Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants

Abstract

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

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.

Fig. 2.. Engineering and characterization of SpCas9 variants capable of targeting NRN PAMs.

(A) Crystal structure of SpCas9 to illustrate amino acid side chains of R1333 and selected PAM-proximal residues. The non-target strand (NTS) is hidden for clarity. Image generated from PDB ID 4UN3(20). (B) Modification of endogenous sites in human cells bearing different NRN PAMs with WT SpCas9, SpG, and SpG derivatives. Editing assessed by targeted sequencing; mean, s.e.m., and individual data points shown for n = 3. (C) HT-PAMDA characterizations of WT SpCas9, SpG, and SpG derivatives to illustrate their NRNN 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). (D) Modification of endogenous sites in human cells bearing different NRN PAMs SpG(L1111R/A1322R/R1333P) and derivatives bearing additional substitutions. See Fig. S8C for all variants tested. Editing assessed by targeted sequencing; mean, s.e.m., and individual data points shown for n = 3.

Fig. 3.. Comparison of WT SpCas9 and SpRY nuclease and base editor activities across NNN PAM sites in human cells.

. (A, B) Mean nuclease activity plots for WT SpCas9 and SpRY on 64 sites with NRN PAMs (panel A) and 31 sites with NYN PAMs (panel B) in human cells. The black line represents the mean of 8 or 3-4 sites (panels a and b, respectively) for each PAM of the indicated class (see also Figs. S9A-C), and the grey outline is a violin plot. (C, D) C-to-T base editing of endogenous sites in human cells bearing NRN and NYN PAMs (panels C and D, respectively) with WT SpCas9 and SpRY-CBE4max constructs. Editing of cytosines in the edit window (positions 3 through 9) assessed by targeted sequencing; the five NYN PAM target sites were selected from high-activity sites in panel B; mean, s.e.m., and individual data points shown for n = 3. (E, F) A-to-G base editing of endogenous sites in human cells bearing NRN and NYN PAMs (panels E and F, respectively) with WT SpCas9 and SpRY-ABEmax constructs. Editing of adenines in the edit window assessed by targeted sequencing; the five NYN PAM target sites were selected from high-activity sites in panel B; mean, s.e.m. and individual data points shown for n = 3. For base editing data in panels C-F, see also Table S5. (G) Relative nuclease activity plots for SpCas9-HF1, SpCas9-NG-HF1, SpG-HF1, and SpRY-HF1 compared to their parental variants across 3-10 sites endogenous sites in HEK 293T cells. Mean modification from sites in Fig. S11A shown as dots, with black line representing the mean of those sites, and the grey outline is a violin plot. The HF1 variants additionally encode N497A, R661A, Q695A, and Q926A substitutions(21). (H) Histogram of the number of GUIDE-seq detected off-target sites for SpCas9 variants across sites with NGG, NGN, and NAN PAMs (see Figs. S12A-C, respectively). (I) Fraction of GUIDE-seq reads attributed to the on- and off-target sites for WT SpCas9, SpG, SpRY, and their respective HF1 variants across 2-6 targets (see also Figs. S12A-C and Table S6).

Fig. 4.. Expanded capabilities of C-to-T base editors with SpG and SpRY to generate protective genetic variants.

(A) Illustration of the relative C-to-T edit activity of CBE4max constructs depending on the position of the cytosine in the spacer (as reviewed in(28)) (B, C) Comparison of the C-to-T editing activities of WT SpCas9, SpG, and SpRY CBE4max constructs across 22 target sites covering ten previously described protective genetic variants accessible or inaccessible with target sites harboring NGG PAMs (panels B and C, respectively). The intended edit highlighted with an orange arrow; C-to-T editing for cytosines within the spacer that are edited above 1% by any variant are plotted for all appropriate variant/guide combinations; SpG was tested only on sites harboring NGN PAMs; editing of cytosines assessed by targeted sequencing with mean C-to-T editing shown for n = 3. The intended edit, bystander synonymous, non-synonymous, and stop codon C-to-T edits are indicated; the PAMs for each target site are shown in the grey arrow annotation; for raw data see Table S5.