Specificity of four SpeCas9 variants.

(A) Schematic of SpeCas9 structure. The amino acid residues important for specificity are shown below. (B) Test of four SpeCas9 variant specificity. Schematic of the GFP activation assay for specificity analysis is shown on the top. A panel of sgRNAs with dinucleotide mutations is shown below. sgRNA activities were measured based on GFP expression. Cells without Cas9 transfection were used as a negative control (NC). Mismatches are shown in red (n = 3). Underlying data for all summary statistics can be found in S1 Data. (TIF)</p

Analysis of sgRNAs.

(A) Alignment of sgRNA scaffolds for six SaCas9 orthologs. The GAAA linker are indicated by the black box. (B) Analysis of SaCas9 orthologs’ secondary RNA structures. These structures were generated by an online tool named RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). (TIF)</p

Evaluation of Sha2Cas9-HF and SpeCas9-HF on-target activities.

(A) Comparison of activities of high-fidelity Cas9s to the wild-type Cas9s (n = 3). The target sequences are shown on the left. PAM is underlined. If the first nucleotide is C or T, additional “g” is added for U6 promoter transcription. Underlying data for all summary statistics can be found in S1 Data. (B) Quantification of editing efficiency for SaCas9, SmiCas9, Sha2Cas9, and SpeCas9. Underlying data for all summary statistics can be found in S1 Data.</p

Analysis of five SaCas9 ortholog activities.

(A) Amino acid sequences of the SaCas9 ortholog PI domain are aligned. The residues that are important for PAM recognition are indicated at the top; the conserved residues among newly identified SaCas9 orthologs are shown in red; the names of newly identified Cas9s are shown in green. (B) Design of the GFP activation reporter construct. A target sequence (protospacer) containing a 7-bp random sequence is inserted between ATG and the GFP-coding sequence. The library DNA is stably integrated into HEK293T cells by lentivirus. (C) Transfection of SaCas9 orthologs induced GFP expression. Percentage of GFP-positive cells was shown. The cells without transfection of Cas9 were used as a negative control.</p

Target sites used in this study.

A list of the endogenous target sites of human and their downstream PAM. PAM, protospacer adjacent motif. (DOCX)</p

Analysis of the SaCas9 variant PAMs.

(A) Amino acid sequence of the SaCas9 variant PI domains. The residues that are important for PAM recognition are marked at the top; the mutations are highlighted in red. (B) SaCas9 variant PAMs were analyzed by the GFP activation assay. WebLogos generated by analyzing the deep sequencing data. (TIF)</p

Raw images of Figs 3 and S4.

(JPG)</p

Protein sequence alignment of SaCas9, Sha2Cas9, and SpeCas9.

The amino acid residues important for specificity are indicated by vertical lines above. The amino acid residue positions are shown on the right. (TIF)</p

Analysis of the PAM sequence of Cas9.

(A) Deep sequencing reveals that SmiCas9, Sha2Cas9, and SpeCas9 generated indels on the targets. (B) WebLogos were generated based on the deep sequencing data. (C) PAM wheels were generated based on the deep sequencing data.</p

Primers used in this study.

A list of oligonucleotide pairs and primers used for deep sequencing. (DOCX)</p