A novel thermal Cas12b from a hot spring bacterium with high target mismatch tolerance and robust DNA cleavage...

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas), such as Cas9 and Cpf1, are RNA-guided endonucleases that target and degrade nucleic acids, providing powerful genomic editing and molecular diagnostic tools. Cas12b enzymes are distinct effectors; however, their features and catalytic boundaries require further characterization. We identified BrCas12b from the thermophile bacterium Brevibacillus sp. SYSU G02855 as a novel ortholog of cas12b. Biochemical analyses revealed that BrCas12b is a dual-RNA-guided endonuclease with higher optimum reaction temperature than that of other reported members of Cas12b. The seed sequence of BrCas12b is only 4 nt in length, indicating that it has greater target mismatch tolerance than that of previously reported Cas effectors; however, it contains a compensatory effect at the position of the cleavage site. Using fluorescence-based detection method to evaluate target cleavage efficiency, we showed that BrCas12b has robust enzymatic cleavage activity (K_cat/K_m (s^-1 M^-1) = 8.80 × 10¹¹), which is significantly higher than that of AacCas12b (K_cat/K_m (s^-1 M^-1) = 7.56 × 10⁸) from Alicyclobacillus acidoterrestris. The results increase our understanding of the catalytic mechanism of Cas12b family members and suggest that BrCas12b might be useful in the application of genomic editing and molecular diagnosis.