PAM-flexible genome editing with an engineered chimeric Cas9

Abstract

CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.

Fig. 1. Engineering, modeling, and PAM Characterization of SpRYc.

A Homology model of SpRYc generated in SWISS-MODEL from PDB 4UN3 and visualized in PyMol. Original domain coordinates are indicated in parentheses above while SpRYc coordinates are indicated below. PAM is indicated in yellow, loop in purple, Sc + + N-terminus in red, and SpRY PID in blue. B PAM enrichment for indicated dCas9 enzymes utilizing PAM-SCANR. Each dCas9 plasmid was electroporated in duplicates, subjected to FACS analysis, and gated for GFP expression based on a negative “No Cas9” control and a positive dSpCas9 control. All samples were performed in independent transformation replicates, and the PAMs of the GFP-positive cells were sequenced via Sanger sequencing. C PAM profiles of SpCas9, Sc + +, SpRY, and SpRYc proteins as determined by HT-PAMDA. Rate constants corresponding to Cas cleavage activity are illustrated as log10 values and are the mean of cleavage reactions against two unique spacer sequences.

Fig. 2. Genome editing capabilities of SpRYc.

A Quantitative analysis of indel formation with indicated Cas9 variants. Indel frequencies were determined via batch analysis following PCR amplification of indicated genomic loci, in comparison to unedited controls for each gene target. All samples were performed in independent transfection replicates and the mean of the quantified indel formation values was calculated. All gRNA sequences can be found in Supplementary Table 1. B Quantitative analysis of A-to-G with indicated ABE8e variants. Base editing conversion rates were determined via BEEP following PCR amplification of indicated genomic loci, in comparison to unedited controls for each gene target. All samples were performed in independent transfection replicates and the mean of the quantified base editing formation values was calculated. All gRNA sequences can be found in Supplementary Table 1. C Off-targets as identified by GUIDE-seq genome-wide for SpCas9, Sc + +, SpRY, and SpRYc each paired with two sgRNAs targeting either EMX1 or VEGFA. Only sites that harbored a sequence with ≤10 mismatches relative to the gRNA were considered potential off-target sites. D Efficiency heatmap of mismatch tolerance assay on genomic targets. Quantified indel frequencies are exhibited for each labeled single or double mismatch (number of bases 5’ upstream of the PAM) in the sgRNA sequence for the indicated Cas9 variant and indicated PAM sequence. All samples were performed in independent transfection replicates and the mean of the quantified indel formation values was calculated.

Fig. 3. Potential disease-associated loci editing applications and structural mechanisms of SpRYc.

A Schematic of SpRYc MECP2 cell line generation and SpRYc-ABE8e editing. Figure was created with BioRender.com. B Base editing conversion rates were determined via CRISPResso2 NGS analysis following PCR amplification of MECP2-integrated loci, in comparison to SpCas9-ABE8e and SpRYc-ABE8e for the C502T installed mutation. Samples were performed in independent nucleofection triplicates (n = 3) ± SD, with the center of error bars depicting the mean. For individual samples, statistical significance was determined by a two-tailed Student’s t test, as compared to the SpCas9-ABE8e control. The exact p values for SpRY-ABE8e and SpRYc-ABE8e compared to SpCas9-ABE8e are both <0.0001. Calculated p values are represented as follows: *, p  <  0.05; **, p  <  0.01; ***, p  <  0.001; ****, p  <  0.0001; ns, not significant. C SpRYc-BE4Max was nucleofected into TruHD cells alongside an sgRNA targeting the HTT repeat. Base editing conversion rate was determined via CRISPResso2 NGS analysis following PCR amplification of the HTT loci. Samples were performed in independent nucleofection triplicates (n = 3) ± SD, with the center of error bars depicting the mean. For individual samples, statistical significance was determined by a two-tailed Student’s t test, as compared to the SpCas9 control. The exact p values for SpRY-BE4Max and SpRYc-BE4Max compared to SpCas9-BE4Max are 0.001 and 0.0028, respectively. Calculated p values are represented as follows: *, p  <  0.05; **, p  <  0.01; ***, p  <  0.001; ****, p  <  0.0001; ns, not significant. The sgRNA and PAM sequences are annotated within their relative positions to the CAG repeat. The red annotation indicates the base to be mutated. The figure was made via Geneious Prime 2023.1.2. D Structural insights via homology modeling in SWISS-MODEL. (i) Interaction of the engineered Sc + + loop (purple) with the backbone of the target strand (TS) PAM region. The REC1 loop from wild-type SpCas9 is indicated in green. (ii) Potential interaction of residue R1331 with the non-target strand (NTS) backbone. (iii) Multiple mutations within the PAM interaction loop allow for a more flexible PAM readout. (iv) The potential van der Waals interaction of W1145 with the ribose moieties of nontarget strand residues could further stabilize the PAM interaction.