Session 8, Abstract 45

OPTIMIZING THE CORRECTIVE DONOR CONSTRUCT FOR CRISPR/CAS9 MEDIATED SITE-SPECIFIC GENE MODIFICATION OF CD40 LIGAND THROUGH CRISPR-BLOCKING MUTATIONS

Brian C. Tulloh* (Dr. Cindy Malone), California State University - Northridge

X-Linked Hyper-IgM (XHIM) is a primary immunodeficiency characterized by the inability to undergo immunoglobulin class switching in B cells. As a result, minimal levels of IgG, IgE, and IgA are present in XHIM patients, which leads to an increased susceptibility to pathogenic infections. A defect in the CD40L gene on the X-chromosome is responsible for XHIM. It is our goal to use the CRISPR/Cas9 gene editing platform to make a site-specific cut within the endogenous CD40L gene and subsequently utilize homology-directed repair (HDR) to insert a corrective cDNA donor sequence, to wholly restore CD40L function while maintaining its control under the endogenous promoter. Although we are able to achieve high rates of correction at CD40L (up to 50%), we also detect significant re-cleavage of our inserted donor in both K562 erythroleukemia cells as well as CD34​+​ peripheral blood stem cells (PBSC’s). We hypothesize this occurs because the Protospacer Adjacent Motif (PAM) which the Cas9 uses as one of the recognition patterns to cleave dsDNA still exists in the corrective donor. By changing the ​S. pyogenes​ Cas9 PAM sequence within our donor from canonical 5’-NGG-3’ to 5’-NHH-3’, we could will mitigate re-cleavage events of the integrated donor by CRISPR/Cas9. Preliminary data in K562 cells demonstrated that the incidence of re-cleavage events decrease from 98.4% to 7.0% when the endogenous PAM is altered to NAA. Additional work is being done to investigate other PAM modifications and to determine if gene correction rates are affected by these CRISPR-blocking mutations in the donor.

 

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