Combinatorial CRISPR/Cas12a excises targeted loci in the mammalian genome at very low efficiency

The CRISPR/Cas12a system proves to be a valuable tool for genetic manipulation due to its capability to handle multiple guide RNAs (gRNAs) simultaneously from a single promoter. In this research, the scientists investigated the potential of employing combinatorial CRISPR/Cas12a to target and excise specific regions within the mammalian genome, with the aim of inducing double-strand breaks (DSBs) upstream and downstream of chosen exons.

To achieve this, they developed a collection of gRNA pairs designed to target introns adjacent to 54 exons from 18 crucial genes, as well as 17 exons of 10 less vital genes. The main objective was to evaluate the efficiency with which Cas12 could excise the targeted loci. Although single gRNAs aimed directly at essential exons caused the anticipated fitness issues in cells, when they utilized pairs of guides targeting the surrounding intronic regions, the impact on cell viability remained minimal, indicating that excision scarcely occurred.

Further examination revealed no discernible relationship between the efficiency of individual gRNAs, their orientation, GC content, or the cut site geometry, and the success of locus excision. These results suggest that, under the specific experimental conditions utilized, the Cas12a system does not achieve the level of locus excision necessary for precise negative selection assays.