Premium
Development of Highly Efficient Dual‐AAV Split Adenosine Base Editor for In Vivo Gene Therapy
Author(s) -
Chen Yuxi,
Zhi Shengyao,
Liu Weiliang,
Wen Jinkun,
Hu Sihui,
Cao Tianqi,
Sun Hongwei,
Li Yang,
Huang Li,
Liu Yizhi,
Liang Puping,
Huang Junjiu
Publication year - 2020
Publication title -
small methods
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.66
H-Index - 46
ISSN - 2366-9608
DOI - 10.1002/smtd.202000309
Subject(s) - genetic enhancement , reporter gene , gene , biology , rna splicing , in vivo , adeno associated virus , gene delivery , vectors in gene therapy , viral vector , computational biology , vector (molecular biology) , genetics , gene expression , recombinant dna , rna
The adenosine base editor (ABE) is able to catalyze A•T to C•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 (≈4800 bp), it is difficult to package ABE (≈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 of PCSK9 in mouse liver and the splicing donor of NR2E3 in 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.