Precise DNA cleavage using CRISPR-SpRYgests

Abstract

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.

Figure 1:. Characterization of SpRYgest in vitro cleavage efficiencies.

(a) Comparison of restriction enzymes (REs) that require fixed 4-8 nt motifs and the near-PAMless Cas9 variant, named SpRY, that can target and cleave DNA substrates without sequence constraints. (b) Illustration of SpRYgest in vitro cleavage reaction workflow. Categorization of substrate cleavage is determined at the final timepoint, judged as near-complete (>80%), partial (40-80%), or incomplete (<40%) digests. (c, d) Initial SpRYgest experiments assessing the DNA cleavage efficiencies of WT SpCas9 and SpRY against a linearized plasmid substrate by targeting either a specific region at 1 bp intervals using 12 gRNAs (panel c), or 8 gRNAs designed to be distributed across the substrate (panels d). (e) Comparison of the in vitro cleavage efficiencies of WT, SpG, and SpRY across 64 target sites representing all 2^nd/3^rd/4^th position combinations of an NNNN PAM. Sites with a shifted NNGG PAM are indicated with an asterisk. (f, g) SpRYgest results using additional secondary gRNAs for primary sites in panels 1c and 1e for which partial or incomplete cleavage was observed. Secondary gRNAs were designed to target the opposite DNA strand placing the DSB at the same position as the primary gRNA (panel f), or to target different sites (spacers) but bearing the same PAMs as the primary gRNA (panel g). (h) Summary of the proportion of gRNAs that led to near-complete, partial, or incomplete substrate cleavage when using WT, SpG and SpRY. For panels c-g, cleavage of DNA substrates was quantified by capillary electrophoresis; mean shown for n = 3.

Figure 2:. Molecular cloning via SpRYgest.

(a) Comparison of the DNA cleavage efficiencies of TtAgo and SpRY across 5 sites designed to adhere to TtAgo guide requirements. TtAgo reactions were performed with pairs of 5’P-ssDNA guides. TtAgo and SpRYgest reactions were performed for 60 and 216 minutes, respectively. Individual datapoints, mean, and s.e.m. shown for n = 3. (b) Proportion of TtAgo and SpRY guides that led to nearly complete, partial, or incomplete cleavage on the 20 target sites from Figs. 1c and 1d (see TtAgo results in Supplementary Fig. 9f) and the 5 sites from Fig. 2a. For panels a and b, cleavage of DNA substrates was quantified by capillary electrophoresis; mean shown for n = 3. (c, d) Schematics of the SpRYgest strategies to add P2A-EGFP sequences to SaCas9-ABE8e and PE2 via single and double SpRYgests, panels c and d, respectively. (e, f) Proportion of clones for which correct addition of the P2A-EGFP sequence was confirmed by Sanger sequencing for SaCas9-ABE8e and PE2 strategies, panels e and f, respectively. (g) Schematic of the SpRYgest strategy to add an NLS to the N-terminal end of SpCas9. (h) Proportion of clones for which correct addition of the N-terminal NLS was confirmed by Sanger sequencing.

Figure 3:. Rapid generation of saturation mutagenesis libraries via SpRYgest.

(a,b) Schematics of SpRYgest strategies to generate saturation mutagenesis libraries of SpCas9 residues important for catalytic activity (panel a) and NGG PAM preference (panel b). (c,d) Sanger sequencing traces and next-generation sequencing results from the libraries, illustrating the nucleotide diversity at mutated residues for the HNH-catalytic and PAM-interacting (PI) domain libraries, panels c and d, respectively. Recoded silent substitutions were intentionally included in the library to assess construction efficiency (highlighted in purple and indicated with a triangle). (e) Schematic of the bacterial positive selection assay^,,, which permits selection of cleavage competent SpCas9 enzymes from saturation mutagenesis libraries. Colonies survive on selective media only when SpCas9 and a gRNA cleave a target site on the toxic plasmid. Mutated regions of SpCas9 can be sequenced from the plasmids harbored within surviving colonies. (f,g) Post-selection results for cleavage competent SpCas9 variants from the catalytic domain HNH residue library (panel f), or from the PI domain library selected against toxic plasmids harboring target sites with NGG and NGAG PAMs (left and right sides of panels g, respectively). Pie charts illustrate the distribution of amino acids at each position in the pre-selection libraries (via NGS) and post-selection libraries (via Sanger sequencing of individual clones) in the top and bottom panels, respectively.

Figure 4:. Optimization of rapid, efficient, and specific SpRYgest reactions.

(a) Comparison of the hands-on and hands-off times of optimized SpRYgests (with gRNAs generated via one-pot reactions) versus RE digests. Approximate times in minutes are shown; lines not drawn to scale. The incubation times can vary for one-pot gRNA generation (see Sup. Fig. 17b), SpRYgest, or restriction enzyme digests. (b) Agarose gel of SpRYgest reactions performed with gRNAs taken directly from standard or scaled-down one-pot gRNA generation reactions (20 μL or 5 μL one-pot reactions, respectively). Plasmid conformations are: R, relaxed; L, linear; S, supercoiled; 1 kb Plus DNA Ladder (N.E.B.); n = 1. (c) Proportion of unique DSBs that were successfully generated via SpRYgest in all experiments of this study. (d) Proportion of gRNAs for which off-target cleavage products were detected during SpRYgests with gRNAs used in all experiments of this study.