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
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TLDR
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.Abstract:
Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2-5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.read more
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Adenine base editing and prime editing of chemically derived hepatic progenitors rescue genetic liver disease.
Yohan Kim,Sung-Ah Hong,Jihyeon Yu,Jeongyun Eom,Kiseok Jang,Sangtae Yoon,Da Hee Hong,Daekwan Seo,Seu-Na Lee,Jae Sung Woo,Jaemin Jeong,Sangsu Bae,Dongho Choi +12 more
TL;DR: In this paper, a mouse model of hereditary tyrosinemia type 1 (HT1) was successfully corrected using both adenine base editors (ABEs) and prime editors (PEs).
Journal ArticleDOI
Massively parallel phenotyping of coding variants in cancer with Perturb-seq
Oana Ursu,James T. Neal,Emily Shea,Pratiksha I. Thakore,Livnat Jerby-Arnon,Lan Nguyen,Danielle Dionne,Celeste Diaz,J. Bauman,Mariam Mounir Mosaad,Christian R. Fagre,April Lo,Maria V. McSharry,Andrew O. Giacomelli,Seav Huong Ly,Orit Rozenblatt-Rosen,William C. Hahn,Andrew J. Aguirre,Alice H. Berger,Aviv Regev,Jesse S. Boehm +20 more
TL;DR: This work developed an approach to functionally assess variant impact in single cells by pooled Perturb-seq and discovered that KRAS variants did not merely fit into discrete functional categories, but spanned a continuum of gain-of-function phenotypes, and that their functional impact could not have been predicted solely by their frequency in patient cohorts.
Journal ArticleDOI
A Cas-embedding strategy for minimizing off-target effects of DNA base editors.
Yajing Liu,Yajing Liu,Changyang Zhou,Shisheng Huang,Shisheng Huang,Lu Dang,Yu Wei,Jun He,Jun He,Yingsi Zhou,Shaoshuai Mao,Wanyu Tao,Wanyu Tao,Yu Zhang,Hui Yang,Xingxu Huang,Tian Chi +16 more
TL;DR: It is reported that the off-target effects of both A > G and C T editors can be dramatically reduced without compromising the on-target editing simply by inserting the editing enzymes into the middle of nCas9 at tolerant sites identified using a transposon-based genetic screen.
Journal ArticleDOI
Evolving AAV-delivered therapeutics towards ultimate cures.
Xiangjun He,Brian Anugerah Urip,Zhenjie Zhang,Chun Christopher Ngan,Chun Christopher Ngan,Bo Feng +5 more
TL;DR: In this article, the major challenges and safety concerns associated with AAV delivery and CRISPR therapeutics, and highlight the recent achievement and toxicity issues reported from clinical applications are discussed.
Journal ArticleDOI
Current trends in gene recovery mediated by the CRISPR-Cas system.
TL;DR: This review focuses on currently available gene recovery strategies that use CRISPR nucleases, particularly for the treatment of genetic disorders, and discusses the pros and cons of different procedures.
References
More filters
Journal ArticleDOI
limma powers differential expression analyses for RNA-sequencing and microarray studies
Matthew E. Ritchie,Belinda Phipson,Di Wu,Yifang Hu,Charity W. Law,Wei Shi,Gordon K. Smyth,Gordon K. Smyth +7 more
TL;DR: The philosophy and design of the limma package is reviewed, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.
Journal ArticleDOI
RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome
Bo Li,Colin N. Dewey +1 more
TL;DR: It is shown that accurate gene-level abundance estimates are best obtained with large numbers of short single-end reads, and estimates of the relative frequencies of isoforms within single genes may be improved through the use of paired- end reads, depending on the number of possible splice forms for each gene.
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.