引导RNA
清脆的
Cas9
转导(生物物理学)
转基因
质粒
生物
基因组编辑
腺相关病毒
遗传增强
基因
核糖核酸
载体(分子生物学)
抄写(语言学)
基因传递
病毒载体
计算生物学
遗传学
重组DNA
生物化学
语言学
哲学
作者
Lewis E Fry,Caroline F Peddle,Marta Stevanovic,Alun R. Barnard,Michelle E. McClements,Robert E. MacLaren
出处
期刊:The CRISPR journal
[Mary Ann Liebert]
日期:2020-08-01
卷期号:3 (4): 276-283
被引量:8
标识
DOI:10.1089/crispr.2020.0021
摘要
Adeno-associated virus (AAV) vectors have been widely adopted for delivery of CRISPR-Cas components, especially for therapeutic gene editing. For a single vector system, both the Cas9 and guide RNA (gRNA) are encoded within a single transgene, usually from separate promoters. Careful design of this bi-cistronic construct is required due to the minimal packaging capacity of AAV. We investigated how placement of the U6 promoter expressing the gRNA on the reverse strand to SaCas9 driven by a cytomegalovirus promoter affected gene editing rates compared to placement on the forward strand. We show that orientation in the reverse direction reduces editing rates from an AAV vector due to reduced transcription of both SaCas9 and guide RNA. This effect was observed only following AAV transduction; it was not seen following plasmid transfection. These results have implications for the design of AAV-CRISPR vectors, and suggest that results from optimizing plasmid transgenes may not translate when delivered via AAV.
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