IMPROVED CRISPR-CAS NUCLEASES FOR GENOME EDITING

In recent years, genome editing has transformed medical therapies. Scientists can now treat diseases at root cause through use of new genome editing technology. The discovery of CRISPR-Cas9 has enabled easy programmable genome editing. In recent clinical trials patients have shown to be permanently cured from a one dose-treatment because permanent changes can be made to the DNA.

Although Crisper is a powerful gene editing technique, it has limitations for therapeutic use. The specificity required from the PAM as well as the overall size of the enzyme prevent crisper from broad use as a human therapy. There has been interest in creating Cas-9 variants that relax PAM or give different PAM specifications. ​​The other significant restraint of CRISPR-Cas9 is the size of the enzyme because of delivery to patients. Viral vectors have a limited cargo capacity and therefore these small CRISPR enzymes are very attractive.

In an attempt to resolve these limitations, I used a newly discovered NNG(where N is any nucleotide base) PAM variant to address the PAM limitation, and a much smaller WTCas12f(wildtype) to address the size constraint. The NNG PAM variant showed robust editing at all NNG sites and minimal editing at non NNG sites, a very encouraging result. WTCas12f showed little to no editing in human cells, but with further engineering this is a promising enzyme due to its 529aa size.

eloliver@eckerd.edu