High efficiency CRISPR/Cas9-mediated gene editing in primary human T-cells using mutant adenoviral E4orf6/E1b55k |[ldquo]|helper|[rdquo]| proteins

Many future therapeutic applications of CRISPR/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components – Cas9, guide RNAs and recombination templates – to primary cells rendered proficient for homology–directed repair. Here, we demonstrate an electroporation/transduction co–delivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with AAV vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T–cells, illustrating its broad potential for application in translational gene editing.