Fig. 1. Engineering and characterization of SpCas9 variants capable of targeting NGN PAMs.

(A) Schematic of SpCas9, highlighting the PAM-interacting (PI) domain along with R1333 and R1335 that make base-specific contacts to the guanines of the NGG PAM. (B) Rendering of a crystal structure of SpCas9 with amino acid side chains proximal to the second guanine of the NGG PAM shown in yellow. In the zoomed image the non-target strand (NTS) is hidden for clarity. Image generated from PDB ID 4UN3(20). (C) HT-PAMDA characterization of wild-type (WT) SpCas9 and engineered variants to illustrate their NGNN PAM preferences. The log₁₀ rate constants (k) are the mean of at least two replicates against two distinct spacer sequences (see also Figs. S2A-C, E). (D) Modification of endogenous sites in human cells bearing a canonical and noncanonical PAMs with WT SpCas9 and SpG. Editing assessed by targeted sequencing; mean, s.e.m., and individual data points shown for n = 3. (E) Mean nuclease activity plots for WT, xCas9(23), SpCas9-NG(22), and SpG on 78 sites with NGN PAMs in human cells. The black line represents the mean of 19-20 sites for each PAM class (see also Fig. S5A), and the grey outline is a violin plot. (F) HT-PAMDA characterization of WT, xCas9, SpCas9-NG, and SpG to illustrate their NGNN PAM preferences. The log₁₀ rate constants (k) are the mean of at least two replicates against two distinct spacer sequences. (G) Mean C-to-T editing plots for WT, xCas9, SpCas9-NG, and SpG cytosine base editors (CBEs) on 57 cytosines within the editing windows (positions 3 through 9) of 20 target sites harboring NGN PAMs in human cells. The black line represents the mean of 12-16 cytosines for each PAM class (see also Fig. S6A), and the grey outline is a violin plot. (H) CBE-HT-PAMDA data for WT, xCas9, SpCas9-NG, and SpG to illustrate their NGNN PAM preferences. The log₁₀ rate constants (k) are single replicates against one spacer sequence (see also Figs. S6C, D). (I) Mean A-to-G editing plots for WT, xCas9, SpCas9-NG, and SpG adenine base editors (ABEs) on 24 adenines within the editing windows (positions 5 through 7) of 21 target sites harboring NGN PAMs in human cells. The black line represents the mean of 3-9 adenines for each PAM class (see also Fig. S7A), and the grey outline is a violin plot.