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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Journal ArticleDOI
Enhancement of prime editing via xrRNA motif-joined pegRNA
Guiquan Zhang,Yao Liu,Shisheng Huang,Shiyuan Qu,Daolin Cheng,Yuan Yao,Quanjiang Ji,Xiaolong Wang,Xingxu Huang,Jianghuai Liu +9 more
TL;DR: In this article , an improved PE platform (xrPE) was developed with substantially enhanced editing efficiencies in multiple cell lines for base conversions, small deletions, and small insertions, respectively.
Journal ArticleDOI
CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury
Marcus Keatinge,Themistoklis M. Tsarouchas,Tahimina Munir,Nicola J. Porter,Juan Larraz,Davide Gianni,Hui-Hsin Tsai,Catherina G. Becker,David A. Lyons,Thomas Becker +9 more
TL;DR: In this article, a rapid in-vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury was conducted, using acute injection of synthetic RNA Oligo CRISPR guide RNAs that were pre-screened for high activity in vivo.
Journal ArticleDOI
Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment
Seung Hun Kang,Seung Hun Kang,Wi Jae Lee,Wi Jae Lee,Ju Hyun An,Ju Hyun An,Jong Hee Lee,Young-Hyun Kim,Young-Hyun Kim,Hanseop Kim,Hanseop Kim,Yeounsun Oh,Yeounsun Oh,Young-Ho Park,Yeung Bae Jin,Bong-Hyun Jun,Junho K. Hur,Junho K. Hur,Sun-Uk Kim,Sun-Uk Kim,Seung-Hwan Lee +20 more
TL;DR: A method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification is reported, which will allow detection of genome editor-induced off- target mutations with high sensitivity and in a non-biased manner.
Journal ArticleDOI
Treating Cystic Fibrosis with mRNA and CRISPR
TL;DR: This work applies learnings from a subset of CF gene therapies that have already been tested in patients to cystic fibrosis and highlights the scientific advances that are still required for non-viral mRNA or CRISPR-based drugs to treat CF successfully in patients.
Journal ArticleDOI
Applicability of the EFSA Opinion on site-directed nucleases type 3 for the safety assessment of plants developed using site-directed nucleases type 1 and 2 and oligonucleotide-directed mutagenesis.
Hanspeter Naegeli,Jean-Louis Bresson,Tamas Dalmay,Ian Crawford Dewhurst,Michelle M. Epstein,Leslie George Firbank,Philippe Guerche,Jan Hejatko,Francisco Javier Moreno,Ewen Mullins,Fabien Nogué,Jose Juan Sánchez Serrano,Giovanni Savoini,Eve Veromann,Fabio Veronesi,Josep M. Casacuberta,Andrea Gennaro,Konstantinos Paraskevopoulos,Tommaso Raffaello,Nils Rostoks +19 more
TL;DR: The GMO Panel concludes that those considerations which are specifically related to the presence of a transgene, intragene or cisgene included in section 4 and the conclusions of the Opinion on SDN‐3 are not relevant to plants obtained viaSDN‐1, SDN-2 or ODM as defined in this Opinion.
References
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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.
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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
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