Cas12a Ultra enables efficient genome editing in human multipotent and pluripotent stem cells

Abstract Background The development of the CRISPR-Cas12a platform has generated considerable interest in the genome editing community. Due to its AT-rich protospacer-adjacent motif (PAM, 5’-TTTV), Cas12a increased the potential number of targetable sites for gene editing beyond that of the prototypical Streptococcus pyogenes CRISPR-Cas9 system. Moreover, evaluation of the off-target activity of CRISPR-Cas12a nucleases suggested high specificity of the platform. Broad application of the CRISPR-Cas12a platform in primary human cells was recently enabled by the development of a re-engineered version of the natural Acidaminococcus Cas12a, called Cas12a Ultra. Methods We transferred the CRISPR-Cas12a Ultra system in the form of ribonucleoprotein complexes into clinically relevant human cells, including T cells, multipotent hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). Allelic gene editing frequencies were determined at various target sites using standard genotyping and next-generation sequencing. Furthermore, we evaluated targeted integration of transgenes into the AAVS1 safe harbor site and the CSF2RA locus of patient-derived iPSCs. Results We achieved allelic gene disruption frequencies of over 90% at various target sites in multiple primary human cell types. In addition, we demonstrated efficient knock-in of a GFP marker gene into the AAVS1 locus, and achieved targeted integration of a therapeutic DNA template into 90% of CSF2RA alleles in iPSCs without selection. Clonal analysis revealed bi-allelic integration in > 50% of the screened iPSC clones without compromising their pluripotency and genome integrity. Conclusions Herein, we demonstrate that the CRISPR-Cas12a Ultra system provides a highly efficient genome editing platform for human stem cell applications, expanding the toolbox for clinical applications.