Programmed DNA destruction by miniature CRISPR-Cas14 enzymes

Abstract

CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools use RNA-guided Cas proteins whose large size (950 to 1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400 to 700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.

Fig. 1.. Architecture and phylogeny of CRISPR-Cas14 genomic loci.

(A) Phylogenetic tree of Type V CRISPR systems. Newly identified miniature CRISPR systems are highlighted in orange. (B) Representative loci architectures for C2c10 and CRISPR-Cas14 systems. (C) Length distribution of Cas14a–c systems compared to Cas12a-e and Cas9. (D) Domain organization of Cas14a compared to Cas9 and Cas12a with the nuclease domains (RuvC and HNH) indicated. Protein lengths are drawn to scale.

Fig. 2.. CRISPR-Cas14a actively adapts and encodes a tracrRNA.

(A) Spacer diversity for Cas14b4 and Cas14b14 with CRISPR repeats diagramed in tan and unique spacers shown in different colors. (B) Metatranscriptomics reads mapped to Cas14a1 and Cas14a3. Inset shows expansion of most abundant repeat and spacer sequence. (C) In silico predicted structure of Cas14a1 crRNA and tracrRNA. Notably, RNase III orthologs were not identified in host genomes (fig. S5A). (D) Fraction of various CRISPR complexes mass made up of by RNA and protein.

Fig. 3.. CRISPR-Cas14a is an RNA-guided DNA-endonuclease.

(A) Cleavage kinetics of Cas14a1 targeting ssDNA, dsDNA, ssRNA and off-target ssDNA. (B) Diagram of Cas14a RNP bound to target ssDNA and Cas14a1 cleavage kinetics of radiolabeled ssDNA in the presence of various RNA components. (C) Tiling of a ssDNA substrate by Cas14a1 guide sequences. (D) Cleavage of the ssDNA viral M13 genome with activated Cas14a1.

Fig. 4.. High fidelity ssDNA SNP detection by CRISPR-Cas14a.

(A) Fluorophore-quencher (FQ) assay for detection of ssDNA by Cas14a1 and the cleavage kinetics for various length FQ substrates. (B) Cleavage kinetics for Cas14a1 with mismatches tiled across the substrate (individual points represent replicate measurements). (C) Diagram of Cas14-DETECTR strategy and HERC2 eye color SNP. (D) Titration of T7 exonuclease and impact on Cas14a-DETECTR. (E) SNP detection using Cas14a-DETECTR with a blue-eye targeting guide for a blue-eyed and brown-eyed saliva sample compared to ssDNA detection using Cas12a.