Recent advances with the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as genome editing tools have opened a new avenue for targeting disease-causing mutations. The programmability of the Cas9 endonuclease by RNA makes it a potentially powerful therapeutic tool to correct such mutations. The CRISPR-Cas9 system consists of a Cas9 endonuclease that is guided by RNA (sgRNA) to create double-stranded breaks in a target DNA segment complementary to the guide. This process is dependent on a 2-8 nucleotide sequence (called PAM) that is adjacent to the target and functions as a Cas9 binding signal. Each Cas9 ortholog recognizes a unique PAM.
However, factors such as the size of Cas9 or the frequency of its PAM sequence in the genome have hindered its clinical use. The Cas9 from Streptococcus pyogenes (SpyCas9) is commonly used in research because its PAM (NGG, where “N” symbolizes any nucleotide) is present every ~8 bp in the genome, providing robust targeting potential. However, it is too large to fit into typical viral vectors used for in vivo delivery, namely adeno-associated vectors (AAV). While several Cas9 orthologs have been characterized, none satisfied the need for a compact, accurate Cas9 with a short PAM.
In this thesis, we use two approaches to identify new compact Cas9 orthologs with small PAMs, one using anti-CRISPR proteins and one by searching through closely related Cas9s. First, we use the presence of anti-CRISPRs (naturally occurring, phage-encoded peptides that inhibit CRISPR-Cas9 described in chapter 2) in a genome as indicators of Cas9s that may be highly active. These orthologs come with the added advantage of having inhibitors that can be used as off-switches. We characterize four Cas9s that are targeted by anti-CRISPR proteins and show that they recognize diverse PAMs in vitro. One of the four Cas9’s, namely HpaCas9 from Haemophilus parainfluenzae, induces efficient genome editing in mammalian cells. However, its long N4GATTT PAM does not satisfy the short PAM criterion.
For our second approach, we asked whether closely related Cas9 orthologs with drastically different PAM-interacting domains (PIDs, the domain responsible for PAM recognition) recognize different PAMs, and if so, can be used for genome editing. To this end, we exploited natural variation in the PID of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis (Nme2Cas9). Nme2Cas9 recognizes a simple dinucleotide PAM (N4CC) that provides a high target site density. All-in-one
AAV delivery of Nme2Cas9 with a guide RNA into adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Finally, we show preliminary data on how CRISPR-Cas9 can be used for therapeutic genome editing for Amytrophic Lateral Sclerosis. Our new findings promise to accelerate the development of genome editing tools for biomedical and therapeutic applications
