CRISPR-Cas12a exhibits metal-dependent specificity switching.

Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+ concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+ concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+ concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+ concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+ concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.

CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes.

CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution.

Maize Glossy2 and Glossy2-like Genes Have Overlapping and Distinct Functions in Cuticular Lipid Deposition.

Plant epidermal cells express unique molecular machinery that juxtapose the assembly of intracellular lipid components and the unique extracellular cuticular lipids that are unidirectionally secreted to plant surfaces. In maize (Zea mays), mutations at the glossy2 (gl2) locus affect the deposition of extracellular cuticular lipids. Sequence-based genome scanning identified a new Gl2 homolog in the maize genome, namely Gl2-like Both the Gl2-like and Gl2 genes are members of the BAHD superfamily of acyltransferases, with close sequence similarity to the Arabidopsis (Arabidopsis thaliana) CER2 gene. Transgenic experiments demonstrated that Gl2-like and Gl2 functionally complement the Arabidopsis cer2 mutation, with differential influences on the cuticular lipids and the lipidome of the plant, particularly affecting the longer alkyl chain acyl lipids, especially at the 32-carbon chain length. Site-directed mutagenesis of the putative BAHD catalytic HXXXDX-motif indicated that Gl2-like requires this catalytic capability to fully complement the cer2 function, but Gl2 can accomplish complementation without the need for this catalytic motif. These findings demonstrate that Gl2 and Gl2-like overlap in their cuticular lipid function, but have evolutionarily diverged to acquire nonoverlapping functions.

An adaptable defense.

The response of bacteria to the threat posed by phages depends on their local environment.