Programmable RNA-guided nucleases are facilitating top-down genome editing endeavors throughout the biomedical research community, and show great potential for use in therapeutic applications. The sequence specificity of commonly used RNA-guided nucleases, including Streptococcus pyogenes Cas9 (SpCas9), is jointly enabled by RNA-DNA base pairing and essential interactions with a target-abutting DNA sequence known as a protospacer adjacent motif (PAM). Although SpCas9 can accommodate virtually any guide RNA sequence, its sequence-specific interactions with PAMs are invariably determined by protein-DNA contacts. SpCas9?s overall programmability is therefore constrained by its intrinsic PAM requirement, and this can limit editing applications that require very precise target site positioning. Previous studies have employed procedures for protein engineering, including directed evolution, to alter the protein component of RNA-guided nucleases such as SpCas9 in various ways. While not exhaustive, some of these studies have successfully produced variants of SpCas9 with certain alternative PAM specificities. Among these, the directed evolution regimens tested to date involved custom microbial systems engineered to impose positive selection on SpCas9?s PAM specificity in vivo. Positive selection is sufficient to drive the evolution of protein variants with relaxed specificity, as well as variants with truly altered, orthogonal specificity. Whereas relaxed PAM specificities that broaden the genomic sequence space accessible for targeting may be ideal for typical editing applications, orthogonal variants are still of use in certain allele-specific editing applications where the PAM requirement can be exploited to discriminate between two alleles that differ by only a single nucleotide. To favor the evolution of orthogonal variants, directed evolution procedures may incorporate negative selection pressures that counterselect against variants which retain their parental substrate preferences. Whether such strategies would be effective for engineering orthogonal PAM specificities has not been investigated, however. The goal of this proposed study is to establish and evaluate microbial directed evolution regimens that impose simultaneous positive and negative selection, or positive selection alone, on SpCas9?s PAM specificity. Side- by-side evaluation of these procedures will be enabled by high-throughput profiling of variant pools evolved in the absence or presence of negative selection, along with mutational dissections and clonal benchmarking assays. Collectively, these experiments will ensure identification of the most desirable SpCas9 variants, and guide the systematic refinement of directed evolution procedures intended to generate orthogonal protein functions.
Directed evolution experiments have been successfully employed for improvement of biologically derived reagents with therapeutic potential, including RNA-guided nucleases. This proposed work will establish and evaluate in vivo directed evolution strategies designed to re-engineer the intrinsic sequence specificity of the most widely used RNA-guided nuclease, SpCas9. Successful execution of this study will yield novel SpCas9 variants with immediate potential in genome editing applications, and more widely embolden future efforts aimed at systematically optimizing selection regimens to facilitate the evolution of protein orthogonality.
