Journal ArticleDOI
MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems
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TLDR
A streamlined protocol for PITCh knock-in is described, including the design and construction of the PITCh vectors, and their delivery to either human cell lines by transfection or to frog embryos by microinjection.Abstract:
Programmable nucleases enable engineering of the genome by utilizing endogenous DNA double-strand break (DSB) repair pathways. Although homologous recombination (HR)-mediated gene knock-in is well established, it cannot necessarily be applied in every cell type and organism because of variable HR frequencies. We recently reported an alternative method of gene knock-in, named the PITCh (Precise Integration into Target Chromosome) system, assisted by microhomology-mediated end-joining (MMEJ). MMEJ harnesses independent machinery from HR, and it requires an extremely short homologous sequence (5-25 bp) for DSB repair, resulting in precise gene knock-in with a more easily constructed donor vector. Here we describe a streamlined protocol for PITCh knock-in, including the design and construction of the PITCh vectors, and their delivery to either human cell lines by transfection or to frog embryos by microinjection. The construction of the PITCh vectors requires only a few days, and the entire process takes ∼ 1.5 months to establish knocked-in cells or ∼ 1 week from injection to early genotyping in frog embryos.read more
Citations
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Journal ArticleDOI
CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing
Kornel Labun,Tessa G. Montague,Maximilian Krause,Yamila N. Torres Cleuren,Håkon Tjeldnes,Eivind Valen +5 more
TL;DR: This major update of CHOPCHOP introduces functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions, and incorporates new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes.
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Applications of CRISPR technologies in research and beyond
TL;DR: Programmable DNA cleavage using CRISPR–Cas9 enables efficient, site-specific genome engineering in single cells and whole organisms and is being used to expedite crop and livestock breeding, engineer new antimicrobials and control disease- carrying insects with gene drives.
Journal ArticleDOI
The dTAG system for immediate and target-specific protein degradation
Behnam Nabet,Justin M. Roberts,Dennis L. Buckley,Dennis L. Buckley,Joshiawa Paulk,Joshiawa Paulk,Shiva Dastjerdi,Annan Yang,Alan L. Leggett,Michael A. Erb,Matthew A. Lawlor,Amanda Souza,Amanda Souza,Thomas G. Scott,Sarah Vittori,Jennifer A. Perry,Jun Qi,Georg E. Winter,Georg E. Winter,Kwok-Kin Wong,Nathanael S. Gray,James E. Bradner,James E. Bradner +22 more
TL;DR: The dTAG system pairs potent heterobifunctional degraders and extensible tagging strategies to achieve immediate and reversible degradation of divergent proteins, facilitating biological investigation and drug target validation in cells and in mice.
Journal ArticleDOI
Cornerstones of CRISPR-Cas in drug discovery and therapy
Christof Fellmann,Benjamin G. Gowen,Pei-Chun Lin,Pei-Chun Lin,Jennifer A. Doudna,Jacob E. Corn +5 more
TL;DR: How CRISPR–Cas can affect the next generation of drugs by accelerating the identification and validation of high-value targets, uncovering high-confidence biomarkers and developing differentiated breakthrough therapies is discussed.
Journal ArticleDOI
Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage
Jian-Ping Zhang,Xiao-Lan Li,Guo-Hua Li,Wanqiu Chen,Cameron Arakaki,Gary D. Botimer,David J. Baylink,Lu Zhang,Wei Wen,Ya-Wen Fu,Jing Xu,Noah Chun,Weiping Yuan,Tao Cheng,Xiao-Bing Zhang +14 more
TL;DR: It is shown that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors.
References
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Journal ArticleDOI
Genome engineering using the CRISPR-Cas9 system
F. Ann Ran,Patrick D. Hsu,Jason Wright,Vineeta Agarwala,Vineeta Agarwala,David A. Scott,Feng Zhang +6 more
TL;DR: A set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies are described.
Journal ArticleDOI
One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.
Haoyi Wang,Hui Yang,Chikdu S. Shivalila,Meelad M. Dawlaty,Albert W. Cheng,Feng Zhang,Feng Zhang,Rudolf Jaenisch +7 more
TL;DR: The CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.
Journal ArticleDOI
A TALE nuclease architecture for efficient genome editing
Jeffrey C. Miller,Siyuan Tan,Guijuan Qiao,Kyle A. Barlow,Jianbin Wang,Danny F Xia,Xiangdong Meng,David Paschon,Elo Leung,Sarah J. Hinkley,Gladys P Dulay,Kevin Hua,Irina Ankoudinova,Gregory J. Cost,Fyodor D. Urnov,H. Steve Zhang,Michael C. Holmes,Lei Zhang,Philip D. Gregory,Edward J. Rebar +19 more
TL;DR: This study identifies TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and uses them to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%.
Book ChapterDOI
Genome engineering using CRISPR-Cas9 system.
Le Cong,Feng Zhang +1 more
TL;DR: This chapter presents all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination.
Book
Early development of Xenopus laevis : a laboratory manual
TL;DR: This book discusses the development of Xenopus laevis Embryos and Temperature Dependence, and the role of radiolysis in this development.