Search-and-replace genome editing without double-strand breaks or donor DNA

被引:2773
作者
Anzalone, Andrew V. [1 ,2 ,3 ]
Randolph, Peyton B. [1 ,2 ,3 ]
Davis, Jessie R. [1 ,2 ,3 ]
Sousa, Alexander A. [1 ,2 ,3 ]
Koblan, Luke W. [1 ,2 ,3 ]
Levy, Jonathan M. [1 ,2 ,3 ]
Chen, Peter J. [1 ,2 ,3 ]
Wilson, Christopher [1 ,2 ,3 ]
Newby, Gregory A. [1 ,2 ,3 ]
Raguram, Aditya [1 ,2 ,3 ]
Liu, David R. [1 ,2 ,3 ]
机构
[1] Broad Inst Harvard & MIT, Merkin Inst Transformat Technol Healthcare, Cambridge, MA 02142 USA
[2] Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
[3] Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USA
关键词
REVERSE-TRANSCRIPTASE; PRION PROTEIN; RNA; BASE; ENDONUCLEASE; CRISPR-CAS9; EXPRESSION; EFFICIENCY; BINDING; VARIANT;
D O I
10.1038/s41586-019-1711-4
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Most genetic variants that contribute to disease(1) are challenging to correct efficiently and without excess by products(2-5). 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 by products, 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 by products 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 expandsthe scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.
引用
收藏
页码:149 / +
页数:30
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