Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
Alexis C. Komor,Yongjoo Kim,Yongjoo Kim,Michael S. Packer,Michael S. Packer,John A. Zuris,John A. Zuris,David R. Liu,David R. Liu +8 more
TLDR
E engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution.Abstract:
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.read more
Citations
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Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
Nicole M. Gaudelli,Alexis C. Komor,Holly A. Rees,Holly A. Rees,Holly A. Rees,Michael S. Packer,Ahmed H. Badran,Ahmed H. Badran,Ahmed H. Badran,David I. Bryson,David R. Liu,David R. Liu,David R. Liu +12 more
TL;DR: Adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA are described and a transfer RNA adenosine deaminase is evolved to operate on DNA when fused to a catalytically impaired CRISPR–Cas9 mutant.
Journal ArticleDOI
Search-and-replace genome editing without double-strand breaks or donor DNA
Andrew V. Anzalone,Andrew V. Anzalone,Andrew V. Anzalone,Peyton B. Randolph,Peyton B. Randolph,Peyton B. Randolph,Jessie Rose Davis,Jessie Rose Davis,Jessie Rose Davis,Alexander A. Sousa,Alexander A. Sousa,Alexander A. Sousa,Luke W. Koblan,Luke W. Koblan,Luke W. Koblan,Jonathan M. Levy,Jonathan M. Levy,Jonathan M. Levy,Peter J. Chen,Peter J. Chen,Peter J. Chen,Christine D. Wilson,Christine D. Wilson,Christine D. Wilson,Gregory A. Newby,Gregory A. Newby,Gregory A. Newby,Aditya Raguram,Aditya Raguram,Aditya Raguram,David R. Liu,David R. Liu,David R. Liu +32 more
TL;DR: A new DNA-editing technique called prime editing offers improved versatility and efficiency with reduced byproducts compared with existing techniques, and shows potential for correcting disease-associated mutations.
Journal ArticleDOI
Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing
Zhen Liu,Zongyang Lu,Guang Yang,Shisheng Huang,Guanglei Li,S. M. Feng,Yajing Liu,Jianan Li,Wenxia Yu,Yu Zhang,Jia Chen,Qiang Sun,Xingxu Huang,Xingxu Huang +13 more
TL;DR: In vivo generation of mouse models carrying clinically relevant mutations using C→T and A→G editors is demonstrated, making it feasible to model and potentially cure relevant genetic diseases.
Journal ArticleDOI
Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements
TL;DR: It is shown that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases, and the observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.
Journal ArticleDOI
Evolved Cas9 variants with broad PAM compatibility and high DNA specificity
Johnny Hao Hu,Shannon M. Miller,Shannon M. Miller,Shannon M. Miller,Maarten H. Geurts,Maarten H. Geurts,Maarten H. Geurts,Weixin Tang,Weixin Tang,Weixin Tang,Liwei Chen,Liwei Chen,Liwei Chen,Ning Sun,Ning Sun,Ning Sun,Christina M. Zeina,Christina M. Zeina,Christina M. Zeina,Xue Gao,Xue Gao,Xue Gao,Holly A. Rees,Holly A. Rees,Holly A. Rees,Zhi Lin,Zhi Lin,Zhi Lin,David R. Liu,David R. Liu,David R. Liu +30 more
TL;DR: In this article, the authors used phage-assisted continuous evolution to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA and GAT.
References
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Journal ArticleDOI
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Martin Jinek,Krzysztof Chylinski,Krzysztof Chylinski,Ines Fonfara,Michael H. Hauer,Jennifer A. Doudna,Emmanuelle Charpentier +6 more
TL;DR: This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
Journal ArticleDOI
Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,Le Cong,F. Ann Ran,F. Ann Ran,David M. Cox,David M. Cox,Shuailiang Lin,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +15 more
TL;DR: The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage as discussed by the authors.
Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,F. A. Ran,David Benjamin Turitz Cox,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +10 more
TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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
CRISPR-Cas systems for editing, regulating and targeting genomes
Jeffry D. Sander,J. Keith Joung +1 more
TL;DR: A modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells, which will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.