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BME 280B Seminar

Speaker Name: 
Alireza Eeraki
Speaker Organization: 
University of Massachusetts Medical School
Start Time: 
Thursday, January 23, 2020 - 11:40am
End Time: 
Thursday, January 23, 2020 - 1:15pm
BioMed 200
Ali Shariati


Recent advances with the bacterial CRISPR defense system as genome editing tools have opened a new avenue for targeting disease-causing mutations. The programmability of the Cas9 endonuclease makes it a powerful therapeutic tool to correct such mutations. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we use two approaches to identify new compact Cas9s with short PAMs that also demonstrated highly accurate genome editing. First, we use the presence of anti-CRISPRs (naturally occurring, phage-encoded peptides that inhibit Cas9) in a genome as indicators of Cas9s that may be highly active. We show that the Cas9 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. 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 dinucleotide PAM that provides a high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA into adult mice and zygotes induces efficient genome editing. Our new findings promise to accelerate the development of genome editing tools for biomedical and therapeutic applications.

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