title: Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome.
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.
authors.affiliation: Institute for Cell and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.|Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Müller
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.|Department of Bioengineering, Rice University, Houston, Texas, USA.
authors.forename: Ciaran M
authors.initials: CM
authors.lastname: Lee
authors.affiliation: Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Giedrius
authors.initials: G
authors.lastname: Gasiunas
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.|Department of Bioengineering, Rice University, Houston, Texas, USA.
authors.forename: Timothy H
authors.initials: TH
authors.lastname: Davis
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.|Current address: CRISPR Therapeutics, Cambridge, Massachusetts, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Cradick
authors.affiliation: Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.|Department of Bioengineering, Rice University, Houston, Texas, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
authors.affiliation: Institute for Cell and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.|Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.affiliation: Institute for Cell and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.|Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany.
authors.forename: Claudio
authors.initials: C
authors.lastname: Mussolino
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D064113:CRISPR-Cas Systems; D002460:Cell Line; D004720:Endonucleases / Q000378:metabolism; D004789:Enzyme Activation; D000072669:Gene Editing; D005822:Genetic Vectors; D015894:Genome, Human; D006801:Humans; D011485:Protein Binding; D000094704:RNA, Guide, CRISPR-Cas Systems; D048249:Streptococcus thermophilus / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D004720:Endonucleases