Development of Highly Efficient Dual-AAV Split Adenosine Base Editor for In Vivo Gene Therapy

被引：52

作者：

Chen, Yuxi ^{[1
,2
]}

Zhi, Shengyao ^{[1
]}

Liu, Weiliang ^{[1
]}

Wen, Jinkun ^{[1
,3
]}

Hu, Sihui ^{[1
]}

Cao, Tianqi ^{[1
]}

Sun, Hongwei ^{[1
]}

Li, Yang ^{[1
]}

Huang, Li ^{[2
]}

Liu, Yizhi ^{[2
]}

Liang, Puping ^{[1
]}

Huang, Junjiu ^{[1
,4
,5
]}

机构：

[1] Sun Yat Sen Univ, Sch Life Sci, State Key Lab Biocontrol, MOE,Key Lab Gene Funct & Regulat, Guangzhou 510275, Peoples R China

[2] Sun Yat Sen Univ, Zhongshan Ophthalm Ctr, State Key Lab Ophthalmol, Guangzhou 510060, Peoples R China

[3] Sun Yat Sen Univ, Affiliated Jiangmen Hosp, Jiangmen Cent Hosp, Guangzhou 510150, Peoples R China

[4] Sun Yat Sen Univ, Affiliated Hosp 1, Key Lab Reprod Med Guangdong Prov, Guangzhou 510275, Peoples R China

[5] Sun Yat Sen Univ, Sch Life Sci, Guangzhou 510275, Peoples R China

来源：

SMALL METHODS | 2020年 / 4卷 / 09期

基金：

中国国家自然科学基金;

关键词：

adeno-associated viruses; adenosine base editors; gene editing; gene therapy; split-ABE; GYRA INTEIN; DNAE INTEIN; OFF-TARGET; RNA; CRISPR-CAS9; EXPRESSION; PROTEINS; RETINA; VECTOR; LIVER;

D O I：

10.1002/smtd.202000309

中图分类号：

O64 [物理化学（理论化学）、化学物理学];

学科分类号：

070304 ; 081704 ;

摘要：

The adenosine base editor (ABE) is able to catalyze A center dot T to C center dot G conversion efficiently and precisely in vivo, representing a new method for gene therapy. Adeno associated virus (AAV) is a well-studied vector for gene delivery in vivo. However, due to the limited loading capacity of AAV vector (approximate to 4800 bp), it is difficult to package ABE (approximate to 5400 bp) into a single AAV. To tackle this problem, ABE can be split into two smaller parts through intein-mediated protein trans-splicing. Here, 14 different split sites of nCas9 (Cas9 nickase) in combination with three different inteins (Mxe, Npu, and Rma) are screened through a GFP-based reporter system to identify novel split-ABEs. After infecting HEK293T and HeLa cells with dual AAVs, two split-ABEs (split-ABE-Rma573 and split-ABE-Rma674) that can edit the target gene efficiently are identified. Furthermore, these dual-AAV split-ABEs can effectively disrupt the splicing acceptor ofPCSK9in mouse liver and the splicing donor ofNR2E3in mouse retina through AI-MAST strategy. This study provides two new split-ABEs to investigate gene function in vivo and in gene therapy, representing a new method to treat diseases by precisely repairing point mutations or inactivating genes through the AI-MAST strategy.

引用

页数：13

共 64 条

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