Characterization of CRISPR‐Cas systems in Leptospira reveals potential application of CRISPR in genotyping of Leptospira interrogans

Abstract

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira. However, understanding of the pathogenic mechanism of Leptospira is still elusive due to the limited number of genetic tools available for this microorganism. Currently, the reason for the genetic inaccessibility of Leptospira is still unknown. It is well known that as an acquired immunity of bacteria, Clustered Regularly Interspaced Short Palindromic Repeat‐CRISPR‐associated gene (CRISPR‐Cas) systems can help bacteria against invading mobile genetic elements. In this study, the occurrence and diversity of CRISPR‐Cas systems in 41 genomes of Leptospira strains were investigated. Three subtypes (subtype I‐B, subtype I‐C and subtype I‐E) of CRISPR‐Cas systems were identified in both pathogenic and intermediate Leptospira species but not in saprophytic species. Noteworthy, the majority of pathogenic species harbor two different types of CRISPR‐Cas systems (subtype I‐B and subtype I‐E). Furthermore, Cas2 protein of subtype I‐C in L. interrogans exhibited a metal‐dependent DNase activity in a nonspecific manner. CRISPR spacers in subtype I‐B are highly conserved within the same serovars and hypervariable across different serovars of L. interrogans. Based on the subtype I‐B CRISPR arrays, the serotypes of different L. interrogans strains were easily identified. Investigation of the origin of CRISPR spacers showed that 192 spacers (23.5%) matched to mobile genetic elements, indicating CRISPR‐Cas systems may play an important role in the defense of foreign invading DNA.

PAM-repeat associations and spacer selection preferences in single and co-occurring CRISPR-Cas systems

“…1B). For more than 25,000 of these, the best hit was completely identical to the spacer, and for the vast majority (60,294), the total number of mismatched nucleotides was three or less (Fig. 1C).…”

“…Table of all unique type I PAMs found for the different subtypes and representative genera that contain the repeat cluster for which each PAM was determined. For previously described PAMs, a reference ID has been added which correspond to the following: 1[60], 2[61], 3[62], 4[63], 5[45], 6[64], 7[65], 8[46], 9[14], 10 [66], 11[67], 12[49], 13[68], 14[69], and 15[70]…”

Transcriptional analysis of CRISPR I-B arrays of Leptospira interrogans serovar Lai and its processing by Cas6

“…In the genus Leptospira and the genome of its infectious serovars (svs. ), three variants of CRISPR/Cas type I systems (subtypes I-B, -C, and -E) are prevalent (Makarova et al, 2011;Xiao et al, 2019). In addition, recently CRISPR-Cas type V was recorded in a saprophytic Leptospira strain (L. biflexa) (Martínez Arbas et al, 2021).…”

“…In addition, recently CRISPR-Cas type V was recorded in a saprophytic Leptospira strain (L. biflexa) (Martínez Arbas et al, 2021). Serovars of L. interrogans harbor two subtypes (I-B and I-C) of the type I system; however, CRISPR arrays were identified only at the I-B locus (Xiao et al, 2019). Thus, it was speculated that the CRISPR-Cas I-B in L. interrogans might be sufficient for CRISPR immunity.…”

“…Thus, it was speculated that the CRISPR-Cas I-B in L. interrogans might be sufficient for CRISPR immunity. In contrast, type I-C possibly carries out other unknown functions in L. interrogans (Xiao et al, 2019). In an in silico study of 41 Leptospira strains, 42% (48 out of 114) of the total identified CRISPR arrays were found more than 10 kb away from any cas gene (Xiao et al, 2019).…”