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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PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing.
TL;DR: In this article, the authors present two web-based tools for prime editing guide RNAs (pegRNAs), which contain a primer binding site and a reverse-transcription template at the 3' end.
Journal ArticleDOI
Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC-Cas9.
Shengxing Wang,Yuan Zong,Qiupeng Lin,Huawei Zhang,Zhuangzhuang Chai,Dandan Zhang,Kunling Chen,Jin-Long Qiu,Caixia Gao +8 more
TL;DR: A series of APOBEC–Cas9 fusion-induced deletion systems (AFIDs) that combine Cas9 with human APOBec3A (A3A), uracil DNA-glucosidase and apurinic or apyrimidinic site lyase are reported, which could be applied to study regulatory regions and protein domains to improve crop plants.
Journal ArticleDOI
Current Status and Applications of Adaptive Laboratory Evolution in Industrial Microorganisms
S.-J. Lee,Pil Kim +1 more
TL;DR: This review delineates the basics of the experimental design of ALE based on several ALE studies of industrial microbial strains and updates current strategies combined with progressed metabolic engineering, in silico modeling and automation to maximize the evolution efficiency.
Journal ArticleDOI
A split prime editor with untethered reverse transcriptase and circular RNA template
Bin Liu,X.L. Dong,Hao-Feng Cheng,Chunwei Zheng,Zexiang Chen,Tomás Rodríguez,Shunqing Liang,Wen Xue,Erik J. Sontheimer +8 more
TL;DR: A split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT) showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion-deletion (indel) byproducts.
Journal ArticleDOI
In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in β-YAC mice.
Chang Li,Aphrodite Georgakopoulou,Arpit Mishra,Sucheol Gil,R. David Hawkins,Evangelia Yannaki,André Lieber +6 more
TL;DR: The observations demonstrate that base editors delivered by HDAd5/35++ vectors represent a promising strategy for precise in vivo genome engineering for the treatment of hemoglobinopathies and no detectable editing was found at top-scored potential off-target genomic sites.
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
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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
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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.