Identification of mesophilic Cas12b nucleases

A 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. 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. We chose 14 uncharacterized Cas12b genes spanning the diversity of the computationally identified candidates for experimental study (Supplementary Fig. 1a), avoiding previously described members and those from recognized thermophiles. 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 site9. 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. coli and challenged transformed cells with a randomized 5′ PAM library followed by deep sequencing (Supplementary Fig. 1b,c). We detected depletion in 4 of the 14 tested Cas12b systems (AkCas12b, BhCas12b, EbCas12b, and LsCas12b), indicating functional DNA interference in a heterologous host. Depleted PAMs were T-rich at positions 1–4 bp upstream of the protospacer, consistent with the preferences observed for previously studied Cas12b members10. We performed small RNA-Seq on E. coli lysates to identify the required RNA components and found putative tracrRNA mapping to the region between Cas12b and the CRISPR array (Supplementary Fig. 2a–d).

To biochemically characterize Cas12b, we tested for in vitro DNA cleavage activity of purified Cas12b proteins with corresponding tracrRNA and crRNA components (Fig. 1b, Supplementary Fig. 2e). We observed only minimal activity with EbCas12b and LsCas12b; however, both AkCas12b and BhCas12b exhibited strong cleavage at 37 °C, warranting further investigation in human cells. 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. 2f). 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. Indels were detected for both Cas12b proteins, but the rates were below 1% (Fig. 1c, d). 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. 1c–e, Supplementary Fig. 3). Although alterations in the AkCas12b sgRNA had little effect, a 5-nt truncation on the 5′ end of the BhCas12b sgRNA substantially improved activity (up to 30-fold) across multiple targets (sgRNA design 2).

Rational engineering of BhCas12b

We frequently observed a slower migrating band during gel electrophoresis of in vitro cleavage reactions, most notably, with AkCas12b, which suggested that Cas12b can nick double-stranded DNA (dsDNA) substrates (Fig. 1b). Reactions with differentially labeled DNA strands revealed that AkCas12b and BhCas12b preferentially cut the non-target strand, and that this behavior is more pronounced at lower temperatures (Fig. 2a). As the inability to cleave the target strand reduces the potential of BhCas12b as an effective nuclease for genome editing, we sought to address this limitation through protein engineering.

Fig. 2 Rational engineering of BhCas12b. a In vitro Cas12b reactions with differentially labeled DNA strands. A slower migrating product is observed during native PAGE separation and separation by denaturing PAGE reveals a preference for AkCas12b and BhCas12b to preferentially cut the non-target strand at lower temperatures. b Location of 10 of the 12 tested residues in the pocket between the target strand and the RuvC active site (purple). BhCas12b residues are highlighted in the structure of the highly similar BthCas12b (PDB: 5wti [10.2210/pdb5WTI/pdb]). c Indel activity of 176 BhCas12b mutations at DNMT1 (target 5) and VEGFA (target 7) normalized to wild type (gray circles). Error bars represent s.d. from n = 2 replicates. d Location of surface-exposed residues mutated to glycine. e Indel activity of 66 BhCas12b mutations at DNMT1 (target 5) and VEGFA (target 7) normalized to wild type (gray circles). Error bars represent s.d. from n = 2 replicates. f Summary of BhCas12b hyperactive variants. g Indel activity of BhCas12b variants at four target sites. Error bars represent s.d. from n = 3–6 replicates. h In vitro cleavage with increasing concentrations of BhCas12b WT and v4 variant. Gel is a representative image from n = 2 experiments. Source data are provided as a Source Data file Full size image

In contrast to the flexible non-target strand, we reasoned that the target strand might be poorly accessible to the RuvC active site of BhCas12b. A crystal structure of a supplementary target strand in AacCas12b revealed several key residues that contact DNA in the pocket between the guide RNA:DNA duplex and the RuvC active site12, and we hypothesized that altering the properties of this pocket in BhCas12b might improve target-strand accessibility and DNA cleavage. We mutated 12 BhCas12b residues identified through alignments with AacCas12b, residues which were also conserved in the structure of the nearly identical Cas12b from Bacillus thermoamylovorans (BthCas12b)(BthCas12b also exhibits activity in cells, but with lower efficiency than BhCas12b [Supplementary Fig. 4a])13. 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 (Fig. 2b, c, Supplementary Fig. 4b, c). As positively charged arginine side chains often interact with the backbone of nucleic acids14, it is possible that increased DNA-binding affinity of the mutants helps pull the target strand toward the RuvC active site and promote DNA cleavage.

As an orthogonal approach, we sought to address the temperature dependence of target-strand cleavage. One common attribute of cold-adapted enzymes is the presence of surface-exposed glycine residues, which can increase protein flexibility and enzymatic activity by acting locally near a catalytic site15 or through allosteric mechanisms16. We generated glycine substitutions at 66 surface-exposed residues and again tested for indel activity at two target sites. Strikingly, we observed over twofold improvement relative to wild type with the E837G variant, a position that is located between the guide RNA:DNA duplex and the RuvC active site (Fig. 2d, e). 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 (Fig. 2f, g, Supplementary Fig. 4d–f). In agreement with these results in human cells, purified BhCas12b v4 protein exhibited increased dsDNA cleavage activity at 37 °C and a clear reduction of nicked dsDNA (Fig. 2h, Supplementary Fig. 4g–j).

BhCas12b v4 mediates genome editing in human cells

Robust genome editing tools should be effective and specific across a range of targets, and therefore, we investigated Cas12b activity more thoroughly in comparison to previously studied Cas nucleases. We tested BhCas12b v4 at 56 targets across five genes in 293T cells and found robust cleavage at ATTN PAMs using AsCas12a at TTTV PAMs as a positive control (Fig. 3a). We also observed high BhCas12b v4 activity at a subset of TTTN and GTTN PAMs, although this activity was less robust than activity at ATTN PAMs (Supplementary Fig. 5a). Analysis of ATTN prevalence in the human genome revealed a similar number of targetable sites, as can be achieved with Cas12a enzymes (Supplementary Fig. 5b). In contrast to SpCas9 and AsCas12a, analysis of the indel patterns formed by BhCas12b revealed prominent larger deletions of 5–15 bp (Fig. 3b). Co-transfection of BhCas12b v4 with single-strand oligonucleotide (ssODN) donors led to comparable editing efficiency as SpCas9 and AsCas12a at a TTTC PAM target (Fig. 3c–e), and higher editing efficiency than SpCas9 at an ATTC PAM target (Supplementary Fig. 5c–e). To further evaluate the efficacy of BhCas12b v4 in human cells, we tested the ability of Cas12b ribonucleoproteins (RNPs) to edit primary human T cells. We generated BhCas12b v4-sgRNA complexes and delivered them into human CD4+ T cells by electroporation. BhCas12b v4 RNPs exhibited indel rates of 32–49% across three tested targets (Fig. 3f). 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.

Fig. 3 BhCas12b v4 mediates genome editing in human cells. a Indel activity in 293T cells of AsCas12a at 28 TTTV targets and BhCas12b v4 at 33 ATTN targets. Each dot represents a single target site, averaged from n = 4 replicates. b Average indel length during genome editing with 30 active BhCas12b guides, 45 active AsCas12a guides, and 39 active SpCas9 guides. c Schematic of a DNMT1 region targetable by SpCas9 and Cas12a/b nucleases and a 120-nt ssODN donor containing a TG-to-CA mutation and PAM-disrupting mutations. d Indel activity of each nuclease at the DNMT1 locus. Error bars represent s.d. from n = 8 replicates. e Frequency of homology-directed repair (HDR) using a target strand (T) or non-target strand (NT) donor. Gray bars indicate the frequency of TG-to-CA mutation, while red bars indicate perfect edits with no detectable mutations in the 36-nt sequence shown in panel c. Error bars represent s.d. from n = 6 replicates. f Indel activity in CD4+ human T cells following BhCas12b v4 RNP delivery. Each dot represents an individual electroporation (n = 2). Source data are provided as a Source Data file Full size image

BhCas12b v4 is a highly specific nuclease

We next sought to determine the genome-wide targeting specificity of BhCas12b in human cells. We chose nine target sites with comparable indel activity between different Cas nucleases (Fig. 4a) and performed Guide-Seq17 analysis. We did not detect any off-target sites for BhCas12b v4 or AsCas12a, whereas SpCas9 led to prominent off-target cleavage in six of the nine tested guides (Fig. 4b, Supplementary Fig. 6), consistent with other reports18,19. For example, for DNMT1 (target 16), we detected 101 insertion sites with SpCas9, with only 10% of reads mapping to the target site, but no off-target sites with BhCas12b v4. Additional Guide-Seq experiments at unmatched target sites detected off-target cleavage at only 2 of 14 sites for BhCas12b v4 (Supplementary Fig. 7 and 8). Consistent with these findings, we observed limited indel activity with double mismatches between the guide RNA and target DNA in positions 1–20, and even a low tolerance for single mismatches (Fig. 4c). These results agree with the reported specificity of AacCas12b in vitro20 and suggest a molecular explanation for the low off-target activity observed in cells.