Directed Evolution of Targeted Cas9 Cleavage to the LCA10 Splice Donor Mutation Barrett Steinberg, Derek Cerchione, Morgan Maeder, Max Skor, Hari Jayaram, Vic Myer

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Directed evolution of targeted Cas9 cleavage to the LCA10 splice donor mutation Barrett Steinberg, Derek Cerchione, Morgan Maeder, Max Skor, Hari Jayaram, Vic Myer

Editas Medicine Inc., Cambridge, MA 02142

Abstract FIGURE 2. Adapting Directed Evolution Method for CEP290 FIGURE 4. Inducible Toxin Allows for Tunable Selection Stringency

Disease-conferring mutation CRISPR/Cas9 has wide reaching scientific A single point mutation at the and therapeutic applications, but exactly A CEP290 locus introduces a A disease causing splice donor Growth curves were measured targeting cutting sites efficiently remains + toxin site in patients with Leber’s under varying conditions to difficult. Several cas9 variants have been + toxin Selection window with Cas expression assess the selection stringency engineered to target alternate PAMs; TRRCNN congenital amaurosis (LCA10) - toxin however, generation of new variants is often (Figure 2A). Using SaCas9 to of our system. The inducible + tox. toxin greatly reduces the laborious. Additionally, the cutting ability of generate a nearby indel would - tox. nucleases is often highly guide-dependent disrupt the cryptic exon. Robust toxin allows wide selection window growth rate of cells in liquid and shows a large amount of variation However, there are few media, as indicated by the between sites. In order to quickly engineer B canonical PAMs close to the green arrow (Figure 4A). new nuclease variants targeted to cleave a mutation, making access to the Successful Cas9 cleavage of given site, we developed a directed region difficult. Currently, we the target provides a evolution platform to select for DNA are engineering an SaCas9 to competitive advantage, as cleavage in bacteria. Our system allows recognize an NNCRRT PAM B shown by the black arrow. rapid mutation of all amino acids throughout near the mutation site. Our Robust toxin activity the protein, expanding potential diversity of directed evolution setup + toxin Subjecting our evolved mutants functional mutants. These mutant libraries includes an SaCas9 library and + toxin to the toxin yields similar are challenged with phage containing target gRNA on one plasmid and a - toxin results to that of WTCas9. sites in a competitive pool. Using this toxin and target site on a + tox. Inducing SMART libraries also method, we are developing S. aureus cas9 second plasmid (Figure 2B). + tox. provides selective advantage to Successful cleavage of the Wide positive - tox. variants which have altered PAM selection window cells as demonstrated by the preferences to target cleavage to the LCA10 target site by a mutant SaCas9 purple arrow, indicating library IVS26 splice donor mutation. This will ensure that it outcompetes functionality (Figure 4B). engineered mutant can be efficiently other variants in each round of packaged into AAV, allowing potential directed evolution. therapeutic use. FIGURE 3. SMART Library Generation FIGURE 5. Identifying and Characterizing Novel Enzymes FIGURE 1. Directed Evolution After three rounds of directed A Overview Mutant SaCas9 libraries A Cas9 B evolution, libraries were subjected to were engineered using the NGS (Figure 5A). Multiple residues scanning mutagenesis at were enriched in distinct regions of random targets (SMART) the protein, most interestingly in the protocol (Figure 3A). The PI domain. process begins with a modified PCR step, in Deep We are currently synthesizing and sequence which forward oligos, testing the efficiency of these containing codon mutants by cleaving at the CEP290 mismatches, introduce Mutate Select Pick site with an NNCRRT PAM. variants degeneracy at every amino acid position across the C PAM for evolved enzymes will be Directed Evolution protein (Figure 3C). Undesired reaction verified by our PAM validation assay Platform products were removed (Figure 5B). Here mutants will be from the library with a exposed to a target with a B C Screen in Synthesize completely degenerate PAM vitro cocktail of nucleases. Libraries were then sequence. After ligating on a Miseq transformed into E. coli and adapter, the sequencing results can Purify infected with phage be used to generate PAM logos, containing a toxic gene and which can validate PAM preferences a target site with a of our evolved variants (Figure 5C). Our directed evolution platform first begins noncanonical NNCRRT Conclusion with generating SaCas9 libraries and PAM (Figure 3B). subjecting them to several rounds of Our directed evolution platform allows for high yielding rapid generation of improved Cas9 variants. The SMART selection. After deep sequencing of our method provides considerable diversity and unbiased access across the entire SaCas9 gene, as opposed to single libraries, we synthesize and purify mutants of Acknowledgement point mutation mutagenesis methods, such as error prone PCR. Rigorous selection parameters ensure high quality interest for further downstream Special thanks to the Editas Scientific team for discussions around various aspects of the project. mutants that can be characterized quickly and efficiently in our downstream processing. The method can be easily characterization. adapted to other directed evolution projects, such as increasing cleavage efficiency, improving specificity, and introducing new enzyme functions.