To increase efficiency, we tested the effect of changes in the sgRNA scaffolds by altering the tracrRNA and crRNA linkage, removing hairpin mismatches, and modifying the 5′ start site and spacer length (Fig. Indels were detected for both Cas12b proteins, but the rates were below 1% (Fig. We then transfected 293T cells with plasmids expressing NLS-tagged Cas12b and sgRNA driven by a U6 promoter and measured nuclease activity through the formation of insertion or deletion (indel) mutations by targeted deep sequencing. Given that the tracrRNA and crRNA for Cas9 can be fused to form a single-guide RNA (sgRNA) 11 to simplify delivery, we explored whether sgRNAs can be designed for both AkCas12b and BhCas12b and found that they supported DNA cleavage activity in vitro (Supplementary Fig.
We observed only minimal activity with EbCas12b and LsCas12b however, both AkCas12b and BhCas12b exhibited strong cleavage at 37 ☌, warranting further investigation in human cells. To biochemically characterize Cas12b, we tested for in vitro DNA cleavage activity of purified Cas12b proteins with corresponding tracrRNA and crRNA components (Fig. coli lysates to identify the required RNA components and found putative tracrRNA mapping to the region between Cas12b and the CRISPR array (Supplementary Fig. Depleted PAMs were T-rich at positions 1–4 bp upstream of the protospacer, consistent with the preferences observed for previously studied Cas12b members 10. We detected depletion in 4 of the 14 tested Cas12b systems (AkCas12b, BhCas12b, EbCas12b, and LsCas12b), indicating functional DNA interference in a heterologous host. coli and challenged transformed cells with a randomized 5′ PAM library followed by deep sequencing (Supplementary Fig. To confirm that each of the identified loci are functional CRISPR–Cas systems and to identify their PAMs, we expressed a human codon-optimized Cas12b with their natural flanking sequence in E. All known class 2 DNA-targeting CRISPR–Cas nucleases require a protospacer-adjacent motif (PAM) 6, 8 for DNA cleavage, and the initial characterization of the Cas12b family revealed a PAM on the 5′ side of the target site 9. 1a), avoiding previously described members and those from recognized thermophiles. We chose 14 uncharacterized Cas12b genes spanning the diversity of the computationally identified candidates for experimental study (Supplementary Fig. The type V–B systems are widely scattered among bacteria, and topology of the phylogenetic tree of Cas12b generally does not follow the bacterial taxonomy, suggestive of extensive horizontal mobility. Identification of mesophilic Cas12b nucleasesĪ BLAST search of the updated sequence databases using previously detected Cas12b proteins as queries identified 27 members of the Cas12b family that are encoded within type V–B loci. We sought to identify Cas12b family members that would be active at lower temperatures and thus could be adapted for human genome editing. Although Cas12b proteins are often smaller than Cas9 and Cas12a and therefore attractive from the standpoint of intracellular delivery via viral vectors, the best characterized Cas12b nuclease from Alicyclobacillus acidoterrestris (AacCas12b) exhibits optimal DNA cleavage activity at 48 ☌, precluding its use in mammalian cells 9. Here, we focus on a third family of class 2 effector, Cas12b, a dual-RNA-guided nuclease containing a single RuvC domain and requiring both crRNA and tracrRNAs 9, 10 (Fig. However, only two families of class 2 nucleases have been harnessed for genome editing in human cells to date: Cas9 3, 4, a dual-RNA-guided nuclease which requires both CRISPR RNA (crRNA) and tracrRNA 5 and contains both HNH and RuvC nuclease domains 6, 7, and Cas12a 8, a single-RNA-guided nuclease which only requires crRNA and contains a single RuvC domain. Current genome editing technologies have focused on class 2 CRISPR–Cas systems, which contain single-protein effector nucleases for DNA cleavage. Enzymes from the prokaryotic clustered, regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR–Cas) systems have been harnessed as reprogrammable and highly specific genome editing tools 1, 2.