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
Reads0
Chats0
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
Citations
More filters
Journal ArticleDOI
Increasing the efficiency and precision of prime editing with guide RNA pairs.
TL;DR: The homologous 3' extension mediated prime editor (HOPE) as discussed by the authors uses paired prime editing guide RNAs (pegRNAs) encoding the same edits in both sense and antisense DNA strands to achieve high editing efficiency.
Journal ArticleDOI
In vivo prime editing of a metabolic liver disease in mice
Désirée Böck,Tanja Rothgangl,Lukas Villiger,Lukas Schmidheini,Mai Matsushita,Nicolas Mathis,Eleonora I. Ioannidi,Nicole Rimann,Hiu Man Grisch-Chan,Susanne Kreutzer,Zacharias Kontarakis,Manfred Kopf,Beat Thöny,Gerald Schwank +13 more
TL;DR: A proof-of-principle in vivo prime editing strategy corrects phenylketonuria in mice and allows for more versatile and precise editing than CRISPR-based strategies but is still challenging to apply in vivo.
Journal ArticleDOI
Efficient targeted insertion of large DNA fragments without DNA donors
Jinlin Wang,Zhou He,Guo-zhu Wang,Ruiwen Zhang,Ju Duan,Pan Gao,Xinlin Lei,Houyuan Qiu,Chuanping Zhang,Ying Zhang,Hao Yin +10 more
TL;DR: Efficient insertion in multiple genomic loci of several cell lines and non-dividing cells is confirmed, which expands the scope of genome editing to enable donor-free insertion of large DNA sequences.
Journal ArticleDOI
RNA-targeting CRISPR systems from metagenomic discovery to transcriptomic engineering.
TL;DR: The rapid development of the RNA-targeting CRISPR–Cas engineering system is discussed and how this can be leveraged to further understand RNA biology is highlighted.
Journal ArticleDOI
Gene drive and resilience through renewal with next generation Cleave and Rescue selfish genetic elements
TL;DR: It is shown that gene drive-mediated population modification in Drosophila can be overwritten with new content while eliminating old, using Cleave and Rescue (ClvR) selfish genetic elements.
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.