Structure-guided discovery of ancestral CRISPR-Cas13 ribonucleases

Abstract

The RNA-guided ribonuclease CRISPR-Cas13 enables adaptive immunity in bacteria and programmable RNA manipulation in heterologous systems. Cas13s share limited sequence similarity, hindering discovery of related or ancestral systems. To address this, we developed an automated structural-search pipeline to identify an ancestral clade of Cas13 (Cas13an) and further trace Cas13 origins to defense-associated ribonucleases. Despite being one-third the size of other Cas13s, Cas13an mediates robust programmable RNA depletion and defense against diverse bacteriophages. However, unlike its larger counterparts, Cas13an uses a single active site for both CRISPR RNA processing and RNA-guided cleavage, revealing that the ancestral nuclease domain has two modes of activity. Discovery of Cas13an deepens our understanding of CRISPR-Cas evolution and expands opportunities for precision RNA editing, showcasing the promise of structure-guided genome mining.

Fig. 1. Structural homology search enabled discovery of ancestral Cas13 systems.

(A) Schematic of automated structure-based discovery pipeline. AFDB, AlphaFold Database. (B) Comparison of Cas13a (PDBID: 5XWY) and Cas13an (AFDBID: A0A7C5SD50) structure and domain architecture. (C) Maximum-likelihood phylogenetic tree of Cas13 subtypes. Sequences are provided in table S6, and alignment and tree files are provided in data S1. (D) Structural comparison of HEPN domains with canonical organization (HEPT; PDBID: 5YEP) and shared rearrangements (Cas13an HEPN1 and HEPN2 domains; AFDBID: A0A7C5SD50, and AbiD/F gene; AFDBID: A0A3S5XYX8). (E) Maximum-likelihood phylogenetic tree of Cas13an HEPN1 and HEPN2 domains and their structural homologs. Sequences and annotations of these proteins are available in table S9, and alignment and tree files are provided in data S1.

Fig. 2.. Cas13an systems provide targeted RNA knockdown and defense against phages.

(A) Small RNA-sequencing of CRISPR-Cas13an1 locus heterologously expressed in E. coli. Inset shows reads of length 40–80 nucleotides (nt) corresponding to processed crRNA. Black squares denote CRISPR-repeat, and green diamond denotes spacer sequence. (B) Schematic of green-fluorescent protein (GFP) depletion assay in E. coli. (C) Serial dilutions of E. coli in GFP depletion assays. Each spot progression represents a 10-fold dilution. (D) Schematic of phage challenge assays in E. coli. (E) Phage challenge assay results for lytic T4-phage using Cas13an8. Each spot progression represents a 10-fold dilution of phage stock. (F) Efficiency of plaquing (EOP) summary of Cas13an8 targeting phages of unrelated, diverse genera and labeled by genome nucleic acid composition. Labels I, II, III, and IV represent Podovirus, Myovirus, Siphovirus, and Jumbo Myoviruses respectively.

Fig. 3.. Complementary RNA triggers both cis- and trans-cleavage in Cas13an.

(A) End point measurement (60 min) of Cas13an2 ribonucleoprotein complex (RNP) cleavage of 5’-fluorescein (FAM)-labeled guide complementary (target RNA) and non-complementary (non-target RNA) substrates. In mutants, alanine substitutions were introduced in HEPN Rx4H motifs: R127A/H132A for dHEPN1, R363A/H368A for dHEPN2, and all four for dHEPN1/2. (B) Kinetics of Cas13an2 RNP mediated target RNA cleavage (cis-cleavage). (C) Schematic of fluorophore-quencher assay to measure collateral RNA cleavage (trans-cleavage) induced by Cas13an2 target RNA recognition (top). Trans-cleavage induced fluorescence traces for Cas13an RNP with target RNA and controls (bottom). AU, arbitrary units. (D) Schematic of mismatch tolerance assay in E. coli. (E) Heatmap representation of Cas13an2 mismatch tolerance for single and double mismatch spacers.

Fig. 4.. Multifunctional HEPN domains enable RNA-guided cleavage and pre-crRNA processing in Cas13an.

(A) Substrates used for RNA processing assays. Top: pre-crRNA and processing site (black triangle) inferred from RNA-sequencing (fig. S12). Bottom: full-length crRNA and processing sites inferred from denaturing gels (fig. S11C). (B) In vitro pre-crRNA processing by Cas13an2. Cas13an processes pre-crRNA in vitro only in the presence of Mg2+ and catalytic residues of both HEPN domains are necessary for processing. Gel with the ladder is attached in fig. S11A. (C) In vitro RNA processing of 5’-FAM-labeled full-length crRNA. Same gel was first examined for FAM signal, and then for SYBR-GOLD stain signal. Gel with the ladder attached is shown in fig. S11B. (D) Parallels in evolutionary paths between two unrelated Class 2 CRISPR-Cas effectors, Cas13 and Cas12.