Abstract
The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, 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. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.
Highlights
The type-V CRISPR effector Cas12b has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members
Only two families of class 2 nucleases have been harnessed for genome editing in human cells to date: Cas[93,4], a dual-RNA-guided nuclease which requires both CRISPR RNA and tracrRNA5 and contains both HNH and RuvC nuclease domains[6,7], and Cas12a8, a single-RNAguided nuclease which only requires crRNA and contains a single RuvC domain
To confirm that each of the identified loci are functional CRISPR–Cas systems and to identify their protospaceradjacent motif (PAM), we expressed a human codon-optimized Cas12b with their natural flanking sequence in E. coli and challenged transformed cells with a randomized 5′ PAM library followed by deep sequencing (Supplementary Fig. 1b,c)
Summary
The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. This work establishes a third RNA-guided nuclease platform, in addition to Cas[9] and Cpf1/Cas12a, for genome editing in human cells. Current genome editing technologies have focused on class 2 CRISPR–Cas systems, which contain single-protein effector nucleases for DNA cleavage. Only two families of class 2 nucleases have been harnessed for genome editing in human cells to date: Cas[93,4], a dual-RNA-guided nuclease which requires both CRISPR RNA (crRNA) and tracrRNA5 and contains both HNH and RuvC nuclease domains[6,7], and Cas12a8, a single-RNAguided nuclease which only requires crRNA and contains a single RuvC domain. We sought to identify Cas12b family members that would be active at lower temperatures and could be adapted for human genome editing
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