Landscape of target:guide homology effects on Cas9-mediated cleavage.
Nucleic Acids Res
; 42(22): 13778-87, 2014 Dec 16.
Article
in En
| MEDLINE
| ID: mdl-25399416
ABSTRACT
To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in 'seed' sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guidetarget duplex (positions 13-18 relative to the PAM). Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. In addition, the first-base Guanine constraint commonly imposed by gRNA expression systems has little effect on overall cleavage efficiency. Taken together, these studies provide an in vitro understanding of the complexities of Cas9-gRNA interaction and cleavage beyond the general paradigm of site determination based on the 'seed' sequence and PAM.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Endodeoxyribonucleases
/
DNA Cleavage
/
CRISPR-Associated Proteins
Language:
En
Journal:
Nucleic Acids Res
Year:
2014
Document type:
Article
Affiliation country:
United States