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
Citations
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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.
Therapeutic genome editing: prospects and challenges
TL;DR: Current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges are discussed.
Journal ArticleDOI
Prime genome editing in rice and wheat
Qiupeng Lin,Yuan Zong,Chenxiao Xue,Shengxing Wang,Shuai Jin,Zixu Zhu,Yanpeng Wang,Andrew V. Anzalone,Andrew V. Anzalone,Andrew V. Anzalone,Aditya Raguram,Aditya Raguram,Aditya Raguram,Jordan L. Doman,Jordan L. Doman,Jordan L. Doman,David R. Liu,David R. Liu,David R. Liu,Caixia Gao +19 more
TL;DR: The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts through codon, promoter, and editing-condition optimization.
Journal ArticleDOI
The promise and challenge of therapeutic genome editing
TL;DR: The scientific, technical and ethical aspects of using CRISPR technology for therapeutic applications in humans are discussed, highlighting both opportunities and challenges of this technology to treat, cure and prevent genetic disease.
Journal ArticleDOI
Applications of CRISPR–Cas in agriculture and plant biotechnology
Haocheng Zhu,Chao Li,Caixia Gao +2 more
TL;DR: The most important applications of CRISPR-Cas in increasing plant yield, quality, disease resistance and herbicide resistance, breeding and accelerated domestication, and prospective applications of this game-changing technology are discussed.
References
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Journal ArticleDOI
Therapeutic genome editing: prospects and challenges
TL;DR: In this article, the authors discuss current progress toward developing programmable nuclease-based therapies as well as future prospects and challenges, and discuss the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies.
Therapeutic genome editing: prospects and challenges
TL;DR: Current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges are discussed.
Journal ArticleDOI
CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response.
Emma Haapaniemi,Emma Haapaniemi,Sandeep Kumar Botla,Jenna Persson,Bernhard Schmierer,Jussi Taipale,Jussi Taipale,Jussi Taipale +7 more
TL;DR: It is reported that genome editing by CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway, suggesting that p53 inhibition may improve the efficiency of genome editing of untransformed cells.
Journal ArticleDOI
Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9
Dominik Paquet,Dylan Kwart,Antonia F. Chen,Andrew A. Sproul,Samson T. Jacob,Shaun Teo,Kimberly Moore Olsen,Andrew Gregg,Andrew Gregg,Scott Noggle,Marc Tessier-Lavigne +10 more
TL;DR: A CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy is described and HDR accuracy is increased dramatically, and a method termed ‘CORRECT’ for scarless genome editing is established.
Journal ArticleDOI
CRISPResso2 provides accurate and rapid genome editing sequence analysis.
Kendell Clement,Holly A. Rees,Matthew C. Canver,Matthew C. Canver,Jason Michael Gehrke,Rick Farouni,Rick Farouni,Jonathan Y. Hsu,Jonathan Y. Hsu,Mitchel A. Cole,David R. Liu,David R. Liu,David R. Liu,J. Keith Joung,Daniel E. Bauer,Luca Pinello,Luca Pinello +16 more
TL;DR: In this article, the authors present CRISPResso2 to analyze base editors, perform allele-specific quantification or incorporate biologically-informed and scalable alignment approaches, and demonstrate its functionality by experimentally measuring and analyzing the editing properties of six genome editing agents.