In:
RNA, Cold Spring Harbor Laboratory, Vol. 21, No. 9 ( 2015-09), p. 1683-1689
Abstract:
The in vivo application of CRISPR/Cas-based DNA editing technology will require the development of efficient delivery methods that likely will be dependent on adeno-associated virus (AAV)-based viral vectors. However, AAV vectors have only a modest, ∼4.7-kb packaging capacity, which will necessitate the identification and characterization of highly active Cas9 proteins that are substantially smaller than the prototypic Streptococcus pyogenes Cas9 protein, which covers ∼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current ∼360 bp size. Here, we report that small, ∼70-bp tRNA promoters can be used to express high levels of tRNA:sgRNA fusion transcripts that are efficiently and precisely cleaved by endogenous tRNase Z to release fully functional sgRNAs. Importantly, cells stably expressing functional tRNA:sgRNA precursors did not show a detectable change in the level of endogenous tRNA expression. This novel sgRNA expression strategy should greatly facilitate the construction of effective AAV-based Cas9/sgRNA vectors for future in vivo use.
Type of Medium:
Online Resource
ISSN:
1355-8382
,
1469-9001
DOI:
10.1261/rna.051631.115
Language:
English
Publisher:
Cold Spring Harbor Laboratory
Publication Date:
2015
detail.hit.zdb_id:
1475737-0
SSG:
12
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