Cas1 and Cas2 From the Type II-C CRISPR-Cas System of Riemerella anatipestifer Are Required for Spacer Acquisition

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide acquired genetic immunity against the entry of mobile genetic elements (MGEs). The immune defense provided by various subtypes of the CRISPR-Cas system has been confirmed and is closely associated with the formation of immunological memory in CRISPR arrays, called CRISPR adaptation or spacer acquisition. However, whether type II-C CRISPR-Cas systems are also involved in spacer acquisition remains largely unknown. This study explores and provides some definitive evidence regarding spacer acquisition of the type II-C CRISPR-Cas system from Riemerella anatipestifer (RA) CH-2 (RA-CH-2). Firstly, introducing an exogenous plasmid into RA-CH-2 triggered spacer acquisition of RA CRISPR-Cas system, and the acquisition of new spacers led to plasmid instability in RA-CH-2. Furthermore, deletion of cas1 or cas2 of RA-CH-2 abrogated spacer acquisition and subsequently stabilized the exogenous plasmid, suggesting that both Cas1 and Cas2 are required for spacer acquisition of RA-CH-2 CRISPR-Cas system, consistent with the reported role of Cas1 and Cas2 in type I-E and II-A systems. Finally, assays for studying Cas1 nuclease activity and the interaction of Cas1 with Cas2 contributed to a better understanding of the adaptation mechanism of RA CRISPR-Cas system. This is the first experimental identification of the naïve adaptation of type II-C CRISPR-Cas system.

Keywords: CRISPR-Cas; Cas1; Cas2; Riemerella anatipestifer; spacer acquisition.

Figure 1

Organization of the type II-C CRISPR-Cas system from RA-CH-2. The CRISPR1 locus from RA-CH-2 comprises three cas genes (cas9, cas1, and cas2), 16 repeats (purple or blue rectangles, 47 bp long) and 15 spacers (colored diamonds, 30 bp long). The tracrRNA (trans-activating crRNA, brown arrow) containing the region with complementarity to the repeats is located upstream of cas9. Between cas2 and the first repeat (purple rectangle), the A/T-rich region is the presumed leader sequence. The primers (short red arrows) were used for PCR amplification to detect CRISPR1 expansion.

Figure 2

Spacer acquisition assays. The 5′ end of the CRISPR1 locus in RA-CH-2 population (harboring the shuttle plasmid pLMF03) was monitored by PCR amplification using the primers indicated in Figure 1 for the culture at the indicated growth cycle. Each growth cycle represents a 3:1,000 dilution of a previous 16-h culture grown for an additional 16 h in TSB without antibiotics. The red solid arrow represents a new spacer integrated into the CRISPR1 array (~280 bp), while the green dotted arrow represents the unexpanded array (~200 bp). The image is representative of multiple experiments.

Figure 3

Plasmid loss assays. (A) Colony count of the cultures at growth cycles 1, 5, 10, 15, and 20 on TSA plates with or without antibiotics. (B) Plasmid loss rates of the cultures. The plasmid loss rate was calculated by the following equation: 1-(the number of CFUs on the TSA-Cfx plate)/(the number of CFUs on the TSA plate). Data were obtained from three independent experiments and analyzed using Student's t-test. The asterisks above the line indicate significance between the two indicated groups. ^***p < 0.001.

Figure 4

Cas1 and Cas2 were essential for RA-CH-2 spacer acquisition. Cultures of RA-CH-2 strains (wild-type and cas gene deletion mutant strains) harboring the shuttle plasmid pLMF03 were grown in TSB without antibiotics for 10 growth cycles. A PCR assay of the cultures was performed with the primers indicated in Figure 1 to detect spacer acquisition. The image is representative of multiple experiments.

Figure 5

Deletion of RA-CH-2 cas1 or cas2 stabilized the shuttle plasmid. Plasmid loss from RA-CH-2 strains (wild-type and cas gene deletion mutant strains harboring pLMF03) were analyzed by counting the colonies on plates with or without antibiotics (Cfx). The rate of plasmid loss was calculated, and data obtained from three independent experiments were analyzed using Student's t-test. ^***p < 0.001.

Figure 6

Distribution of the protospacers. The protospacers were numbered based on the sequenced spacers (as listed in Table 3) and are indicated on the pLMF03 plasmid map according to their positions and orientations (pink arrows). Arrows pointing in the clockwise direction represent protospacers matching the plus strand of the pLMF03 vector, and those pointing in the counterclockwise direction represent protospacers matching the minus strand.

Figure 7

Prediction of the PAM using WebLogo. The flanking sequences upstream of the protospacers (10 nt) were aligned using WebLogo. The first nucleotide (at position −1) was adjacent to the 5′ end of the protospacer. The sizes of the letters indicate the relative frequency of the corresponding base at that position, and the conserved PAM was predicted to be 5′-GWATTN-3′ (W represents A or T).

Figure 8

Interaction of RA Cas1 and Cas2 in vitro. (A,B) Western blotting using anti-FLAG and anti-Cas1 antibodies, respectively. Lanes 1 and 4, the samples immunoprecipitated by anti-Cas1. Lane 2, the pull-down sample obtained by using Ni-NTA agarose. Lanes 3 and 5, the untreated control samples (cell lysates before assay).

Figure 9

Nuclease activity assays for RA Cas1. (A) The recombinant RA Cas1 protein degraded circular and linear dsDNA. Lanes 1–5, circular dsDNA substrate. Lanes 6–10, linear dsDNA substrate. Lanes 1 and 6, no protein (no Cas1). Lanes 2 and 7, no metal ion (no Mn²⁺). Lanes 3, 4, 8, and 9, nuclease reactions of the recombinant Cas1 protein in the presence of Mn²⁺; Lanes 4 and 9, reaction products extracted with phenol. Lanes 5 and 10, deoxyribonuclease I (1 mg/ml DNase I) used for the nuclease reaction as a positive control. (B) Nuclease activity assays for Cas1 mutants; reaction products were extracted with phenol before electrophoresis. All nuclease reaction products were analyzed by 1.5% agarose gel electrophoresis.