NEW YORK (GenomeWeb) – Researchers led by the Broad Institute's Feng Zhang have engineered an ortholog of the CRISPR nuclease Cas12b, making its genome editing activity in human cell lines and ex vivo in primary human T cells more robust.
As they reported today in Nature Communications, the researchers explored the Cas12b family, and found that the nuclease from the Bacillus hisashii bacterium, BhCas12b, was the most promising for human genome editing. However, they also found that at 37° C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency.
The researchers proceeded with protein engineering in order to mutate BhCas12b at several sites and measured the resulting activity. "We measured indel activity at two target sites with a total of 176 BhCas12b single mutants and found increased activity with several mutations, including K846R and S893R, which exhibited additive effects as a double mutant," the authors wrote. "Testing combinations of mutations led to progressively active variants with a final BhCas12b v4 mutant containing K846R/ S893R/E837G that exhibited the highest activity across multiple targets."
When testing the mutant BhCas12b in human cells, the researchers found that it exhibited increased dsDNA cleavage activity at 37° C and a clear reduction of nicked dsDNA. They also compared the activity of the BhCas12b mutant to that of SpCas9 and AsCas12a and found that BhCas12b revealed prominent larger deletions of five to 15 base pairs at ATTN PAM sites. Further, co-transfection of BhCas12b v4 with single-strand oligonucleotide donors led to comparable editing efficiency as SpCas9 and AsCas12a at a TTTC PAM target, and higher editing efficiency than SpCas9 at an ATTC PAM target.
The researchers then tested the ability of Cas12b ribonucleoproteins to edit primary human T cells by generating BhCas12b v4-sgRNA complexes and delivering them into human CD4+ T cells by electroporation, and found that BhCas12b v4 RNPs exhibited indel rates of 32 percent to 49 percent across three tested targets.
"Together, these data indicate that BhCas12 v4 can be harnessed as an effective programmable nuclease in a variety of genome editing contexts, including in a therapeutically relevant human cell type," they wrote. "BhCas12b v4 is a highly specific nuclease."
These findings, they concluded, emphasize the benefit of multiple orthologs and the need for a thorough investigation of the targeting rules of Cas nucleases. The engineering of BhCas12b described in this study led to a "substantial increase" in the efficiency of dsDNA cleavage and provides a way forward for the engineering of other CRISPR nucleases as genome editing tools, the team added, noting that "BhCas12b is a comparatively compact protein (at 1,108 amino acids), and therefore, in contrast to SpCas9 and AsCas12a, is suitable for efficient packaging into adeno-associated virus."