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The Expanding Dissemination and Distribution Patterns of Diverse CRISPR Plasmids by Addgene. CRISPR J. (IF 3.7) Pub Date : 2023-11-22 Brook Pyhtila,Seth Kasowitz,Rachel Leeson,Rodolphe Barrangou
CRISPR-based technologies have rapidly enabled the democratization of genome editing in academic institutions through distribution by Addgene over the past decade. Recently, several distribution milestones have been reached, with a collection of >15,000 plasmids deposited by >1,000 laboratories spanning ∼40 countries now shipped 300,000 times to ∼5,000 organizations traversing ∼100 countries. Yet,
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The CRISPR Toolbox: The End of the Beginning. CRISPR J. (IF 3.7) Pub Date : 2023-10-01 Rodolphe Barrangou
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Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE): An Experimental and Computational Approach for Analyzing CRISPR-Edited Cells. CRISPR J. (IF 3.7) Pub Date : 2023-10-01 Peng Zhang,Laurent Abel,Jean-Laurent Casanova,Rui Yang
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) creates double-stranded breaks, the repair of which generates indels around the target sites. These repairs can be mono-/multi-allelic, and the editing is often random and sometimes prolonged, resulting in considerable intercellular heterogeneity. The genotyping of CRISPR-Cas9-edited cells is challenging
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Advances in Cas12a-Based Amplification-Free Nucleic Acid Detection. CRISPR J. (IF 3.7) Pub Date : 2023-09-25 Shixin Ji,Xueli Wang,Yangkun Wang,Yingqi Sun,Yingying Su,Xiaosong Lv,Xiangwei Song
In biomedicine, rapid and sensitive nucleic acid detection technology plays an important role in the early detection of infectious diseases. However, most traditional nucleic acid detection methods require the amplification of nucleic acids, resulting in problems such as long detection time, complex operation, and false-positive results. In recent years, clustered regularly interspaced short palindromic
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Enhancing Precision and Efficiency of Cas9-Mediated Knockin Through Combinatorial Fusions of DNA Repair Proteins. CRISPR J. (IF 3.7) Pub Date : 2023-09-15 Ryan R Richardson,Marilyn Steyert,Saovleak N Khim,Garrett W Crutcher,Cheryl Brandenburg,Colin D Robertson,Andrea J Romanowski,Jeffrey Inen,Bekir Altas,Alexandros Poulopoulos
Cas9 targets genomic loci with high specificity. For knockin with double-strand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. In this study, we demonstrate a high-throughput workflow to comparatively assess on-target efficiency and precision of editing
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Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells. CRISPR J. (IF 3.7) Pub Date : 2023-09-07 Kaivalya Molugu,Namita Khajanchi,Cicera R Lazzarotto,Shengdar Q Tsai,Krishanu Saha
Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited iPSC clones. We developed a strategy to improve the
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APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels. CRISPR J. (IF 3.7) Pub Date : 2023-09-06 Amanda E Rieffer,Yanjun Chen,Daniel J Salamango,Sofia N Moraes,Reuben S Harris
Precision genome editing has become a reality with the discovery of base editors. Cytosine base editor (CBE) technologies are improving rapidly but are mostly optimized for TC dinucleotide targets. Here, we report the development and implementation of APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels (ARSENEL) in living cells. The ARSENEL panel is comprised of four constructs that
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CRISPR Milestones for Sustainable Agriculture and Forestry. CRISPR J. (IF 3.7) Pub Date : 2023-08-01 Rodolphe Barrangou
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Fanzors: Mysterious TnpB-Like Bacterial Transposon-Related RNA-Guided DNA Nucleases of Eukaryotes. CRISPR J. (IF 3.7) Pub Date : 2023-08-01 Tautvydas Karvelis,Virginijus Siksnys
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CRISPR and the Plant Pathologists' Holy Grail. CRISPR J. (IF 3.7) Pub Date : 2023-08-01 Matthew R Willmann
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CRISPR Empowers Tree Bioengineering for a Sustainable Future. CRISPR J. (IF 3.7) Pub Date : 2023-07-31 Gen Li,Yiping Qi
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CRISPR-Cas-Based Biomonitoring for Marine Environments: Toward CRISPR RNA Design Optimization Via Deep Learning. CRISPR J. (IF 3.7) Pub Date : 2023-07-12 Benjamín Durán-Vinet,Karla Araya-Castro,Anastasija Zaiko,Xavier Pochon,Susanna A Wood,Jo-Ann L Stanton,Gert-Jan Jeunen,Michelle Scriver,Anya Kardailsky,Tzu-Chiao Chao,Deependra K Ban,Maryam Moarefian,Kiana Aran,Neil J Gemmell
Almost all of Earth's oceans are now impacted by multiple anthropogenic stressors, including the spread of nonindigenous species, harmful algal blooms, and pathogens. Early detection is critical to manage these stressors effectively and to protect marine systems and the ecosystem services they provide. Molecular tools have emerged as a promising solution for marine biomonitoring. One of the latest
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Rapid and Technically Simple Detection of SARS-CoV-2 Variants Using CRISPR Cas12 and Cas13. CRISPR J. (IF 3.7) Pub Date : 2023-06-21 Gabriel Lamothe,Julie Carbonneau,Charles Joly Beauparlant,Thierry Vincent,Patrik Quessy,Anthony Guedon,Gary Kobinger,Jean-Francois Lemay,Guy Boivin,Arnaud Droit,Nathalie Turgeon,Jacques P Tremblay
The worldwide proliferation of the SARS-CoV-2 virus in the past 3 years has allowed the virus to accumulate numerous mutations. Dangerous lineages have emerged one after another, each leading to a new wave of the pandemic. In this study, we have developed the THRASOS pipeline to rapidly discover lineage-specific mutation signatures and thus advise the development of Clustered Regularly Interspaced
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Predicting Mutations Generated by Cas9, Base Editing, and Prime Editing in Mammalian Cells. CRISPR J. (IF 3.7) Pub Date : 2023-06-20 Juliane Weller,Ananth Pallaseni,Jonas Koeppel,Leopold Parts
The first fruits of the CRISPR-Cas revolution are starting to enter the clinic, with gene editing therapies offering solutions to previously incurable genetic diseases. The success of such applications hinges on control over the mutations that are generated, which are known to vary depending on the targeted locus. In this review, we present the current state of understanding and predicting CRISPR-Cas
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Discovery and Characterization of Novel Type V Cas12f Nucleases with Diverse Protospacer Adjacent Motif Preferences. CRISPR J. (IF 3.7) Pub Date : 2023-06-02 Allison Sharrar,Luisa Arake de Tacca,Trevor Collingwood,Zuriah Meacham,David Rabuka,Johanna Staples-Ager,Michael Schelle
Small Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector.
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Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability. CRISPR J. (IF 3.7) Pub Date : 2023-06-01 Lisa M Alexander,Daniela S Aliaga Goltsman,Jason Liu,Jyun-Liang Lin,Morayma M Temoche-Diaz,Sarah M Laperriere,Andreas Neerincx,Christien Bednarski,Philipp Knyphausen,Andre Cohnen,Justine Albers,Liliana Gonzalez-Osorio,Rodrigo Fregoso Ocampo,Jennifer Oki,Audra E Devoto,Cindy J Castelle,Rebecca C Lamothe,Gregory J Cost,Cristina N Butterfield,Brian C Thomas,Christopher T Brown
Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved
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The Transposon-Encoded Protein TnpB Processes Its Own mRNA into ωRNA for Guided Nuclease Activity. CRISPR J. (IF 3.7) Pub Date : 2023-06-01 Suchita P Nety,Han Altae-Tran,Soumya Kannan,F Esra Demircioglu,Guilhem Faure,Seiichi Hirano,Kepler Mears,Yugang Zhang,Rhiannon K Macrae,Feng Zhang
TnpB is a member of the Obligate Mobile Element Guided Activity (OMEGA) RNA-guided nuclease family, is harbored in transposons, and likely functions to maintain the transposon in genomes. Previously, it was shown that TnpB cleaves double- and single-stranded DNA substrates in an RNA-guided manner, but the biogenesis of the TnpB ribonucleoprotein (RNP) complex is unknown. Using in vitro purified apo
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Search for Origins of Anti-CRISPR Proteins by Structure Comparison. CRISPR J. (IF 3.7) Pub Date : 2023-06-01 Harutyun Sahakyan,Kira S Makarova,Eugene V Koonin
Many bacterial and archaeal viruses encode anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas systems via various mechanisms. The majority of the Acrs are small, non-enzymatic proteins that abrogate CRISPR activity by binding to Cas effector proteins. The Acrs evolve fast, due to the arms race with the respective CRISPR-Cas systems, which hampers the elucidation of their evolutionary
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Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering. CRISPR J. (IF 3.7) Pub Date : 2023-05-23 Rebecca C Lamothe,Meghan D Storlie,Diego A Espinosa,Rachel Rudlaff,Patrick Browne,Jason Liu,Andres Rivas,Audra Devoto,Jennifer Oki,Ashcon Khoubyari,Daniela S Aliaga Goltsman,Jyun-Liang Lin,Cristina N Butterfield,Christopher T Brown,Brian C Thomas,Gregory J Cost
Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable
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The Promises and Pitfalls of CRISPR-Mediated Base Editing in Stem Cells. CRISPR J. (IF 3.7) Pub Date : 2023-05-19 Poh Kuan Wong,Nurul Nadia Mohamad Zamberi,Saiful Effendi Syafruddin,Fook Choe Cheah,Norazrina Azmi,Jia Xian Law,Eng Wee Chua
Stem cells such as induced pluripotent stem cells, embryonic stem cells, and hematopoietic stem and progenitor cells are growing in importance in disease modeling and regenerative medicine. The applications of CRISPR-based gene editing to create a mélange of disease and nondisease stem cell lines have further enhanced the utility of this innately versatile group of cells in the studies of human genetic
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Model System to Analyze RNA-Mediated DNA Repair in Mammalian Cells. CRISPR J. (IF 3.7) Pub Date : 2023-05-18 Lisa Tschage,Eric Kowarz,Rolf Marschalek
"RNA-templated/directed DNA repair" is a biological mechanism that has been experimentally demonstrated in bacteria, yeast, and mammalian cells. Recent study has shown that small noncoding RNAs (DDRNAs) and/or newly RNAPII transcribed RNAs (dilncRNAs) are orchestrating the initial steps of double-strand break (DSB) repair. In this study, we demonstrate that also pre-mRNA could be used as direct or
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Efficient Genome and Base Editing in Human Cells Using ThermoCas9. CRISPR J. (IF 3.7) Pub Date : 2023-05-03 Despoina Trasanidou,Patrick Barendse,Evgenios Bouzetos,Laura de Haan,Hans Bouwmeester,Raymond H J Staals,Ioannis Mougiakos,John van der Oost
Most genetic engineering applications reported thus far rely on the type II-A CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpyCas9), limiting the genome-targeting scope. In this study, we demonstrate that a small, naturally accurate, and thermostable type II-C Cas9 ortholog from Geobacillus thermodenitrificans (ThermoCas9) with alternative target site preference is active in human cells, and it
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Clarifying CRISPR: Why Repeats Identified in the Human Genome Should Not Be Considered CRISPRs. CRISPR J. (IF 3.7) Pub Date : 2023-04-11 Murat Buyukyoruk,William S Henriques,Blake Wiedenheft
Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated genes (cas) are essential components of adaptive immune systems that protect bacteria and archaea from viral infection. CRISPR-Cas systems are found in about 40% of bacterial and 85% of archaeal genomes, but not in eukaryotic genomes. Recently, an article published in Communications Biology reported the identification
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CRISPR-Cas9 Long-Read Sequencing for Mapping Transgenes in the Mouse Genome. CRISPR J. (IF 3.7) Pub Date : 2023-04-01 W Bart Bryant,Allison Yang,Susan H Griffin,Wei Zhang,Ashiq M Rafiq,Weiping Han,Ferenc Deak,Mary Katherine Mills,Xiaochun Long,Joseph M Miano
Microinjected transgenes, both large and small, are known to insert randomly into the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. As the vast majority of transgenic mouse lines remain unmapped, we developed CRISPR-Cas9
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Special Issue: Manipulating the Microbiome with CRISPR. CRISPR J. (IF 3.7) Pub Date : 2023-04-01 Brady Cress,Rodolphe Barrangou
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CLASH of the Titans: How CAR-T Cells Can Triumph Over Tumors. CRISPR J. (IF 3.7) Pub Date : 2023-03-21 Rianne Opstelten,Julian J Freen-van Heeren
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Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a. CRISPR J. (IF 3.7) Pub Date : 2023-03-21 Melissa D Morales-Moreno,Erick G Valdés-Galindo,Mariana M Reza,Tatiana Fiordelisio,Jorge Peon,Armando Hernandez-Garcia
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves
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Periodontal Disease Pathogens, Pathogenesis, and Therapeutics: The CRISPR-Cas Effect. CRISPR J. (IF 3.7) Pub Date : 2023-03-20 Madhurya N Kedlaya,Lakshmi Puzhankara,Rohit Prasad,Akshatha Raj
Periodontal disease (PD) is an immune-inflammatory disease affecting the supporting structures of the teeth, which results in progressive destruction of the hard and soft tissues surrounding teeth, ultimately resulting in tooth loss. The primary etiological factor for this disease is the presence of pathogenic microorganisms. Pathogenic bacteria face antagonistic conditions and foreign DNA components
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Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2. CRISPR J. (IF 3.7) Pub Date : 2023-03-13 Cathryn M Mayes,Joshua L Santarpia
CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with
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Functional and Phylogenetic Diversity of Cas10 Proteins. CRISPR J. (IF 3.7) Pub Date : 2023-03-13 Tanner Wiegand,Royce Wilkinson,Andrew Santiago-Frangos,Mackenzie Lynes,Roland Hatzenpichler,Blake Wiedenheft
Cas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10
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Specific High-Sensitivity Enzymatic Reporter UnLOCKing-Mediated Detection of Oncogenic BCR::ABL1 and EGFR Rearrangements. CRISPR J. (IF 3.7) Pub Date : 2023-03-13 Grégoire Cullot,Samuel Amintas,Laura Karembé,Valérie Prouzet-Mauléon,Julie Rébillard,Lisa Boureau,David Cappellen,Aurélie Bedel,François Moreau-Gaudry,Stéphanie Dulucq,Sandrine Dabernat,Béatrice Turcq
Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior
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PASTE: The Way Forward for Large DNA Insertions. CRISPR J. (IF 3.7) Pub Date : 2023-01-30 Muhammad Arslan Mahmood,Shahid Mansoor
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Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies CRISPR J. (IF 3.7) Pub Date : 2023-01-20 Guillermo Ureña-Bailén, Milena Block, Tommaso Grandi, Faidra Aivazidou, Jona Quednau, Dariusz Krenz, Alberto Daniel-Moreno, Andrés Lamsfus-Calle, Thomas Epting, Rupert Handgretinger, Stefan Wild, Markus Mezger
Cellular therapies hold enormous potential for the cure of severe hematological and oncological disorders. The forefront of innovative gene therapy approaches including therapeutic gene editing and hematopoietic stem cell transplantation needs to be processed by good manufacturing practice to ensure safe application in patients. In the present study, an effective transfection protocol for automated
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In Vitro CRISPR-Cas12a-Based Detection of Cancer-Associated TP53 Hotspot Mutations Beyond the crRNA Seed Region CRISPR J. (IF 3.7) Pub Date : 2023-01-13 Kavish A.V. Kohabir, Lars O. Nooi, Arjen Brink, Ruud H. Brakenhoff, Erik A. Sistermans, Rob M.F. Wolthuis
Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis, exhibits indiscriminate ssDNase activity in trans,
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Gene Editing Corrects In Vitro a G > A GLB1 Transition from a GM1 Gangliosidosis Patient CRISPR J. (IF 3.7) Pub Date : 2023-01-11 Delphine Leclerc, Louise Goujon, Sylvie Jaillard, Bénédicte Nouyou, Laurence Cluzeau, Léna Damaj, Christèle Dubourg, Amandine Etcheverry, Thierry Levade, Roseline Froissart, Stéphane Dréano, Xavier Guillory, Leif A Eriksson, Erika Launay, Frédéric Mouriaux, Marc-Antoine Belaud-Rotureau, Sylvie Odent, David Gilot
Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced β-galactosidase (β-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim
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Miniature CRISPR-Cas12f1-Mediated Single-Nucleotide Microbial Genome Editing Using 3′-Truncated sgRNA CRISPR J. (IF 3.7) Pub Date : 2022-12-23 Ho Joung Lee, Hyun Ju Kim, Sang Jun Lee
The CRISPR-Cas system has been used as a convenient tool for genome editing because the nuclease that cuts the target DNA and the guide RNA that recognizes the target are separated into modules. Cas12f1, which has a smaller size than that of other Cas nucleases, is easily loaded into vectors and is emerging as a new genome editing tool. In this study, AsCas12f1 was used to negatively select only Escherichia
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Replication Protein Rep Provides Selective Advantage to Viruses in the Presence of CRISPR-Cas Immunity CRISPR J. (IF 3.7) Pub Date : 2022-12-22 Weijia Zhang, Yuvaraj Bhoobalan-Chitty, Xichuan Zhai, Yan Hui, Lars Hestbjerg Hansen, Ling Deng, Xu Peng
Anti-Clustered regularly interspaced small palindromic repeat (CRISPR) (Acr) phages cooperate to establish a successful infection in CRISPR-containing host. We report here the selective advantage provided by a replication initiator, Rep, toward cooperative host immunosuppression by viruses encoding Acrs. A rep knockout mutant (Δgp16) of Sulfolobus islandicus rod-shaped virus 2 produced around fourfold
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Prime Editing in Mammals: The Next Generation of Precision Genome Editing CRISPR J. (IF 3.7) Pub Date : 2022-12-12 Dawei Wang, Xiude Fan, Mengzhu Li, Tianbo Liu, Peng Lu, Guangxin Wang, Yuan Li, JunMing Han, JiaJun Zhao
The recently established prime editor (PE) system is regarded as next-generation gene-editing technology. This methodology can install any base-to-base change as well as insertions and deletions without the requirement for double-stranded break formation or donor DNA templates; thus, it offers more targeting flexibility and greater editing precision than conventional CRISPR-Cas systems or base editors
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A Novel CRISPR Interference Effector Enabling Functional Gene Characterization with Synthetic Guide RNAs CRISPR J. (IF 3.7) Pub Date : 2022-12-12 Clarence Mills, Andrew Riching, Ashleigh Keller, Jesse Stombaugh, Amanda Haupt, Elena Maksimova, Sarah M. Dickerson, Emily Anderson, Kevin Hemphill, Chris Ebmeier, John A. Schiel, Josien Levenga, Matthew Perkett, Anja van Brabant Smith, Zaklina Strezoska
While CRISPR interference (CRISPRi) systems have been widely implemented in pooled lentiviral screening, there has been limited use with synthetic guide RNAs for the complex phenotypic readouts enabled by experiments in arrayed format. Here we describe a novel deactivated Cas9 fusion protein, dCas9-SALL1-SDS3, which produces greater target gene repression than first or second generation CRISPRi systems
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Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity CRISPR J. (IF 3.7) Pub Date : 2022-12-12 Alyssa Hoy, Ya Ying Zheng, Jia Sheng, Maksim Royzen
The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential toward the treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on the incorporation of modifications into the single-guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation
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Natural Nucleoside Modifications in Guide RNAs Can Modulate the Activity of the CRISPR-Cas9 System In Vitro CRISPR J. (IF 3.7) Pub Date : 2022-12-12 Daria V. Prokhorova, Ivan P. Vokhtantsev, Polina O. Tolstova, Evgenii S. Zhuravlev, Lilia M. Kulishova, Dmitry O. Zharkov, Grigory A. Stepanov
At the present time, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has been widely adopted as an efficient genomic editing tool. However, there are some actual problems such as the off-target effects, cytotoxicity, and immunogenicity. The incorporation of modifications into guide RNAs permits enhancing both the efficiency and the specificity
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CRISPR-Cas9 Genome Editing Uncovers the Mode of Action of Methoprene in the Yellow Fever Mosquito, Aedes aegypti CRISPR J. (IF 3.7) Pub Date : 2022-12-12 Guan-Heng Zhu, Sharath Chandra Gaddelapati, Yaoyu Jiao, Jinmo Koo, Subba Reddy Palli
Methoprene, a juvenile hormone (JH) analog, is widely used for insect control, but its mode of action is not known. To study methoprene action in the yellow fever mosquito, Aedes aegypti, the E93 (ecdysone-induced transcription factor) was knocked out using the CRISPR-Cas9 system. The E93 mutant pupae retained larval tissues similar to methoprene-treated insects. These insects completed pupal ecdysis
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Impact of KIT Editing on Coat Pigmentation and Fresh Meat Color in Yorkshire Pigs CRISPR J. (IF 3.7) Pub Date : 2022-12-12 Xinyu Liang, Jin Lan, Meina Xu, Ke Qin, Hongbo Liu, Guanjie Sun, Xiaohong Liu, Yaosheng Chen, Zuyong He
The white coat color of Yorkshire pigs is caused by the dominant white I allele, which has been associated with at least one copy of the 450-kb duplication encompassing the entire KIT gene and a splice mutation (G > A) at the first base of intron 17. The splice mutation in KIT has an adverse effect on pigmentation in mice. Therefore, removing the 450 kb duplications harboring the KIT copy with splice
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Highly Efficient One-Step Tagging of Endogenous Genes in Primary Cells Using CRISPR-Cas Ribonucleoproteins CRISPR J. (IF 3.7) Pub Date : 2022-12-12 Yao Yao, Jiaxuan Cao, Wentian Wang, Boya Liu, Xiaolei Pei, Lei Zhang, Shuquan Rao
Genome editing tools have simplified the generation of knock-in gene fusions, which are widely used to study proteins in their natural context. However, strategies for tagging endogenous genes in primary cells are few and inefficient. In this study, we developed a one-step endogenous gene-tagging strategy by co-delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein
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Increasing Genome Editing Efficiency of Cas9 Nucleases by the Simultaneous Use of Transcriptional Activators and Histone Acetyltransferase Activator CRISPR J. (IF 3.7) Pub Date : 2022-12-12 Junhao Liu, Bo Li, Lele Yang, Naixia Ren, Meichen Xu, Qilai Huang
The CRISPR-Cas9 system shows diverse levels of genome editing activities on eukaryotic chromatin, and high-efficiency sgRNA targets are usually desired in application. In this study, we show that chromatin open status is a pivotal determinant of the Cas9 editing activity in mammalian cells, and increasing chromatin accessibility can efficiently improve Cas9 genome editing. However, the strategy that
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CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits CRISPR J. (IF 3.7) Pub Date : 2022-12-12 Ioanna Morianou, Andrea Crisanti, Tony Nolan, Andrew M. Hammond
The introduction of small unmarked edits to the genome of insects is essential to study the molecular underpinnings of important biological traits, such as resistance to insecticides and genetic control strategies. Advances in CRISPR genome engineering have made this possible, but prohibitively laborious for most laboratories due to low rates of editing and the lack of a selectable marker. To facilitate
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A CRISPR-dCas9 System for Assaying and Selecting for RNase III Activity In Vivo in Escherichia coli CRISPR J. (IF 3.7) Pub Date : 2022-12-09 Pricila Hauk, Ryan Weeks, Marc Ostermeier
Ribonuclease III (RNase III) and RNase III-like ribonucleases have a wide range of important functions and are found in all organisms, yet a simple and high-throughput in vivo method for measuring RNase III activity does not exist. Typical methods for measuring RNase III activity rely on in vitro RNA analysis or in vivo methods that are not suitable for high-throughput analysis. In this study, we describe
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A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance CRISPR J. (IF 3.7) Pub Date : 2022-11-11 Maturada Patchsung, Aimorn Homchan, Kanokpol Aphicho, Surased Suraritdechachai, Thanyapat Wanitchanon, Archiraya Pattama, Khomkrit Sappakhaw, Piyachat Meesawat, Thanakrit Wongsatit, Artittaya Athipanyasilp, Krittapas Jantarug, Niracha Athipanyasilp, Juthamas Buahom, Supapat Visanpattanasin, Nootaree Niljianskul, Pimchai Chaiyen, Ruchanok Tinikul, Nuanjun Wichukchinda, Surakameth Mahasirimongkol, Rujipas
Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection—such as multiplexed detection for viral variant surveillance—may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic
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On- and Off-Target Analyses of CRISPR-Cas12b Genome Editing Systems in Rice CRISPR J. (IF 3.7) Pub Date : 2022-11-04 Filiz Gurel, Yuechao Wu, Changtian Pan, Yanhao Cheng, Gen Li, Tao Zhang, Yiping Qi
The CRISPR-associated Cas12b system is the third most efficient CRISPR tool for targeted genome editing in plants after Cas9 and Cas12a. Although the genome editing ability of AaCas12b has been previously investigated in rice, its off-target effects in plants are largely not known. In this study, we first engineered single-guide RNA (sgRNA) complexes with various RNA scaffolds to enhance editing frequency
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Advances in CRISPR Delivery Methods: Perspectives and Challenges CRISPR J. (IF 3.7) Pub Date : 2022-10-13 Selami Demirci, Khaled Essawi, Paula Germino-Watnick, Xiong Liu, Waleed Hakami, John F. Tisdale
With the advent of new genome editing technologies and the emphasis placed on their optimization, the genetic and phenotypic correction of a plethora of diseases sit on the horizon. Ideally, genome editing approaches would provide long-term solutions through permanent disease correction instead of simply treating patients symptomatically. Although various editing machinery options exist, the clustered
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Expression of Cas9 in a Syngeneic Model of Primary Central Nervous System Lymphoma Induces Intracerebral NK and CD8 T Cell-Mediated Lymphoma Cell Lysis Via Perforin CRISPR J. (IF 3.7) Pub Date : 2022-10-13 Manuel Montesinos-Rongen, Monica Sanchez-Ruiz, Susann Siebert, Claudia Winter, Reiner Siebert, Anna Brunn, Martina Deckert
The development of clustered regulatory interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR-Cas9)-mediated gene modification has opened an exciting avenue of targeting genes to study the pathogenesis of diseases and to develop novel therapeutic concepts. However, as the effector protein Cas9 is of bacterial origin, unwanted side effects due to a host immune response against Cas9
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RNA-Responsive gRNAs for Controlling CRISPR Activity: Current Advances, Future Directions, and Potential Applications CRISPR J. (IF 3.7) Pub Date : 2022-10-13 Oana Pelea, Tudor A. Fulga, Tatjana Sauka-Spengler
CRISPR-Cas9 has emerged as a major genome manipulation tool. As Cas9 can cause off-target effects, several methods for controlling the expression of CRISPR systems were developed. Recent studies have shown that CRISPR activity could be controlled by sensing expression levels of endogenous transcripts. This is particularly interesting, as endogenous RNAs could harbor important information about the
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Microfluidic Enrichment and Computational Analysis of Rare Sequences from Mixed Genomic Samples for Metagenomic Mining CRISPR J. (IF 3.7) Pub Date : 2022-10-13 Naiwen Cui, Guihem Faure, Ankita Singh, Rhiannon Macrae, Feng Zhang
Many powerful molecular biology tools have their origins in natural systems, including restriction modification enzymes and the CRISPR effectors, Cas9, Cas12, and Cas13. Heightened interest in these systems has led to mining of genomic and metagenomic data to identify new orthologs of these proteins, new types of CRISPR systems, and uncharacterized natural systems with novel mechanisms. To accelerate
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Optimized Guide RNA Selection Improves Streptococcus pyogenes Cas9 Gene Editing of Human Hematopoietic Stem and Progenitor Cells CRISPR J. (IF 3.7) Pub Date : 2022-10-13 Han J.M.P. Verhagen, Carlijn Kuijk, Laurens Rutgers, Anne M. Kokke, Santhe A. van der Meulen, Gerard van Mierlo, Carlijn Voermans, Emile van den Akker
Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research because the method is fast, viral free, and shows fewest off target effects. We evaluated various parameters to genetically engineer human hematopoietic stem and progenitor cells (HSPCs) using Streptococcus pyogenes Cas9 (spCas9) RNPs, and achieve gene editing efficiencies up to 80%. We find
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Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors CRISPR J. (IF 3.7) Pub Date : 2022-10-13 Sukanya Iyer, Aamir Mir, Joel Vega-Badillo, Benjamin P. Roscoe, Raed Ibraheim, Lihua Julie Zhu, Jooyoung Lee, Pengpeng Liu, Kevin Luk, Esther Mintzer, Dongsheng Guo, Josias Soares de Brito, Charles P. Emerson Jr., Phillip D. Zamore, Erik J. Sontheimer, Scot A. Wolfe
While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce