editing avoid introducing double strand breaks, but low editing efficiencies make those techniques still an arduous process in hPSCs. Here we report that co-delivering of p53DD, a dominant negative fragment of p53, can greatly enhance prime editing and cytosine base editing efficiencies in generating precise mutations in hPSCs. We further apply PE3 in combination with p53DD to efficiently create multiple isogenic hPSC lines, including lines carrying GBA or LRRK2 mutations associated with Parkinson disease and a LMNA mutation linked to Hutchinson-Gilford progeria syndrome. We also correct GBA and LMNA mutations in the patient-specific iPSCs. Our data show that p53DD improves PE3 efficiency without compromising the genome-wide safety, making it feasible for safe and routine generation of isogenic hPSC lines for disease modeling.},
}

Humans
Tumor Suppressor Protein p53/genetics
Cytosine
Gene Editing/methods
*Pluripotent Stem Cells/physiology
*Induced Pluripotent Stem Cells
CRISPR-Cas Systems

@article {pmid36301754,
year = {2022},
author = {Guo, JZ and Su, J and Dai, H and Wang, XY and Wu, WB and Chen, T and Zhang, J and Wang, WH},
title = {Establishment of a mouse model of Netherton syndrome based on CRISPR/Cas9 technology.},
journal = {European journal of dermatology : EJD},
volume = {32},
number = {4},
pages = {459-463},
doi = {10.1684/ejd.2022.4287},
pmid = {36301754},
issn = {1952-4013},
mesh = {Humans ; Mice ; Animals ; *Netherton Syndrome/genetics/pathology ; CRISPR-Cas Systems ; Disease Models, Animal ; Proteinase Inhibitory Proteins, Secretory/genetics ; Mice, Inbred C57BL ; Serine Peptidase Inhibitor Kazal-Type 5/genetics ; Mice, Knockout ; *Dermatitis/genetics ; Technology ; },
abstract = {Background: Netherton syndrome is a rare but severe autosomal recessive disorder with dominant impaired skin barrier function, caused by mutations in the SPINK5 (serine protease inhibitor Kazal-type 5) gene, which encodes LEKTI (lymphoepithelial Kazal-type-related inhibitor).

Objectives: To establish a murine model of Netherton syndrome based on CRISPR/Cas9 gene editing technology.

Materials & Methods: Spink5-sgRNA was designed to target exon 3 of the mouse Spink5 gene. Cas9 mRNA and sgRNA were microinjected into the zygotes of C57BL/6J mice. Spink5 homozygous knockout mice were born from a heterozygous intercross, and the phenotype of these mice was compared with wild-type regarding gross morphology, histopathology and immunofluorescent detection of LEKTI.

Results: Following microinjection of zygotes using the CRISPR/Cas9 system, sequencing demonstrated a 22-bp deletion at exon 3 of the mouse Spink5 gene. Histological investigation revealed complete detachment of the stratum corneum from the underlying granular layer and an absence of LEKTI in skin from Spink5 homozygous knockout mice.

Conclusion: The 22-bp deleted Spink5 transgenic mouse model demonstrates the clinical phenotype and genotype of human Netherton syndrome, representing a useful tool for future gene correction and skin barrier/inflammation studies.},
}

Humans
Mice
Animals
*Netherton Syndrome/genetics/pathology
CRISPR-Cas Systems
Disease Models, Animal
Proteinase Inhibitory Proteins, Secretory/genetics
Mice, Inbred C57BL
Serine Peptidase Inhibitor Kazal-Type 5/genetics
Mice, Knockout
*Dermatitis/genetics
Technology

@article {pmid36301479,
year = {2023},
author = {Tiwari, K and Kumar, R and Saudagar, P},
title = {Design of SaCas9-HF for In Vivo Gene Therapy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2575},
number = {},
pages = {261-268},
pmid = {36301479},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Staphylococcus aureus/metabolism ; Gene Editing/methods ; Genetic Therapy ; Endonucleases/genetics ; },
abstract = {Genome alteration results in several diseases for which therapeutics are limited. Gene editing provides a strong and potential alternative for the treatment of rare and genetic diseases. CRISPR-Cas9-based system is now being envisaged as a potential tool for the cure of genetic diseases. The RNA-guided nuclease, SaCas9 enzyme, along with its HF versions is widely employed for in vivo gene editing because of its small size and high efficiency. The current work summarizes the widely used and improved methods for in vivo manipulation of genes. The potential of CRISPR-Cas9-based systems can be harnessed to treat genetic diseases and holds great promise for therapeutic interventions in gene therapy. The in vivo gene editing poses a caveat in the form of delivery systems, the tissue in question, and several other factors. This work describes the methods which have been optimized to offer high efficiency, delivery, and gene editing in vivo.},
}

*CRISPR-Cas Systems/genetics
*Staphylococcus aureus/metabolism
Gene Editing/methods
Genetic Therapy
Endonucleases/genetics

@article {pmid36301478,
year = {2023},
author = {Kumar, R and Tiwari, K and Saudagar, P},
title = {Simplified CRISPR-Mediated DNA Editing in Multicellular Eukaryotes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2575},
number = {},
pages = {241-260},
pmid = {36301478},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Eukaryota/genetics ; RNA, Guide/genetics/metabolism ; Gene Editing/methods ; DNA/genetics ; },
abstract = {The CRISPR-Cas9 system is becoming an imperative tool to edit the genome of various organisms. The gene-editing study by the CRISPR-Cas9 system has revolutionized the diverse field of biomedical research, genome engineering, and gene therapy. CRISPR-Cas9 system has been modified to induce genome editing by small-guide RNAs, which function together with Cas9 nuclease for sequence-specific cleavage of target sequences. Here, we describe the simplified protocol of CRISPR-Cas9-mediated DNA editing in multicellular eukaryotes, including the construction of gRNA plasmids into vectors, screening of positive clones, transfections into 293FT cell line, and transduction into Jurkat cells. We also describe different bioinformatic tools to design suitable gRNAs with increased efficiency and decreased off-target events. Further, we describe the assessments of DNA editing by indel mutations and sequencing in transduced cells.},
}

Humans
*CRISPR-Cas Systems/genetics
*Eukaryota/genetics
RNA, Guide/genetics/metabolism
Gene Editing/methods
DNA/genetics

@article {pmid36301469,
year = {2023},
author = {Pereira, GC},
title = {Latest Trends in Nucleic Acids' Engineering Techniques Applied to Precision Medicine.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2575},
number = {},
pages = {25-38},
pmid = {36301469},
issn = {1940-6029},
mesh = {Humans ; *Nucleic Acids ; Precision Medicine ; *COVID-19 ; Genetic Engineering/methods ; DNA ; CRISPR-Cas Systems ; },
abstract = {Nucleic acids are paving the way for advanced therapeutics. Unveiling the genome enabled a better understanding of unique genotype-phenotype profiling. Methods for engineering and analysis of nucleic acids, from polymerase chain reaction to Cre-Lox recombination, are contributing greatly to biomarkers' discovery, mapping of cellular signaling cascades, and smart design of therapeutics in precision medicine. Investigating the different subtypes of DNA and RNA via sequencing and profiling is empowering the scientific community with valuable information, to be used in advanced therapeutics, tracking epigenetics linked to disease. Recent results from the application of nucleic acids in novel therapeutics and precision medicine are very encouraging, demonstrating great potential to treat cancer, viral infections via inoculation (e.g., SAR-COV-2 mRNA vaccines), along with metabolic and genetic disorders. Limitations posed by challenges in delivery mode are being addressed to enable efficient guided-gene-programmed precision therapies. With the focus on genetic engineering and novel therapeutics, more precisely, in precision medicine, this chapter discusses the advance enabled by knowledge derived from these innovative branches of biotechnology.},
}

Humans
*Nucleic Acids
Precision Medicine
*COVID-19
Genetic Engineering/methods
DNA
CRISPR-Cas Systems

@article {pmid36219232,
year = {2022},
author = {Liu, M and Ma, W and Zhou, Y and Liu, B and Zhang, X and Zhang, S},
title = {A Label-Free Photoelectrochemical Biosensor Based on CRISPR/Cas12a System Responsive Deoxyribonucleic Acid Hydrogel and "Click" Chemistry.},
journal = {ACS sensors},
volume = {7},
number = {10},
pages = {3153-3160},
doi = {10.1021/acssensors.2c01636},
pmid = {36219232},
issn = {2379-3694},
mesh = {*Cadmium Compounds ; Hydrogels ; CRISPR-Cas Systems ; *Biosensing Techniques ; DNA/genetics ; },
abstract = {A novel label-free photoelectrochemical (PEC) biosensor is presented in this work. As a barrier, the DNA hydrogel could block the coupling between g-C3N4 and CdS quantum dots (QDs). Therefore, extremely low photocurrent signals were obtained. The presence of target microRNA-21 can initiate the rolling circle amplification (RCA) reaction, which in turn produces many repeated sequences to activate the CRISPR/Cas12a system. The trans-cleavage activity of the CRISPR/Cas12a system led to the degradation of DNA hydrogels efficiently. As a result, the g-C3N4/CdS QDs heterojunction was formed through "click" chemistry. Through the amplification of the RCA and CRISPR/Cas12a system, the sensitivity of the PEC biosensor was improved significantly with the detection limit of 3.2 aM. The proposed sensor also showed excellent selectivity and could be used to detect actual samples. In addition, the modular design could facilitate the detection of different objects. Thus, the proposed CRISPR/Cas12a system responsive DNA hydrogel provides a simple, sensitive, and flexible way for label-free PEC analysis.},
}

*Cadmium Compounds
Hydrogels
CRISPR-Cas Systems
*Biosensing Techniques
DNA/genetics

@article {pmid36206316,
year = {2022},
author = {Gao, H and Feng, M and Li, F and Zhang, K and Zhang, T and Zhang, Z and Yang, C and Deng, R and Zhang, J and Jiang, P},
title = {G-Quadruplex DNAzyme-Substrated CRISPR/Cas12 Assay for Label-Free Detection of Single-Celled Parasitic Infection.},
journal = {ACS sensors},
volume = {7},
number = {10},
pages = {2968-2977},
doi = {10.1021/acssensors.2c01104},
pmid = {36206316},
issn = {2379-3694},
mesh = {Mice ; Animals ; *DNA, Catalytic/genetics ; CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *G-Quadruplexes ; *Parasitic Diseases/genetics ; },
abstract = {Early diagnosis of parasitic diseases can dramatically alleviate medical, economic, and social burdens. Herein, we report a sensitive and label-free assay for diagnosing single-celled parasitic infections using G-quadruplex (G4) DNAzyme as a reporter for CRISPR/Cas12. The substitution of a fluorescent DNA reporter with G4 DNAzyme increased the sensitivity for detecting Leishmania donovani (L. donovani) by 5 times and obviated the need for using chemically labeled DNA probes. The G4 DNAzyme-substrated CRISPR/Cas12 (GsubCas12) assay yielded a limit of detection of 3.1 parasites in the detection of cultured L. donovani and was further applied to analyze L. donovani in infected mice. The results showed that the GsubCas12 assay could positively detect L. donovani in spleen samples from infected mice about 2 weeks after low-dose inoculation, nearly 2 weeks earlier than that of parasitological analysis. GsubCas12 assay is promising as a diagnostic tool for parasitic infection in resource-limited regions.},
}

Mice
Animals
*DNA, Catalytic/genetics
CRISPR-Cas Systems
*Biosensing Techniques/methods
*G-Quadruplexes
*Parasitic Diseases/genetics

@article {pmid35951752,
year = {2022},
author = {Wu, J and Liu, H and Ren, S and Li, P and Li, X and Lin, L and Sun, Q and Zhang, L and Lin, C and Wang, Y},
title = {Generating an oilseed rape mutant with non-abscising floral organs using CRISPR/Cas9 technology.},
journal = {Plant physiology},
volume = {190},
number = {3},
pages = {1562-1565},
doi = {10.1093/plphys/kiac364},
pmid = {35951752},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems/genetics ; *Brassica napus/genetics ; Technology ; },
}

*CRISPR-Cas Systems/genetics
*Brassica napus/genetics
Technology

@article {pmid36309609,
year = {2022},
author = {Das, N and Ghosh Dhar, D and Dhar, P},
title = {Editing the genome of common cereals (Rice and Wheat): techniques, applications, and industrial aspects.},
journal = {Molecular biology reports},
volume = {},
number = {},
pages = {},
pmid = {36309609},
issn = {1573-4978},
abstract = {Gene editing techniques have made a significant contribution to the development of better crops. Gene editing enables precise changes in the genome of crops, which can introduce new possibilities for altering the crops' traits. Since the last three decades, various gene editing techniques such as meganucleases, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspersed short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins) have been discovered. In this review, we discuss various gene editing techniques and their applications to common cereals. Further, we elucidate the future of gene-edited crops, their regulatory features, and industrial aspects globally. To achieve this, we perform a comprehensive literature survey using databases such as PubMed, Web of Science, SCOPUS, Google Scholar etc. For the literature search, we used keywords such as gene editing, crop genome modification, CRISPR/Cas, ZFN, TALEN, meganucleases etc. With the advent of the CRISPR/Cas technology in the last decade, the future of gene editing has transitioned into a new dimension. The functionality of CRISPR/Cas in both DNA and RNA has increased through the use of various Cas enzymes and their orthologs. Constant research efforts in this direction have improved the gene editing process for crops by minimizing its off-target effects. Scientists also use computational tools, which help them to design experiments and analyze the results of gene editing experiments in advance. Gene editing has diverse potential applications. In the future, gene editing will open new avenues for solving more agricultural issues and boosting crop production, which may have great industrial prospects.},
}

@article {pmid36309062,
year = {2022},
author = {Phulpoto, IA and Yu, Z and Qazi, MA and Ndayisenga, F and Yang, J},
title = {A comprehensive study on microbial-surfactants from bioproduction scale-up toward electrokinetics remediation of environmental pollutants: Challenges and perspectives.},
journal = {Chemosphere},
volume = {},
number = {},
pages = {136979},
doi = {10.1016/j.chemosphere.2022.136979},
pmid = {36309062},
issn = {1879-1298},
abstract = {Currently, researchers have focused on electrokinetic (EK) bioremediation due to its potential to remove a wide-range of pollutants. Further, to improve their performance, synthetic surfactants are employed as effective additives because of their excellent solubility and mobility. Synthetic surfactants have an excessive position in industries since they are well-established, cheap, and easily available. Nevertheless, these surfactants have adverse environmental effects and could be detrimental to aquatic and terrestrial life. Owing to social and environmental awareness, there is a rising demand for bio-based surfactants in the global market, from environmental sustainability to public health, because of their excellent surface and interfacial activity, higher and stable emulsifying property, biodegradability, non- or low toxicity, better selectivity and specificity at extreme environmental conditions. Unfortunately, challenges to biosurfactants, like expensive raw materials, low yields, and purification processes, hinder their applicability to large-scale. To date, extensive research has already been conducted for production scale-up using multidisciplinary approaches. However, it is still essential to research and develop high-yielding bacteria for bioproduction through traditional and biotechnological advances to reduce production costs. Herein, this review evaluates the recent progress made on microbial-surfactants for bioproduction scale-up and provides detailed information on traditional and advanced genetic engineering approaches for cost-effective bioproduction. Furthermore, this study emphasized the role of electrokinetic (EK) bioremediation and discussed the application of BioS-mediated EK for various pollutants remediation.},
}

@article {pmid36308835,
year = {2022},
author = {Ding, L and Wu, Y and Liu, LE and He, L and Yu, S and Effah, CY and Liu, X and Qu, L and Wu, Y},
title = {Universal DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a bioassay for the synchronous detection of two exosomal proteins and its application in intelligent diagnosis of cancer.},
journal = {Biosensors & bioelectronics},
volume = {219},
number = {},
pages = {114827},
doi = {10.1016/j.bios.2022.114827},
pmid = {36308835},
issn = {1873-4235},
abstract = {Exosomal proteins are considered to be promising indicators of cancer. Herein, a novel DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a strategy was proposed for the synchronous determination of exosomal proteins: serum amyloid A-1 protein (SAA1) and coagulation factor V (FV). In this design, the paired antibodies were used to recognize targets, thereby ensuring the specificity. DNAzyme walkers were employed to convert the contents of SAA1 and FV into activators (P1 and P2), and one target can produce abundant activators, thus achieving an initial amplification of signal. Furthermore, the P1 and P2 can activate CRISPR-Cas12a/Cas13a system, which in turn trans-cleaves the reporters, enabling a second amplification and generating two fluorescent signals. The assay is highly sensitive (limits of detection as low as 30.00 pg/mL for SAA1 and 200.00 pg/mL for FV), highly specific and ideally accurate. More importantly, it is universal and can be used to detect both non-membrane and membrane proteins in exosome. Besides, the method can be successfully applied to detect SAA1 and FV in plasma exosomes to differentiate between lung cancer patients and healthy individuals. To explore the application of the developed method in tumor diagnosis, a deep learning model based on the expressions of SAA1 and FV was developed. The accuracy of this model can achieve 86.96%, which proves that it has a promising practical application capacity. Thus, this study does not only provide a new tool for the detection of exosomal proteins and cancer diagnosis, but also propose a new strategy to detect non-nucleic acid analytes for CRISPR-Cas system.},
}

@article {pmid36308834,
year = {2022},
author = {Duan, M and Li, B and Zhao, Y and Liu, Y and Liu, Y and Dai, R and Li, X and Jia, F},
title = {A CRISPR/Cas12a-mediated, DNA extraction and amplification-free, highly direct and rapid biosensor for Salmonella Typhimurium.},
journal = {Biosensors & bioelectronics},
volume = {219},
number = {},
pages = {114823},
doi = {10.1016/j.bios.2022.114823},
pmid = {36308834},
issn = {1873-4235},
abstract = {CRISPR/Cas-based biosensors were typically used for nucleic-acid targets detection and complex DNA extraction and amplification procedures were usually inevitable. Here, we report a CRISPR/Cas12a-mediated, DNA extraction and amplification-free, highly direct and rapid biosensor (abbreviated as "CATCHER") for Salmonella Typhimurium (S. Typhimurium) with a simple (3 steps) and fast (∼2 h) sensing workflow. Magnetic nanoparticle immobilized anti-S. Typhimurium antibody was worked as capture probe to capture the target and provide movable reaction interface. Colloidal gold labeled with anti-S. Typhimurium antibody and DNase I was used as detection probe to bridge the input target and output signal. First, in the presence of S. Typhimurium, an immuno-sandwich structure was formed. Second, DNase I in sandwich structure degraded the valid, complete activator DNA to invalid DNA fragments which can't trigger the trans-cleavage activity of Cas12a. Finally, the integrity of reporter DNA was preserved presenting a low fluorescence signal. Conversely, in the absence of S. Typhimurium, strong fluorescence recovery appeared owing to the cutting of reporter by activated Cas12a. Significantly, the proposed "CATCHER" showed satisfactory detection performance for S. Typhimurium with the limit of detection (LOD) of 7.9 × 101 CFU/mL in 0.01 M PBS and 6.31 × 103 CFU/mL in spiked chicken samples, providing a general platform for non-nucleic acid targets.},
}

@article {pmid36307966,
year = {2022},
author = {Belshaw, N and Grouneva, I and Aram, L and Gal, A and Hopes, A and Mock, T},
title = {Efficient gene replacement by CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.18587},
pmid = {36307966},
issn = {1469-8137},
abstract = {CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR in a diploid photosynthetic organism. HR was induced in T. pseudonana using sequence specific CRISPR/Cas, paired with a dsDNA donor matrix, generating substitution of the silacidin, nitrate reductase and urease genes by a resistance cassette (FCP:NAT). Up to approximately 85% of NAT resistant T. pseudonana colonies screened positive for HR by nested PCR. Precise integration of FCP:NAT at each locus was confirmed using an inverse PCR approach. The knockout of the nitrate reductase and urease genes impacted growth on nitrate and urea, respectively, while the knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and biotechnological applications targeted on harnessing the metabolic potential of diatoms.},
}

@article {pmid36306795,
year = {2022},
author = {Liu, X and Zhang, L and Wang, H and Xiu, Y and Huang, L and Gao, Z and Li, N and Li, F and Xiong, W and Gao, T and Zhang, Y and Yang, M and Feng, Y},
title = {Target RNA activates the protease activity of Craspase to confer antiviral defense.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2022.10.007},
pmid = {36306795},
issn = {1097-4164},
abstract = {In the type III-E CRISPR-Cas system, a Cas effector (gRAMP) is associated with a TPR-CHAT to form Craspase (CRISPR-guided caspase). However, both the structural features of gRAMP and the immunity mechanism remain unknown for this system. Here, we report structures of gRAMP-crRNA and gRAMP:cRNA:target RNA as well as structures of Craspase and Craspase complexed with cognate target RNA (CTR) or non-cognate target RNA (NTR). Importantly, the 3' anti-tag region of NTR and CTR binds at two distinct channels in Craspase, and CTR with a non-complementary 3' anti-tag induces a marked conformational change of the TPR-CHAT, which allosterically activates its protease activity to cleave an ancillary protein Csx30. This cleavage then triggers an abortive infection as the antiviral strategy of the type III-E system. Together, our study provides crucial insights into both the catalytic mechanism of the gRAMP and the immunity mechanism of the type III-E system.},
}

@article {pmid36306733,
year = {2022},
author = {Pacesa, M and Lin, CH and Cléry, A and Saha, A and Arantes, PR and Bargsten, K and Irby, MJ and Allain, FH and Palermo, G and Cameron, P and Donohoue, PD and Jinek, M},
title = {Structural basis for Cas9 off-target activity.},
journal = {Cell},
volume = {185},
number = {22},
pages = {4067-4081.e21},
doi = {10.1016/j.cell.2022.09.026},
pmid = {36306733},
issn = {1097-4172},
abstract = {The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.},
}

@article {pmid36306216,
year = {2022},
author = {Das, A and Doss, K and Mandal, J},
title = {CRISPR-cas heterogeneity and plasmid incompatibility types in relation to virulence determinants of Shigella.},
journal = {Journal of medical microbiology},
volume = {71},
number = {10},
pages = {},
doi = {10.1099/jmm.0.001607},
pmid = {36306216},
issn = {1473-5644},
abstract = {Introduction. Virulence factors (VFs) are the most potent weapon in the molecular armoury of Shigella. In bacteria, the mobile genetic elements (MGEs) are contributors to the evolution of different types of clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-cas) variants and plasmid incompatibility types. The present study explored the virulence potential of Shigella in relation to the CRISPR-cas pattern and incompatibility types among the isolates.Hypothesis/Gap Statement. The profile of the CRISPR-cas systems among clinical isolates of Shigella in India has not been reported earlier. Limited knowledge is available on the pattern of plasmid incompatibility groups among clinical isolates Shigella. The bias is always towards studying the genetic elements associated with AMR, but the present study highlights CRISPR-cas and incompatibility types among Shigella in association with virulence.Aim. We aimed to investigate the distribution of virulence factors, CRISPR-cas pattern followed by plasmid incompatibility types among Shigella isolates.Methodology. Between 2012-2017, a total of 187 isolates of Shigella were included in the study. The virulence genes' distribution was carried out. CRISPR-cas profiling followed by analysis of the repeats and spacers was carried out. PCR-based replicon typing was used to determine the incompatibility types. The interplay was statistically determined using STATA.Results. The distribution of virulence genes showed varied pattern with ipaH present in all the isolates followed by ompA (93.6 %), virF (66.8 %), ial and sen (60.4 %), set1A (39.6 %) and set1B (39 %). CRISPR 1, CRISPR 3 and Cas6-Cas5 region were dominantly conserved. Twenty-two types of spacers were identified. The CRISPR3 repeat appeared to have a highly conserved sequence. CRISPR2 being the least common CRISPR type showed a strong association with an array of virulence genes (ial-set1A-set1B-virF) while CRISPR1 being the most dominant showed the least association with virulence genes (sen-virF). The dominant plasmids were found to be belonging to the inc FII group. The incompatibility groups FII, IncIγ, U, FIIS, FIIK, K, A/C, I1alpha was found to be associated with a greater number of virulence genes.Conclusion. The isolates showed increasing diversity in their gene content that contributes to increasing heterogeneity among the isolates, which is a known virulence strategy among pathogens.},
}

@article {pmid36305010,
year = {2020},
author = {Gürel, F and Zhang, Y and Sretenovic, S and Qi, Y},
title = {CRISPR-Cas nucleases and base editors for plant genome editing.},
journal = {aBIOTECH},
volume = {1},
number = {1},
pages = {74-87},
pmid = {36305010},
issn = {2662-1738},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) and base editors are fundamental tools in plant genome editing. Cas9 from Streptococcus pyogenes (SpCas9), recognizing an NGG protospacer adjacent motif (PAM), is a widely used nuclease for genome editing in living cells. Cas12a nucleases, targeting T-rich PAMs, have also been recently demonstrated in several plant species. Furthermore, multiple Cas9 and Cas12a engineered variants and orthologs, with different PAM recognition sites, editing efficiencies and fidelity, have been explored in plants. These RNA-guided sequence-specific nucleases (SSN) generate double-stranded breaks (DSBs) in DNA, which trigger non-homologous end-joining (NHEJ) repair or homology-directed repair (HDR), resulting in insertion and deletion (indel) mutations or precise gene replacement, respectively. Alternatively, genome editing can be achieved by base editors without introducing DSBs. So far, several base editors have been applied in plants to introduce C-to-T or A-to-G transitions, but they are still undergoing improvement in editing window size, targeting scope, off-target effects in DNA and RNA, product purity and overall activity. Here, we summarize recent progress on the application of Cas nucleases, engineered Cas variants and base editors in plants.},
}

@article {pmid36305005,
year = {2020},
author = {Li, S and Xia, L},
title = {Precise gene replacement in plants through CRISPR/Cas genome editing technology: current status and future perspectives.},
journal = {aBIOTECH},
volume = {1},
number = {1},
pages = {58-73},
pmid = {36305005},
issn = {2662-1738},
abstract = {CRISPR/Cas, as a simple, versatile, robust and cost-effective system for genome manipulation, has dominated the genome editing field over the past few years. The application of CRISPR/Cas in crop improvement is particularly important in the context of global climate change, as well as diverse agricultural, environmental and ecological challenges. Various CRISPR/Cas toolboxes have been developed and allow for targeted mutagenesis at specific genome loci, transcriptome regulation and epigenome editing, base editing, and precise targeted gene/allele replacement or tagging in plants. In particular, precise replacement of an existing allele with an elite allele in a commercial variety through homology-directed repair (HDR) is a holy grail in genome editing for crop improvement as it has been very difficult, laborious and time-consuming to introgress the elite alleles into commercial varieties without any linkage drag from parental lines within a few generations in crop breeding practice. However, it still remains very challenging in crop plants. This review intends to provide an informative summary of the latest development and breakthroughs in gene replacement using CRISPR/Cas technology, with a focus on achievements, potential mechanisms and future perspectives in plant biological science as well as crop improvement.},
}

@article {pmid36305004,
year = {2020},
author = {Char, SN and Yang, B},
title = {Genome editing in grass plants.},
journal = {aBIOTECH},
volume = {1},
number = {1},
pages = {41-57},
pmid = {36305004},
issn = {2662-1738},
abstract = {Cereal crops including maize, rice, wheat, sorghum, barley, millet, oats and rye are the major calorie sources in our daily life and also important bioenergy sources of the world. The rapidly advancing and state-of-the-art genome-editing tools such as zinc finger nucleases, TAL effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (CRISPR-Cas9-, CRISPR-Cas12a- and CRISPR/Cas-derived base editors) have accelerated the functional genomics and have promising potential for precision breeding of grass crops. With the availability of annotated genomes of the major cereal crops, application of these established genome-editing toolkits to grass plants holds promise to increase the nutritional value and productivity. Furthermore, these easy-to-use and robust genome-editing toolkits have advanced the reverse genetics for discovery of novel gene functions in crop plants. In this review, we document some of important progress in development and utilization of genome-editing tool sets in grass plants. We also highlight present and future uses of genome-editing toolkits that can sustain and improve the quality of cereal grain for food consumption.},
}

@article {pmid36305002,
year = {2020},
author = {Schmidt, C and Schindele, P and Puchta, H},
title = {From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas.},
journal = {aBIOTECH},
volume = {1},
number = {1},
pages = {21-31},
pmid = {36305002},
issn = {2662-1738},
abstract = {In the last years, tremendous progress has been achieved in the field of gene editing in plants. By the induction of single site-specific double-strand breaks (DSBs), the knockout of genes by non-homologous end joining has become routine in many plant species. Recently, the efficiency of inducing pre-planned mutations by homologous recombination has also been improved considerably. However, very little effort has been undertaken until now to achieve more complex changes in plant genomes by the simultaneous induction of several DSBs. Several reports have been published on the efficient induction of deletions. However, the induction of intrachromosomal inversions and interchromosomal recombination by the use of CRISPR/Cas has only recently been reported. In this review, we want to sum up these results and put them into context with regards to what is known about natural chromosome rearrangements in plants. Moreover, we review the recent progress in CRISPR/Cas-based mammalian chromosomal rearrangements, which might be inspiring for plant biologists. In the long run, the controlled restructuring of plant genomes should enable us to link or break linkage of traits at will, thus defining a new area of plant breeding.},
}

@article {pmid36304720,
year = {2020},
author = {Hsieh-Feng, V and Yang, Y},
title = {Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing.},
journal = {aBIOTECH},
volume = {1},
number = {2},
pages = {123-134},
pmid = {36304720},
issn = {2662-1738},
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system (CRISPR/Cas) has recently become the most powerful tool available for genome engineering in various organisms. With efficient and proper expression of multiple guide RNAs (gRNAs), the CRISPR/Cas system is particularly suitable for multiplex genome editing. During the past several years, different CRISPR/Cas expression strategies, such as two-component transcriptional unit, single transcriptional unit, and bidirectional promoter systems, have been developed to efficiently express gRNAs as well as Cas nucleases. Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes, endogenous RNases, and exogenous endoribonuclease (Csy4). Besides being constitutively and ubiquitously expressed, inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible, tissue-specific, and/or synthetic promoters for specific research purposes. Most recently, the emergence of CRISPR/Cas ribonucleoprotein delivery methods, such as engineered nanoparticles, further revolutionized transgene-free and multiplex genome editing. In this review, we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.},
}

@article {pmid36304520,
year = {2022},
author = {Li, J and Yu, X and Zhang, C and Li, N and Zhao, J},
title = {The application of CRISPR/Cas technologies to Brassica crops: current progress and future perspectives.},
journal = {aBIOTECH},
volume = {3},
number = {2},
pages = {146-161},
pmid = {36304520},
issn = {2662-1738},
abstract = {Brassica species are a global source of nutrients and edible vegetable oil for humans. However, all commercially important Brassica crops underwent a whole-genome triplication event, hindering the development of functional genomics and breeding programs. Fortunately, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) technologies, by allowing multiplex and precise genome engineering, have become valuable genome-editing tools and opened up new avenues for biotechnology. Here, we review current progress in the use of CRISPR/Cas technologies with an emphasis on the latest breakthroughs in precise genome editing. We also summarize the application of CRISPR/Cas technologies to Brassica crops for trait improvements. Finally, we discuss the challenges and future directions of these technologies for comprehensive application in Brassica crops. Ongoing advancement in CRISPR/Cas technologies, in combination with other achievements, will play a significant role in the genetic improvement and molecular breeding of Brassica crops.},
}

@article {pmid36302881,
year = {2022},
author = {Yu, G and Wang, X and Zhang, Y and An, Q and Wen, Y and Li, X and Yin, H and Deng, Z and Zhang, H},
title = {Structure and function of a bacterial type III-E CRISPR-Cas7-11 complex.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {36302881},
issn = {2058-5276},
abstract = {The type III-E CRISPR-Cas system uses a single multidomain effector called Cas7-11 (also named gRAMP) to cleave RNA and associate with a caspase-like protease Csx29, showing promising potential for RNA-targeting applications. The structural and molecular mechanisms of the type III-E CRISPR-Cas system remain poorly understood. Here we report four cryo-electron microscopy structures of Cas7-11 at different functional states. Cas7-11 has four Cas7-like domains, which assemble into a helical filament to accommodate CRISPR RNA (crRNA), and a Cas11-like domain facilitating crRNA-target RNA duplex formation. The Cas7.1 domain is critical for crRNA maturation, whereas Cas7.2 and Cas7.3 are responsible for target RNA cleavage. Target RNA binding induces the structural arrangements of Csx29, potentially exposing the catalytic site of Csx29. These results delineate the molecular mechanisms underlying pre-crRNA processing, target RNA recognition and cleavage for Cas7-11, and provide a structural framework to understand the role of Csx29 in type III-E CRISPR system.},
}

@article {pmid36298718,
year = {2022},
author = {Kanojia, A and Sharma, M and Shiraz, R and Tripathi, S},
title = {Flavivirus-Host Interaction Landscape Visualized through Genome-Wide CRISPR Screens.},
journal = {Viruses},
volume = {14},
number = {10},
pages = {},
doi = {10.3390/v14102164},
pmid = {36298718},
issn = {1999-4915},
mesh = {Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Flavivirus/genetics ; CRISPR-Cas Systems ; Reproducibility of Results ; *Viruses/genetics ; Antiviral Agents/pharmacology ; },
abstract = {Flaviviruses comprise several important human pathogens which cause significant morbidity and mortality worldwide. Like any other virus, they are obligate intracellular parasites. Therefore, studying the host cellular factors that promote or restrict their replication and pathogenesis becomes vital. Since inhibiting the host dependency factors or activating the host restriction factors can suppress the viral replication and propagation in the cell, identifying them reveals potential targets for antiviral therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has provided an effective means of producing customizable genetic modifications and performing forward genetic screens in a broad spectrum of cell types and organisms. The ease, rapidity, and high reproducibility of CRISPR technology have made it an excellent tool for carrying out genome-wide screens to identify and characterize viral host dependency factors systematically. Here, we review the insights from various Genome-wide CRISPR screens that have advanced our understanding of Flavivirus-Host interactions.},
}

Humans
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Flavivirus/genetics
CRISPR-Cas Systems
Reproducibility of Results
*Viruses/genetics
Antiviral Agents/pharmacology

@article {pmid36293232,
year = {2022},
author = {Yee, T and Wert, KJ},
title = {Base and Prime Editing in the Retina-From Preclinical Research toward Human Clinical Trials.},
journal = {International journal of molecular sciences},
volume = {23},
number = {20},
pages = {},
doi = {10.3390/ijms232012375},
pmid = {36293232},
issn = {1422-0067},
support = {5T32GM131945/NH/NIH HHS/United States ; P30EY030413/EY/NEI NIH HHS/United States ; N/A//Hamon Center for Regenerative Science and Medicine Fellowship/ ; N/A//Van Sickle Family Foundation Award/ ; },
mesh = {Humans ; Aged ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/metabolism ; Gene Editing ; Retina/metabolism ; *Retinal Diseases/metabolism ; DNA/metabolism ; },
abstract = {Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of diseases that are one of the leading causes of vision loss in young and aged individuals. IRDs are mainly caused by a loss of the post-mitotic photoreceptor neurons of the retina, or by the degeneration of the retinal pigment epithelium. Unfortunately, once these cells are damaged, it is irreversible and leads to permanent vision impairment. Thought to be previously incurable, gene therapy has been rapidly evolving to be a potential treatment to prevent further degeneration of the retina and preserve visual function. The development of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) base and prime editors have increased the capabilities of the genome editing toolbox in recent years. Both base and prime editors evade the creation of double-stranded breaks in deoxyribonucleic acid (DNA) and the requirement of donor template of DNA for repair, which make them advantageous methods in developing clinical therapies. In addition, establishing a permanent edit within the genome could be better suited for patients with progressive degeneration. In this review, we will summarize published uses of successful base and prime editing in treating IRDs.},
}

Humans
Aged
*CRISPR-Cas Systems
CRISPR-Associated Protein 9/metabolism
Gene Editing
Retina/metabolism
*Retinal Diseases/metabolism
DNA/metabolism

@article {pmid36292795,
year = {2022},
author = {Wang, L and Gao, Y and Wang, J and Huang, N and Jiang, Q and Ju, Z and Yang, C and Wei, X and Xiao, Y and Zhang, Y and Yang, L and Huang, J},
title = {Selection Signature and CRISPR/Cas9-Mediated Gene Knockout Analyses Reveal ZC3H10 Involved in Cold Adaptation in Chinese Native Cattle.},
journal = {Genes},
volume = {13},
number = {10},
pages = {},
doi = {10.3390/genes13101910},
pmid = {36292795},
issn = {2073-4425},
support = {2021YFF1000703//National Key Research and Development Program of China/ ; 32102502//National Natural Science Foundation of China/ ; 2019LZGC011//Shandong Agricultural Elite Variety Project/ ; },
mesh = {Animals ; Cattle/genetics ; Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Thermogenesis ; Glucose ; Lipids ; },
abstract = {Cold stress is an important factor affecting cattle health, production performance, and reproductive efficiency. Understanding of the potential mechanism underlying genetic adaptation to local environments, particularly extreme cold environment, is limited. Here, by using FLK and hapFLK methods, we found that the Zinc finger CCCH-type containing 10 (ZC3H10) gene underwent positive selection in the Menggu, Fuzhou, Anxi, and Shigatse humped cattle breeds that are distributed in the cold areas of China. Furthermore, ZC3H10 expression significantly increased in bovine fetal fibroblast (BFF) cells at 28 °C for 4 h. ZC3H10 knockout BFFs were generated using CRISPR/Cas9. Wild and ZC3H10-deleted BFFs were treated at two temperatures and were divided into four groups (WT, wild and cultured at 38 °C; KO, ZC3H10-/- and 38 °C; WT_LT, wild, and 28 °C for 4 h; and KO_LT, ZC3H10-/- and 28 °C for 4 h. A total of 466, 598, 519, and 650 differently expressed genes (two-fold or more than two-fold changes) were identified by determining transcriptomic difference (KO_LT vs. KO, WT_LT vs. WT, KO vs. WT, and KO_LT vs. WT_LT, respectively). Loss of ZC3H10 dysregulated pathways involved in thermogenesis and immunity, and ZC3H10 participated in immunity-related pathways induced by cold stress and regulated genes involved in glucose and lipid metabolism and lipid transport (PLTP and APOA1), thereby facilitating adaptability to cold stress. Our findings provide a foundation for further studies on the function of ZC3H10 in cold stress and development of bovine breeding strategies for combatting the influences of cold climate.},
}

Animals
Cattle/genetics
Gene Knockout Techniques
*CRISPR-Cas Systems
*Thermogenesis
Glucose
Lipids

@article {pmid36292612,
year = {2022},
author = {Cosenza, LC and Zuccato, C and Zurlo, M and Gambari, R and Finotti, A},
title = {Co-Treatment of Erythroid Cells from β-Thalassemia Patients with CRISPR-Cas9-Based β039-Globin Gene Editing and Induction of Fetal Hemoglobin.},
journal = {Genes},
volume = {13},
number = {10},
pages = {},
doi = {10.3390/genes13101727},
pmid = {36292612},
issn = {2073-4425},
support = {208872/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; AIFA-2016-02364887//Italian Medicines Agency/ ; 306201-FP7-HEALTH-2012-INNOVATION-1//European Union/ ; FINOTTI 2021//FAR - Ferrara University/ ; FINOTTI 2018//FIR- Ferrara University/ ; },
mesh = {Adult ; Humans ; Fetal Hemoglobin/genetics/metabolism ; Gene Editing/methods ; *beta-Thalassemia/genetics/therapy ; CRISPR-Cas Systems/genetics ; Erythroid Cells/metabolism ; *Thalassemia/genetics ; Sirolimus ; },
abstract = {Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the β039-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with β-thalassemia. The aim of our study was to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols. The gene editing of the β039-globin mutation was obtained using a CRISPR-Cas9-based experimental strategy; the correction of the gene sequence and the transcription of the corrected gene were analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; the relative content of HbA and HbF was studied by high-performance liquid chromatography (HPLC) and Western blotting. For HbF induction, the repurposed drug rapamycin was used. The data obtained conclusively demonstrate that the maximal production of HbA and HbF is obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from β039-thalassemia patients. In conclusion, GE and HbF induction might be used in combination in order to achieve the de novo production of HbA together with an increase in induced HbF.},
}

Adult
Humans
Fetal Hemoglobin/genetics/metabolism
Gene Editing/methods
*beta-Thalassemia/genetics/therapy
CRISPR-Cas Systems/genetics
Erythroid Cells/metabolism
*Thalassemia/genetics
Sirolimus

@article {pmid36292566,
year = {2022},
author = {Shi, J and Ni, X and Huang, J and Fu, Y and Wang, T and Yu, H and Zhang, Y},
title = {CRISPR/Cas9-Mediated Gene Editing of BnFAD2 and BnFAE1 Modifies Fatty Acid Profiles in Brassica napus.},
journal = {Genes},
volume = {13},
number = {10},
pages = {},
doi = {10.3390/genes13101681},
pmid = {36292566},
issn = {2073-4425},
support = {grant no. 31972875//the National Natural Science Foundation of China/ ; grant no. 2022E10012//Key Laboratory of Digital Upland Crops of Zhejiang Province/ ; },
mesh = {*Brassica napus/genetics ; Gene Editing/methods ; Erucic Acids ; Fatty Acids/genetics ; Fatty Acid Elongases/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Fatty Acids, Unsaturated ; Oleic Acid ; Oxidoreductases/genetics ; },
abstract = {Fatty acid (FA) composition determines the quality of oil from oilseed crops, and thus is a major target for genetic improvement. FAD2 (Fatty acid dehydrogenase 2) and FAE1 (fatty acid elongase 1) are critical FA synthetic genes, and have been the focus of genetic manipulation to alter fatty acid composition in oilseed plants. In this study, to improve the nutritional quality of rapeseed cultivar CY2 (about 50% oil content; of which 40% erucic acid), we generated novel knockout plants by CRISPR/Cas9 mediated genome editing of BnFAD2 and BnFAE1 genes. Two guide RNAs were designed to target one copy of the BnFAD2 gene and two copies of the BnFAE1 gene, respectively. A number of lines with mutations at three target sites of BnFAD2 and BnFAE1 genes were identified by sequence analysis. Three of these lines showed mutations in all three target sites of the BnFAD2 and BnFAE1 genes. Fatty acid composition analysis of seeds revealed that mutations at all three sites resulted in significantly increased oleic acid (70-80%) content compared with that of CY2 (20%), greatly reduced erucic acid levels and slightly decreased polyunsaturated fatty acids content. Our results confirmed that the CRISPR/Cas9 system is an effective tool for improving this important trait.},
}

*Brassica napus/genetics
Gene Editing/methods
Erucic Acids
Fatty Acids/genetics
Fatty Acid Elongases/genetics
CRISPR-Cas Systems
Plants, Genetically Modified/genetics
Fatty Acids, Unsaturated
Oleic Acid
Oxidoreductases/genetics

@article {pmid36291103,
year = {2022},
author = {Karagyaur, M and Primak, A and Efimenko, A and Skryabina, M and Tkachuk, V},
title = {The Power of Gene Technologies: 1001 Ways to Create a Cell Model.},
journal = {Cells},
volume = {11},
number = {20},
pages = {},
doi = {10.3390/cells11203235},
pmid = {36291103},
issn = {2073-4409},
support = {19-75-30007//Russian Science Foundation/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genome ; Cloning, Molecular ; },
abstract = {Modern society faces many biomedical challenges that require urgent solutions. Two of the most important include the elucidation of mechanisms of socially significant diseases and the development of prospective drug treatments for these diseases. Experimental cell models are a convenient tool for addressing many of these problems. The power of cell models is further enhanced when combined with gene technologies, which allows the examination of even more subtle changes within the structure of the genome and permits testing of proteins in a native environment. The list and possibilities of these recently emerging technologies are truly colossal, which requires a rethink of a number of approaches for obtaining experimental cell models. In this review, we analyze the possibilities and limitations of promising gene technologies for obtaining cell models, and also give recommendations on the development and creation of relevant models. In our opinion, this review will be useful for novice cell biologists, as it provides some reference points in the rapidly growing universe of gene and cell technologies.},
}

*Gene Editing
*CRISPR-Cas Systems
Genome
Cloning, Molecular

@article {pmid36289480,
year = {2022},
author = {Zarate, OA and Yang, Y and Wang, X and Wang, JP},
title = {BoostMEC: predicting CRISPR-Cas9 cleavage efficiency through boosting models.},
journal = {BMC bioinformatics},
volume = {23},
number = {1},
pages = {446},
pmid = {36289480},
issn = {1471-2105},
mesh = {*RNA, Guide/genetics ; *CRISPR-Cas Systems ; Gene Editing ; Machine Learning ; },
abstract = {BACKGROUND: In the CRISPR-Cas9 system, the efficiency of genetic modifications has been found to vary depending on the single guide RNA (sgRNA) used. A variety of sgRNA properties have been found to be predictive of CRISPR cleavage efficiency, including the position-specific sequence composition of sgRNAs, global sgRNA sequence properties, and thermodynamic features. While prevalent existing deep learning-based approaches provide competitive prediction accuracy, a more interpretable model is desirable to help understand how different features may contribute to CRISPR-Cas9 cleavage efficiency.

RESULTS: We propose a gradient boosting approach, utilizing LightGBM to develop an integrated tool, BoostMEC (Boosting Model for Efficient CRISPR), for the prediction of wild-type CRISPR-Cas9 editing efficiency. We benchmark BoostMEC against 10 popular models on 13 external datasets and show its competitive performance.

CONCLUSIONS: BoostMEC can provide state-of-the-art predictions of CRISPR-Cas9 cleavage efficiency for sgRNA design and selection. Relying on direct and derived sequence features of sgRNA sequences and based on conventional machine learning, BoostMEC maintains an advantage over other state-of-the-art CRISPR efficiency prediction models that are based on deep learning through its ability to produce more interpretable feature insights and predictions.},
}

*RNA, Guide/genetics
*CRISPR-Cas Systems
Gene Editing
Machine Learning

@article {pmid36241739,
year = {2022},
author = {Varshney, P and Varshney, GK},
title = {Expanded precision genome-editing toolbox for human disease modeling in zebrafish.},
journal = {Lab animal},
volume = {51},
number = {11},
pages = {287-289},
pmid = {36241739},
issn = {1548-4475},
mesh = {Animals ; Humans ; *Zebrafish/genetics ; *Gene Editing ; CRISPR-Cas Systems/genetics ; },
}

Animals
Humans
*Zebrafish/genetics
*Gene Editing
CRISPR-Cas Systems/genetics

@article {pmid35976141,
year = {2022},
author = {Sprink, T and Wilhelm, R and Hartung, F},
title = {Genome editing around the globe: An update on policies and perceptions.},
journal = {Plant physiology},
volume = {190},
number = {3},
pages = {1579-1587},
doi = {10.1093/plphys/kiac359},
pmid = {35976141},
issn = {1532-2548},
abstract = {A decade ago, the CRISPR/Cas system has been adapted for genome editing. Since then, hundreds of organisms have been altered using genome editing and discussions were raised on the regulatory status of genome edited organisms esp. crops. To date, many countries have made decisions on the regulatory status of products of genome editing, by exempting some kinds of edits from the classical GMO regulation. However, the guidance differs between countries even in the same region. Several countries are still debating the issue or are in the progress of updating guidance and regulatory systems to cover products of genome editing. The current global situation of different regulatory systems is putting a harmonized framework on genome-edited crops in the far future. In this update, we summarize the current developments in the field of regulation concerning edited crops and present a short insight into perception of genome editing in the society.},
}

@article {pmid36301532,
year = {2022},
author = {Matsuo, M and Matsuyama, M and Kobayashi, T and Kanda, S and Ansai, S and Kawakami, T and Hosokawa, E and Daido, Y and Kusakabe, TG and Naruse, K and Fukamachi, S},
title = {Retinal Cone Mosaic in sws1-Mutant Medaka (Oryzias latipes), A Teleost.},
journal = {Investigative ophthalmology & visual science},
volume = {63},
number = {11},
pages = {21},
doi = {10.1167/iovs.63.11.21},
pmid = {36301532},
issn = {1552-5783},
abstract = {Purpose: Ablation of short single cones (SSCs) expressing short-wavelength-sensitive opsin (SWS1) is well analyzed in the field of regenerative retinal cells. In contrast with ablation studies, the phenomena caused by the complete deletion of SWS1 are less well-understood. To assess the effects of SWS1 deficiency on retinal structure, we established and analyzed sws1-mutant medaka.

Methods: To visualize SWS1, a monoclonal anti-SWS1 antibody and transgenic reporter fish (Tg(sws1:mem-egfp)) were generated. We also developed a CRISPR/Cas-driven sws1-mutant line. Retinal structure of sws1 mutant was visualized using anti-SWS1, 1D4, and ZPR1 antibodies and coumarin derivatives and compared with wild type, Tg(sws1:mem-egfp), and another opsin (lws) mutant.

Results: Our rat monoclonal antibody specifically recognized medaka SWS1. Sws1 mutant retained regularly arranged cone mosaic as lws mutant and its SSCs had neither SWS1 nor long wavelength sensitive opsin. Depletion of sws1 did not affect the expression of long wavelength sensitive opsin, and vice versa. ZPR1 antibody recognized arrestin spread throughout double cones and long single cones in wild-type, transgenic, and sws1-mutant lines.

Conclusions: Comparative observation of sws1-mutant and wild-type retinas revealed that ZPR1 negativity is not a marker for SSCs with SWS1, but SSCs themselves. Loss of functional sws1 did not cause retinal degeneration, indicating that sws1 is not essential for cone mosaic development in medaka. Our two fish lines, one with visualized SWS1 and the other lacking functional SWS1, offer an opportunity to study neural network synapsing with SSCs and to clarify the role of SWS1 in vision.},
}

@article {pmid36301087,
year = {2022},
author = {Wu, L and Wang, X and Wu, X and Xu, S and Liu, M and Cao, X and Tang, T and Huang, X and Huang, H},
title = {MnO2 Nanozyme-Mediated CRISPR-Cas12a System for the Detection of SARS-CoV-2.},
journal = {ACS applied materials & interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.2c14497},
pmid = {36301087},
issn = {1944-8252},
abstract = {The CRISPR-Cas system was developed into a molecular diagnostic tool with high sensitivity, low cost, and high specificity in recent years. Colorimetric assays based on nanozymes offer an attractive point-of-care testing method for their low cost of use and user-friendly operation. Here, a MnO2 nanozyme-mediated CRISPR-Cas12a system was instituted to detect SARS-CoV-2. MnO2 nanorods linked to magnetic beads via a single-stranded DNA (ssDNA) linker used as an oxidase-like nanozyme inducing the color change of 3,3',5,5'-tetramethylbenzidine, which can be distinguished by the naked eye. The detection buffer color will change when the Cas12a is activated by SARS-CoV-2 and indiscriminately cleave the linker ssDNA. The detection limit was 10 copies per microliter and showed no cross-reaction with other coronaviruses. The nanozyme-mediated CRISPR-Cas12a system shows high selectivity and facile operation, with great potential for molecular diagnosis in point-of-care testing applications.},
}

@article {pmid36298520,
year = {2022},
author = {Rabaan, AA and Mutair, AA and Hajissa, K and Alfaraj, AH and Al-Jishi, JM and Alhajri, M and Alwarthan, S and Alsuliman, SA and Al-Najjar, AH and Al Zaydani, IA and Al-Absi, GH and Alshaikh, SA and Alkathlan, MS and Almuthree, SA and Alawfi, A and Alshengeti, A and Almubarak, FZ and Qashgari, MS and Abdalla, ANK and Alhumaid, S},
title = {A Comprehensive Review on the Current Vaccines and Their Efficacies to Combat SARS-CoV-2 Variants.},
journal = {Vaccines},
volume = {10},
number = {10},
pages = {},
doi = {10.3390/vaccines10101655},
pmid = {36298520},
issn = {2076-393X},
abstract = {Since the first case of Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, SARS-CoV-2 infection has affected many individuals worldwide. Eventually, some highly infectious mutants-caused by frequent genetic recombination-have been reported for SARS-CoV-2 that can potentially escape from the immune responses and induce long-term immunity, linked with a high mortality rate. In addition, several reports stated that vaccines designed for the SARS-CoV-2 wild-type variant have mixed responses against the variants of concern (VOCs) and variants of interest (VOIs) in the human population. These results advocate the designing and development of a panvaccine with the potential to neutralize all the possible emerging variants of SARS-CoV-2. In this context, recent discoveries suggest the design of SARS-CoV-2 panvaccines using nanotechnology, siRNA, antibodies or CRISPR-Cas platforms. Thereof, the present comprehensive review summarizes the current vaccine design approaches against SARS-CoV-2 infection, the role of genetic mutations in the emergence of new viral variants, the efficacy of existing vaccines in limiting the infection of emerging SARS-CoV-2 variants, and efforts or challenges in designing SARS panvaccines.},
}