Expression of CRISPR/Cas single guide RNAs using small tRNA promoters.
RNA
; 21(9): 1683-9, 2015 Sep.
Article
in En
| MEDLINE
| ID: mdl-26187160
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, â¼4.7-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 â¼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current â¼360 bp size. Here, we report that small, â¼70-bp tRNA promoters can be used to express high levels of tRNAsgRNA fusion transcripts that are efficiently and precisely cleaved by endogenous tRNase Z to release fully functional sgRNAs. Importantly, cells stably expressing functional tRNAsgRNA 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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA, Transfer
/
Genetic Engineering
/
Promoter Regions, Genetic
/
RNA, Guide, Kinetoplastida
/
CRISPR-Associated Proteins
Limits:
Animals
/
Humans
Language:
En
Journal:
RNA
Journal subject:
BIOLOGIA MOLECULAR
Year:
2015
Document type:
Article
Affiliation country:
Estados Unidos