567. Enhanced Efficiency of CRISPR-Mediated Gene Knock-Out in Primary Human T-Cells

RNA-guided endonuclease (RGEN) technology has great promise for enabling efficient editing of essentially any target genomic locus. Here, we have evaluated application of S. pyogenes Cas9 for gene editing in primary human T-cells using mRNA-mediated delivery of a first generation spCas9 and adeno-associated virus (AAV) to drive guide RNA expression. We find that spCas9-mediated editing using this approach can achieve targeted gene disruption rates at the TCRa locus of up to 30%. Furthermore, evaluation of the dose response of editing efficiency at different Cas9 mRNA doses and over a range of AAV MOI suggests that editing efficiency is limited primarily by AAV-driven guide RNA expression. Evaluation of several approaches to achieve higher editing efficiencies led to the development of a novel method capable of achieving up to 90% TCRa disruption at reduced AAV MOI in selected cell populations. These results suggest that a Cas9-mRNA/AAV-guide approach can be applied to effectively disrupt multiple individual genes in primary human T-cells, and highlight an innovate method through which CRISPR/Cas9 technology may be applied with enhanced efficiency.