Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements
TLDR
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.Abstract:
CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.read more
Citations
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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
Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors
Andrew V. Anzalone,Luke W. Koblan,Luke W. Koblan,Luke W. Koblan,David R. Liu,David R. Liu,David R. Liu +6 more
TL;DR: This work analyzes key considerations when choosing genome editing agents and identifies opportunities for future improvements and applications in basic research and therapeutics.
Journal ArticleDOI
Base editing: precision chemistry on the genome and transcriptome of living cells
TL;DR: A comprehensive account of the state of the art of base editing of DNA and RNA is provided, including the progressive improvements to methodologies, understanding and avoiding unintended edits, cellular and organismal delivery of editing reagents and diverse applications in research and therapeutic settings.
Journal ArticleDOI
Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects
TL;DR: Recent advances of the three major genome editing technologies are reviewed and the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies are discussed, focusing on eukaryotic cells and animal models.
Journal ArticleDOI
CRISPR-engineered T cells in patients with refractory cancer
Edward A. Stadtmauer,Joseph A. Fraietta,Megan M. Davis,Adam D. Cohen,Kristy L. Weber,Eric Lancaster,Patricia A. Mangan,Irina Kulikovskaya,Minnal Gupta,Fang Chen,Lifeng Tian,Vanessa E. Gonzalez,Jun Xu,In-Young Jung,J. Joseph Melenhorst,Gabriela Plesa,Joanne Shea,Tina Matlawski,Amanda Cervini,Avery L. Gaymon,Stephanie Desjardins,Anne Lamontagne,January Salas-Mckee,Andrew D. Fesnak,Don L. Siegel,Bruce L. Levine,Julie K. Jadlowsky,Regina M. Young,Anne Chew,Wei-Ting Hwang,Elizabeth O. Hexner,Beatriz M. Carreno,Christopher L. Nobles,Frederic D. Bushman,Kevin R. Parker,Yanyan Qi,Ansuman T. Satpathy,Howard Y. Chang,Yangbing Zhao,Simon F. Lacey,Carl H. June +40 more
TL;DR: This first-in-human, phase 1 clinical trial was designed to test the safety and feasibility of multiplex CRISPR-Cas9 gene editing of T cells from patients with advanced, refractory cancer and found the persistence of the T cells expressing the engineered TCR was much more durable than in three previous clinical trials during which T cells were infused.
References
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DNA targeting specificity of RNA-guided Cas9 nucleases
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TL;DR: In this article, the Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing.
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TL;DR: It is found that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches.
Journal ArticleDOI
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
TL;DR: 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.
Journal ArticleDOI
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity
F. Ann Ran,Patrick D. Hsu,Chie Yu Lin,Jonathan S. Gootenberg,Silvana Konermann,Alexandro E. Trevino,David A. Scott,Azusa Inoue,Shogo Matoba,Yi Zhang,Feng Zhang +10 more
TL;DR: In this paper, an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks is described. But the approach is limited to mouse zygotes.
Journal ArticleDOI
High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.
Yanfang Fu,Jennifer A Foden,Cyd Khayter,Morgan L. Maeder,Deepak Reyon,J. Keith Joung,Jeffry D. Sander +6 more
TL;DR: It is found that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface, and off-target cleavage of CRISPR-associated (Cas)9-based RGNs is characterized.