Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells.

Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach to increasing the frequency of HDR in human HSPCs by the delivery of an inhibitor of 53BP1 (named "i53") as a recombinant peptide. We show that the use of i53 peptide effectively increases the frequency of HDR-mediated genome editing at a variety of therapeutically relevant loci in HSPCs as well as other primary human cell types. We show that incorporating the use of i53 recombinant protein allows high frequencies of HDR while lowering the amounts of AAV6 needed by 8-fold. HDR edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in NSG mice, suggesting that i53 recombinant protein might be safely integrated into the standard CRISPR/AAV6-mediated genome editing protocol to gain greater numbers of edited cells for transplantation of clinically meaningful cell populations.

AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines.

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.