An Engineered AsCas12a Nuclease Facilitates the Rapid Generation of Therapeutic Medicines

Dr. Zuris discussed the rationale and approach for generating a new AsCas12a variant or AsCas12 ULTRA, how the newly developed variant compares in activity and specificity against other nucleases, and its application to T and NK cell gene editing.

Dr. Zuris highlighted how the knockout of endogenous TRAC, B2M, and CIITA genes are edits critical to the safety and success of allogeneic T cell therapies. Additionally, modifications such as CAR knockin would be required to enable desirable therapeutic activity. Therefore, multiplexed gene-editing capabilities are critical to engineering the next generation of cellular therapies due to the number of modifications necessary to achieve both desired activity and safety. To enable T and NK cell therapeutics development, Dr. Zuris’ team focused on AsCas12a, which offered the ability to target genomic regions otherwise unreachable by some available Cas9 nucleases (e.g., SpCas9, SaCas9, and SaCas9 KKH). Although AsCas12a has a more limited genomic targeting range, Dr. Zuris emphasized that this could prove advantageous in reducing potential off-target editing. Combined with AsCas12a’s higher intrinsic specific activity and fidelity, this nuclease represented an attractive candidate for engineering cell therapeutics.

Nevertheless, recognizing that AsCas12a is inefficient in achieving knockout and knockin editing, efforts were directed towards engineering AsCas12a for improved editing efficiency. The resulting variant, AsCas12 ULTRA, showed significantly enhanced editing efficiency in T and HSPC cells while preserving specificity. Overall, his team showed that AsCas12a supports multiplexed T cell editing with high efficiency above 90% when targeting TRAC, B2M, and CIITA for knockout and around 60% knockin efficiency when targeting transgenes into the TRAC and B2M loci. Lastly, Dr. Zuris shared how the new AsCas12a ULTRA variant is highly efficient for multiplexed NK cell editing.

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