Deep mutational scanning identifies Cas1 and Cas2 variants that enhance type II-A CRISPR-Cas spacer acquisition

Abstract

A remarkable feature of CRISPR-Cas systems is their ability to acquire short sequences from invading viruses to create a molecular record of infection. These sequences, called spacers, are inserted into the CRISPR locus and mediate sequence-specific immunity in prokaryotes. In type II-A CRISPR systems, Cas1, Cas2 and Csn2 form a supercomplex with Cas9 to integrate viral sequences. While the structure of the integrase complex has been described, a detailed functional analysis of the spacer acquisition machinery is lacking. We developed a genetic system that combines deep mutational scanning (DMS) of Streptococcus pyogenes cas genes with a method to select bacteria that acquire new spacers. Here, we show that this procedure reveals key interactions at the Cas1-Cas2 interface critical for spacer integration, identifies Cas variants with enhanced spacer acquisition and immunity against phage infection, and provides insights into the molecular determinants of spacer acquisition, offering a platform to improve CRISPR-Cas-based applications.

Fig. 1. Reporter strain for type II-A CRISPR-Cas spacer acquisition.

a Schematic representation of the reporter strain. Spacer acquisition from a prespacer (PS) containing a ribosome binding site (RBS) and a start codon (ATG) results in the installation of a translation start site, leading to downstream reporter expression. P_cr, CRISPR array promoter; P_cas, cas operon promoter; R, repeat. b Fluorescence microscopy of S. aureus cells carrying a reporter plasmid expressing mNG (green fluorescence) with a single CRISPR repeat, a spacer with a translation start site (spc1(ATG)) or a spacer with a defective start site in which the ATG start codon was replaced by a stop codon (spc1(TAG)). Each spacer construct was combined with different tracrRNAs or transformed into staphylococci lacking RNase III (Δrnc). Representative images are shown (n = 2 biological replicates). c Spacer acquisition rate as determined by the ratio of CFUs on erythromycin and chloramphenicol (Erm^R/Cm^R) after electroporation of reporter strains with the indicated prespacers (or no prespacer) in the presence or absence of csn2. Individual data points are shown with error bars representing the mean ± s.d. of four technical replicates.

Fig. 2. DMS reveals interacting residues at the Cas1-Cas2 interface.

a Deep mutational scanning (DMS) workflow. Libraries were electroporated with PS1 and plated on erythromycin to select for clones that have acquired a spacer (Erm^R, +spc1), and on chloramphenicol as the unselected control (total CFU). Amino acid preferences were determined by comparative next-generation sequencing (NGS) of the two populations. b Differential selection of amino acids (rows) across the Cas1 and Cas2 proteins (columns), colored by log₂ fold change (log₂FC). Residues 234–260 of Cas1 and 90–102 of Cas2 are highlighted, as well as their secondary structures. c AlphaFold3 model of Cas1-Cas2 integrase complex. A 22-bp prespacer with 4-nt overhangs is shown in black, Cas1 dimers are shown in gray, and the Cas2 dimer is colored by Shannon Entropy (SE). The inset shows the side chains of Cas1 R5, Y261 and Cas2 S99, R102, and the backbone atoms of Cas2 residues 95–101. Cas1 residues 234–260 are highlighted in purple. d, e Spacer acquisition rate as determined by the ratio of CFUs on erythromycin and chloramphenicol (Erm^R/Cm^R) after electroporation of PS1 into reporter strains with indicated cas variants. Individual data points are shown with error bars representing the mean ± s.d. (technical replicates, n = 3 for dCas1, n = 6 for all other conditions).

Fig. 3. Positively selected residue changes result in increased spacer acquisition rate.

a–c Differential selection of all possible amino acids (rows) at tested Cas1 (a), Cas2 (b) and Csn2 (c) sites (columns), colored by log₂ fold change (log₂FC). The amino acids introduced in the variants are marked with a dotted line. d–f Spacer acquisition rate as determined by the ratio of CFUs on erythromycin and chloramphenicol (Erm^R/Cm^R) after electroporation of PS1 into reporter strains with indicated residue changes in Cas1 (d), Cas2 (e) and Csn2 (f). Individual data points are shown with error bars representing the mean ± s.d. (technical replicates, n = 4 for Cas1 and Cas2, n = 3 for Csn2). Significant improvements of variants relative to wt are indicated. g, h Same as (d–f) but with combinations of different residue changes in the reporter strain. Individual data points are shown with error bars representing the mean ± s.d. (technical replicates, n = 4 for all conditions, except n = 27 for wt, and n = 8 for Cas1 M77H Cas2 T16Q and Cas2 Y5F T16Q). Significance and fold changes are indicated for the two best variants. i Spacer acquisition rate as determined by the ratio of CFUs on kanamycin and tetracycline (Kan^R/Tet^R) after electroporation of E. coli reporter strains with the indicated prespacers and variants. Δ indicates deletion of cas1, cas2 and csn2. Individual data points are shown with error bars representing the mean ± s.d. of 14 technical replicates. Significant improvements and fold changes of variants relative to wt are indicated.

Fig. 4. Cas1 and Cas2 variants with enhanced spacer acquisition properties improve phage immunity without observable fitness cost.

a Plate images obtained after lytic infection of S. aureus strains, carrying indicated cas genes of the type II-A CRISPR-Cas system with a single repeat CRISPR array on a plasmid, with phage ΦNM4γ4 in soft agar. Δ indicates gene deletion. b Agarose gel electrophoresis of PCR amplification of the CRISPR array present in plasmids extracted from colonies that survived bacteriophage infection. The number of spacers in the PCR product (0, 1 or 2) is indicated on the right. Representative images of n = 9 biological replicates are shown. c Quantification of CRISPR-resistant colonies in a. Individual data points are shown with error bars representing the mean ± s.d. (n = 9 biological replicates). d Growth of strains carrying different variants of type II-A cas genes measured by OD₆₀₀ of the cultures after infection with phage ΦNM4γ4. Average data is shown (n = 18 individual infections from six biological replicates, each split into three wells and infected separately, with individual data shown in Supplementary Fig. 16a). e Fraction of Erm^R staphylococci after daily passaging of mixed cultures composed of strains expressing either a wt type II-A CRISPR-Cas locus (Cm^R) or the indicated DMS variants (Erm^R and Cm^R) in pairwise growth competition experiments. Individual data points are shown (n = 4 biological replicates). ns: not significant.