Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR-Cas effector complexes

Abstract

Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction-modification (R-M) and CRISPR-Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR-Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR-Cas systems by lowering the affinity of the Cascade and Cas9-crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR-Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems.

Figure 1.

Modifications of nucleobases in phage T4 DNA. (A) Cytosine present in phage T4(C). (B) 5-Hydroxymethylation of cytosine present in phage T4(hmC). (C) Glucosyl-5-hydroxymethylation present in phage T4(ghmC).

Figure 2.

Efficiency of plaquing (EOP) assays of T4 phages on E. coli cells expressing CRISPR–Cas systems targeting T4 genes 19 or 22. (A) Representation of the circular genome of bacteriophage T4. Positions of protospacer sequences and PAMs are shown (indicated by orange bars and purple bars respectively). Glucosyl-5-hydroxymethylcytosines are indicated in red and nucleotides that base pair with crRNA are indicated by black dots. (B) EOP of T4 phages on E. coli cells expressing targeting Cascade complexes normalized to EOP on non-targeting strains. (C) EOP of T4 phages on E. coli cells expressing targeting Cas9 proteins normalized to EOP on non-targeting strains.

Figure 3.

Effect of T4 DNA modifications on type I-E CRISPR–Cas sgRNA mediated DNA targeting. (A) Schematic of DNA targeting and R-loop formation by Cascade. Modified cytosine residues are indicated in red. (B) Cleavage assay of Cas3 in conjunction with Cascade on 98 bp modified targets, indicated by black arrow. The marker is indicated by white arrows. Cascade effector complexes are loaded with either targeting crRNA (T crRNA) or non-targeting crRNA (NT crRNA). Restriction products of Cas3 are of undefined length. (C). Electrophoretic Mobility Shift Assay (EMSA) of Cascade on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows, dotted lines represent separate gels.

Figure 4.

Effect of T4 DNA modifications on type II-A CRISPR–Cas sgRNA mediated DNA targeting. (A) Schematic of DNA targeting by Cas9. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. (B) Cleavage assay of Cas9 on 98 bp modified targets (indicated by black arrow). Cas9 is loaded with either targeting sgRNA (T sgRNA) or non-targeting sgRNA (NT sgRNA). Restriction products of Cas9 are 61 and 37 bp. (C) EMSA of dCas9 on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.

Figure 5.

Effect of T4 DNA modifications of PAM-complementary cytosines on type II-A CRISPR–Cas sgRNA mediated DNA targeting. Cleavage assay of Cas9 on target DNA containing 5-hmC (indicated in red). Cas9 is loaded with either targeting sgRNA (T sgRNA) or non-targeting sgRNA (NT sgRNA). Restriction products of Cas9 are 57 and 33 bp. The marker is indicated by white arrows.

Figure 6.

Effect of T4 DNA modifications on type V-A CRISPR–Cas sgRNA mediated DNA targeting. (A) Schematic of DNA targeting by Cas12a. Modified cytosine residues are indicated in red. Cleavage sites are indicated by black arrows. (B) Cleavage assay of Cas12a on 98 bp modified targets (indicated by black arrow). Cas12a is loaded with either targeting crRNA (C crRNA) or non-targeting crRNA (NC crRNA). Cleavage products of Cas12a are 49 and 44 bp. The marker is indicated by white arrows. (C) Electrophoretic Mobility Shift Assay (EMSA) of Cas12a on target DNA containing C, 5-hmC or 5-ghmC (indicated by black arrow) at increasing protein concentrations [nM]. Fraction of bound target is indicated by white arrows.

Figure 7.

Potential steric clashes between target nucleotide 5-ghmC modifications and CRISPR effector proteins. (A) Multiple clashes (indicated in red) are observed between the polypeptide chains of Cascade and 5-ghmC modifications of nucleotides in the target strand (TS, complementary to the crRNA) and non-target strand (NTS). (B) Clashes are mostly observed between the polypeptide chains of Cas9 and 5-ghmC modifications of nucleotides in the seed region. (C) No clashes are observed in the seed region for Cas12a.