PAM-Flexible Genome Editing with an Engineered Chimeric Cas9

Introduction:: To conduct programmable genome editing, CRISPR-associated (Cas) endonucleases require a protospacer adjacent motif (PAM) to follow the target DNA sequence specified by the guide RNA (gRNA).The widely-utilized Cas9 from Streptococcus pyogenes bacteria (SpCas9) requires a 5'-NGG-3' PAM, which imposes severe accessibility constraints for therapeutically-relevant editing applications requiring precise genomic positioning, such as base editing and homology-directed repair.



To expand the targetable sequence space of CRISPR, we previously engineered Sc++, a variant of ScCas9 which enables a 5'-NNG-3' preference, rather than the canonical 5'-NGG-3'. Concurrent with the development of Sc++, Walton, et al. engineered a near-PAMless Cas9, termed SpRY, which enables strong 5'-NRN-3' specificity, alongside weaker 5'-NYN-3' targeting. Both Sc++ and SpRY thus represent exciting advances in CRISPR-based genome editing due to their robust editing characteristics and unprecedented genomic accessibility.


In this study, we combine Sc++ and SpRY to engineer a chimeric Cas9 enzyme that can induce edits with highly orthogonal PAM targeting. To do this, we employ experimental enzyme engineering and computational modeling to graft the PID of SpRY to the N-terminus of Sc++, generating a chimeric SpRY-Sc++ Cas9 (herein referred to as SpRYc). We demonstrate that SpRYc specifically edit various 5'-NNN-3' PAM targets in human cells, enabling unique editing applications. Finally, we conduct homology modeling to gain insights into the protein-DNA interactions that may enable SpRYc's PAM flexibility. In total, SpRYc’s demonstrated PAM flexibility offers numerous opportunities for broad-targeting genome editing applications and therapeutic translation.

Materials and Methods:: To generate SpRYc, we combined the N-terminal ORF of Sc++ (residues 1-1110) and residues 1111-1368 of SpRY's ORF (Figure 1A). Analogously, the ORFs of SpCas9, SpRY, and SpRYc were integrated within the ABE8e backbones. To characterize PAM of SpRYc, we adapted a positive selection bacterial screen based on green fluorescent protein (GFP) expression conditioned on PAM binding, termed PAM-SCANR. We also performed the HT-PAMDA and GUIDE-Seq as described previously.39,21.To evaluate SpRYc’s activity at diverse gene sequences, we compared the PAM specificities and DNA cleavage capabilities of SpRYc to SpCas9 and SpRY by transfecting HEK293T cells with plasmids expressing each Cas9 alongside one of sixteen sgRNAs which were directed to various genomic loci representing every two-base PAM combination (5'-NNN-3'), and the off target test was performed using unbiased GUIDE-Seq method. At final, we sought to investigate its utility as a potential therapeutic modality for the treatment of genetic diseases, which we took corresponidng mutations in Rett syndrome to test efficiency of base editing.


Results, Conclusions, and Discussions:: Results

The PAM-SCANR results suggest that while SpRY preferentially binds to an A or G at position 2 in the PAM, SpRYc more potently binds adenine bases at position 2, but does not bias against any specific base either (Figure 1B). The HT-PAMDA assay showes that SpRYc exhibited slower cleavage rates than SpRY, suggesting that SpRYc may elicit its optimal activity in either its “dead” or nickase variants, rather than as a nuclease (Figure 1C). The target region genomic editing results demonstrated that SpRYc generates modifications at all tested genomic loci, performing comparably to SpRY, and more optimally on select 5’-NYN-3’ loci (Figure 2A). The similar results were also found with SpRYc-ABE8e , which can base edit at all tested genomic sequences (Figure 2B).



The genome-wide GUIDE-Seq results demonstrate that compared to SpRY, SpRYc has nearly four-fold lower off-target activity with the VEGFA-targeting guide RNA, and two-fold lower off-target activity when directed against the EMX1 site (Figure 2C). We corroborated this data via a mismatch tolerance assay, which SpRYc exhibited decreased activity on mismatched sequences, as compared to SpRY, with slightly lowered on-target activity (Figure 2D). We finally choose a pathogenic mutation in MECP2 to investigate its utility as a potential therapeutic modality for the treatment of genetic diseases (Figure 3A). After subsequent DNA extraction, loci amplification, and sequencing, we demonstrate that SpRYc-ABE8e can effectively mutate MECP2, including over 20% editing efficiency at the C502T mutation (Figure 3B).



Conclusions and discussion

In this study, we engineer a chimeric Cas9 by harnessing the structural properties of SpRY and Sc++ to generate SpRYc, a Cas9 with flexible PAM preference. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique approaches to generate SpRYc, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning. While SpRYc serves as a step forward towards unrestricted, fully programmable genome editing, its development, more importantly, represents a culmination of a variety of state-of-the-art in silico and in vitro PAM engineering methods.


Acknowledgements (Optional): :

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