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Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome.

Authors :
Müller M
Lee CM
Gasiunas G
Davis TH
Cradick TJ
Siksnys V
Bao G
Cathomen T
Mussolino C
Source :
Molecular therapy : the journal of the American Society of Gene Therapy [Mol Ther] 2016 Mar; Vol. 24 (3), pp. 636-44. Date of Electronic Publication: 2015 Dec 14.
Publication Year :
2016

Abstract

RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. However, the nontrivial level of off-target activity reported in several human cells may hamper clinical translation. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We hypothesized that more stringent PAM requirements reduce the occurrence of off-target mutagenesis. To test this postulation, we generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, PRKDC and CARD11. Our results demonstrate that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved. In conclusion, our results show that Cas9 proteins with longer or more restrictive PAM requirements provide a safe alternative to SpCas9-based RGNs and hence a valuable option for future human gene therapy applications.

Details

Language :
English
ISSN :
1525-0024
Volume :
24
Issue :
3
Database :
MEDLINE
Journal :
Molecular therapy : the journal of the American Society of Gene Therapy
Publication Type :
Academic Journal
Accession number :
26658966
Full Text :
https://doi.org/10.1038/mt.2015.218