a, Expanded schematic of the positive selection from (left panel), and validation that SpCas9 behaves as expected in the positive selection (right panel). b, Schematic of how the positive selection was adapted to select for SpCas9 variants that have altered PAM recognition specificities. A library of SpCas9 clones with randomized PAM-interacting (PI) domains (residues 1097-1368) is challenged by a selection plasmid that harbors an altered PAM. Variants that survive the selection by cleaving the positive selection plasmid are sequenced to determine the mutations that enable altered PAM specificity.

a, Sequences of variants that survived >10% when re-tested in the positive selection assay against an NGA PAM site (see Online Methods). Variants were selected from libraries containing randomly mutagenized PAM-interacting (PI) domains (residues 1097–1368) with or without a starting R1335Q mutation. Sequence differences compared with wild-type SpCas9 are highlighted. The histogram represents the number of changes at each position (not counting the starting R1335Q mutation). b, Sequences of variants that survived >10% when re-tested in the positive selection assay against a site containing an NGC PAM. Variants were selected from libraries containing randomly mutagenized PAM-interacting (PI) domains (residues 1097–1368) with starter mutation pairs of R1335E/T1337R or R1335T/T1337R. Sequence differences compared with wild-type SpCas9 (shown at the top) are highlighted. The histogram below illustrates the number of changes at each position (not counting starter mutations at R1335 or T1337).

a, Expanded schematic illustrating the negative selection from (left panel), and validation that wild-type SpCas9 behaves as expected in a screen of sites with functional (NGG) and non-functional (NGA) PAMs (right panel). b, Schematic of how the negative selection was used as a site-depletion assay to screen for functional PAMs by constructing negative selection plasmid libraries containing 6 randomized base pairs in place of the PAM. Selection plasmids that contain PAMs cleaved by a Cas9/sgRNA of interest are depleted while PAMs that are not cleaved (or poorly cleaved) are retained. The frequencies of the PAMs following selection are compared to their pre-selection frequencies in the starting libraries to calculate the post-selection PAM depletion value (PPDV). c, d, A cutoff for statistically significant PPDVs was established by plotting the PPDV of PAMs for catalytically inactive SpCas9 (dCas9) (grouped and plotted by their 2^nd/3^rd/4^th positions) for the two randomized PAM libraries (c). A threshold of 3.36 standard deviations from the mean PPDV for the two libraries was calculated (red lines in (d)), establishing that any PPDV deviation below 0.85 is statistically significant compared to dCas9 treatment (red dashed line in (c)). The gray dashed line in (c) indicates a five-fold depletion in the assay (PPDV of 0.2).

Data points represent the average EGFP disruption of the two NGAN and NGNG PAM sites for the VQR and EQR variants () plotted against the mean PPDV observed for library 1 and 2 () for the corresponding PAM. The red dashed line indicates PAMs that are statistically significantly depleted (PPDV of 0.85, see ), and the gray dashed line represents five-fold depletion (PPDV of 0.2). Mean values are plotted with the 95% confidence interval.

a, Structural representations of the six residues implicated in PAM recognition. The left panel illustrates the proximity of D1135 to S1136, a residue that makes a water-mediated, minor groove contact to the 3^rd base position of the PAM. The right panel illustrates the proximity of G1218, E1219, and T1337 to R1335, a residue that makes a direct, base-specific major groove contact to the 3^rd base position of the PAM. Angstrom distances indicated by yellow dashed lines; non-target strand guanine bases dG2 and dG3 of the PAM are shown in blue; other DNA bases shown in orange; water molecules shown in red; images generated using PyMOL from PDB:4UN3. b, Mutational analysis of six residues in SpCas9 that are implicated in PAM recognition. Clones containing one of three types of mutations at each position were tested for EGFP disruption with two sgRNAs targeted to sites harboring NGG PAMs. For each position, we created an alanine substitution and two non-conservative mutations. S1136 and R1335 were previously reported to mediate contacts to the 3^rd guanine of the PAM, and D1135, G1218, E1219, and T1337 are reported in this study. EGFP disruption activities were quantified by flow cytometry; background control represented by the dashed red line; error bars represent s.e.m., n = 3.

For each target locus, the wild-type sequence is shown at the top with the protospacer highlighted in yellow (highlighted in green if present on the complementary strand) and the PAM is marked as red underlined text. Deletions are shown as red dashes highlighted in gray and insertions as lower case letters highlighted in blue. The net change in length caused by each indel mutation is shown on the right (+, insertion; –, deletion). Note that some alterations have both insertions and deletions of sequence and in these instances the alterations are enumerated in parentheses. The number of times each mutant allele was recovered (if more than once) is shown in brackets.

a, Sequences targeted by wild-type, VQR, and VRER SpCas9 are shown in blue, red, and green, respectively. Sequences of sgRNAs and primers used to amplify these loci for T7E1 are provided in and . b, Mean mutagenesis frequencies detected by T7E1 for wild-type SpCas9 at eight target sites bearing NGG PAMs in the four different endogenous human genes (corresponding to the annotations in the top panel). Error bars represent s.e.m., n = 3.

The intended on-target site is marked with a black square, and mismatched positions within off-target sites are highlighted. a, The specificity of the VQR variant was assessed in human cells by targeting endogenous sites containing NGA PAMs: EMX1 site 4, FANCF site 1, FANCF site 3, FANCF site 4, RUNX1 site 1, RUNX1 site 3, VEGFA site 1, and ZSCAN2. b, The specificity of the VRER variant was assessed in human cells by targeting endogenous sites containing NGCG PAMs: FANCF site 3, FANCF site 4, RUNX1 site 1, VEGFA site 1, and VEGFA site 2.

a, Mutagenesis frequencies detected by T7E1 for wild-type and D1135E SpCas9 at six endogenous sites in human cells. Error bars represent s.e.m., n = 3; mean fold change in activity is shown. b, Titration of the amount of wild-type or D1135E SpCas9-encoding plasmid transfected for EGFP disruption experiments in human cells. The amount of sgRNA plasmid used for all of these experiments was fixed at 250 ng. Two sgRNAs targeting different EGFP sites were used; error bars represent s.e.m., n = 3. c, Targeted deep-sequencing of on- and off-target sites for 3 sgRNAs using wild-type and D1135E SpCas9. The on-target site is shown at the top, with off-target sites listed below highlighting mismatches to the on-target. Fold decreases in activity with D1135E relative to wild-type SpCas9 at off-target sites greater than the change in activity at the on-target site are highlighted in green; control indel levels for each amplicon are reported. d, Mean frequency of GUIDE-seq oligo tag integration at the on-target sites, estimated by restriction fragment length polymorphism analysis. Error bars represent s.e.m., n = 4. e, Mean mutagenesis frequencies at the on-target sites detected by T7E1 for GUIDE-seq experiments. Error bars represent s.e.m., n = 4. f, GUIDE-seq read-count differences between wild-type SpCas9 and D1135E at 3 endogenous human cell sites. The on-target site is shown at the top and off-target sites are listed below with mismatches highlighted. In the table, a ratio of off-target activity to on-target activity is compared between wild-type and D1135E to calculate the normalized fold-changes in specificity (with gains in specificity highlighted in green). For sites without detectable GUIDE-seq reads, a value of 1 has been assigned to calculate an estimated change in specificity (indicated in orange). Off-target sites analyzed by deep-sequencing in panel c are numbered to the left of the EMX1 site 3 and VEGFA site 3 off-target sites

a, PPDV scatterplots for St1Cas9 comparing the sgRNA complementarity lengths of 20 and 21 nucleotides obtained with a randomized PAM library for spacer 1 (top panel) or spacer 2 (bottom panel). PAMs were grouped and plotted by their 3^rd/4^th/5^th/6^th positions. The red dashed line indicates PAMs that are statistically significantly depleted (see ) and the gray dashed line represents five-fold depletion (PPDV of 0.2). b, Table of PAMs with PPDVs of less than 0.2 for St1Cas9 under each of the four conditions tested. PAM numbering shown on the left is the same as in , PPDV scatterplots for SaCas9 comparing the sgRNA complementarity lengths of 21 and 23 nucleotides obtained with a randomized PAM library for spacer 1 (top panel) or spacer 2 (bottom panel). PAM were grouped and plotted by their 3^rd/4^th/5^th/6^th positions. The red and gray dashed lines are the same as in (a). d, Table of PAMs with PPDVs of less than 0.2 for SaCas9 under each of the four conditions tested. PAM numbering is the same as in , Human cell activity of St1Cas9 and SaCas9 across various spacer lengths via EGFP disruption (panel e, data from ) and endogenous gene mutagenesis detected by T7E1 (panel f, data from ). Activity for all replicates shown (n = 3 or 4); bars illustrate mean and 95% confidence interval; number of sites per spacer length indicated.

a, Activity of wild-type and mutant SpCas9s assessed via U2OS human cell-based EGFP disruption. Frequencies were quantified by flow cytometry; error bars represent s.e.m., n = 3; mean level of background EGFP loss represented by the dashed red line for this and subsequent panels (c, g, h, and j). b, Schematic of the positive selection assay (see also ). c, Combinatorial assembly and human cell testing of mutations obtained from the positive selection for SpCas9 variants that can cleave a target site containing an NGA PAM, using the EGFP disruption assay. d, Schematic of the negative selection assay, adapted to profile Cas9 PAM specificity by generating a library of plasmids that contain a randomized sequence adjacent to the 3’ end of the protospacer (see also ). e, Scatterplot of the post-selection PAM depletion values (PPDVs) of wild-type SpCas9 with two randomized PAM libraries (each with a different protospacer). PAMs are plotted by their 2^nd/3^rd/4^th positions. The red dashed line indicates statistically significant depletion (obtained from a dCas9 control experiment, see ), and the gray dashed line represents five-fold depletion (PPDV of 0.2). f, PPDV scatterplots for the VQR and EQR variants. g, EGFP disruption frequencies for wild-type, VQR, and EQR SpCas9 on sites with NGAN and NGNG PAMs. h, Combinatorial assembly and human cell testing of mutations obtained from the positive selection for SpCas9 variants that can cleave a target site containing an NGC PAM, using the EGFP disruption assay. i, PPDV scatterplot for the VRER variant. j, EGFP disruption frequencies for wild-type and VRER SpCas9 on sites with NGCN and NGNG PAMs.

a, Mutagenesis frequencies in zebrafish embryos induced by wild-type or VQR SpCas9 at endogenous gene sites bearing NGAG PAMs. Mutation frequencies were determined using the T7E1 assay; n.d., not detectable by T7E1; error bars represent s.e.m., n = 5 to 9 embryos. b, Endogenous gene disruption activity of the VQR variant quantified by T7E1 assay. Error bars represent s.e.m., n = 3. c, Endogenous gene disruption activity of wild-type SpCas9 against NGA PAM sites quantified by T7E1 assay, where VQR data is re-presented from panel b for ease of comparison. Error bars represent s.e.m., n = 3. d, Mutation frequencies of wild-type, VRER, and VQR SpCas9 at endogenous human cell sites containing NGCG PAMs quantified by T7E1 assay; error bars represent s.e.m., n = 3. e, Representation of the number sites in the human genome with 20 nt spacers targetable by wild-type, VQR, and VRER SpCas9. The 5’-G is included for expression from a U6 promoter. f, Number of off-target cleavage sites identified by GUIDE-seq for the VQR and VRER variants using sgRNAs from panels b and d.

a, PPDV scatterplots for wild-type and D1135E SpCas9 for the two randomized PAM libraries. PAMs are plotted by their 2^nd/3^rd/4^th positions, and wild-type data is the same as shown in for ease of comparison. The red dashed line indicates PAMs that are statistically significantly depleted (see ), and the gray dashed line indicates five-fold depletion (PPDV of 0.2). b, EGFP disruption activities of wild-type and D1135E SpCas9 on sites that contain canonical and non-canonical PAMs in human cells. Disruption frequencies were quantified by flow cytometry; mean background level of EGFP loss represented by the dashed red line; error bars represent s.e.m., n = 3; fold change in activity is shown. c, Summary of targeted deep-sequencing data demonstrating specificity gains at off-target sites when using D1135E (see also ). d, Summary of GUIDE-seq detected changes in specificity between wild-type and D1135E at off-target sites (see also ). Estimated fold-gain in specificity at sites without read-counts for D1135E are not plotted (see ).

a, b, PPDV scatterplots for St1Cas9 (panel a) and SaCas9 (panel b), with PAMs plotted by their 3^rd/4^th/5^th/6^th positions. The red dashed line indicates PAMs that are statistically significantly depleted (), and the gray dashed line represents five-fold depletion (PPDV of 0.2); α, PAM previously predicted by a bioinformatic approach; β, PAMs previously identified under stringent experimental conditions; *, novel PAMs discovered in this study; γ, PAMs previously identified under moderate experimental conditions c, Survival percentages of St1Cas9 and SaCas9 in the bacterial positive selection when challenged with selection plasmids that harbor different target sites and PAMs. d, e, Mutation frequencies of St1Cas9 (panel d) and SaCas9 (panel e) quantified by T7E1 assay at sites in four endogenous human genes. Error bars represent s.e.m., n = 3; n.d., not detectable by T7E1.