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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Predictable and precise template-free CRISPR editing of pathogenic variants
Max W. Shen,Mandana Arbab,Jonathan Y. Hsu,Daniel Worstell,Sannie J. Culbertson,Olga Krabbe,Olga Krabbe,Christopher A. Cassa,Christopher A. Cassa,David R. Liu,David R. Liu,David R. Liu,David K. Gifford,Richard I. Sherwood,Richard I. Sherwood +14 more
TL;DR: This study establishes an approach for precise, template-free genome editing using a machine-learning algorithm to predict the spectrum of CRISPR–Cas9-nuclease-mediated DNA repair outcomes at human genomic target sites.
Journal ArticleDOI
Flap Endonuclease 1: A Central Component of DNA Metabolism
TL;DR: FEN1 is a genome stabilization factor that prevents flaps from equilibrating into structures that lead to duplications and deletions and interacts with other nucleases and helicases that allow it to act efficiently on structured flaps.
Journal ArticleDOI
Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents
Xue Gao,Yong Tao,Yong Tao,Yong Tao,Veronica Lamas,Mingqian Huang,Wei-Hsi Yeh,Bifeng Pan,Yujuan Hu,Yujuan Hu,Johnny Hao Hu,Johnny Hao Hu,Johnny Hao Hu,David B. Thompson,David B. Thompson,Yilai Shu,Yilai Shu,Yamin Li,Hongyang Wang,Hongyang Wang,Shiming Yang,Qiaobing Xu,Daniel B. Polley,M. Charles Liberman,Weijia Kong,Jeffrey R. Holt,Zheng-Yi Chen,David R. Liu,David R. Liu,David R. Liu +29 more
TL;DR: It is shown that cationic lipid-mediated in vivo delivery of Cas9–guide RNA–lipid complexes can ameliorate hearing loss in a mouse model of human genetic deafness, suggesting that protein–RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.
Journal ArticleDOI
Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area.
TL;DR: It is demonstrated that dopaminergic neurons of the substantia nigra do not allow for long-term expression from adenoviral vectors, and strikingly, these neurons appeared to specifically attenuate transgene expression by deleting the adenviral genome.
Journal ArticleDOI
The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction
TL;DR: The naturally split α subunit of the DNA polymerase III (DnaE) intein from Nostoc punctiforme PCC73102 (Npu) is studied using purified proteins and an apparent first‐order rate constant of 1.1 ± 0.2 s−1 is determined, which represents the highest rate reported for the protein trans‐splicing reaction so far.