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Conserved DNA motifs in the type II‐A CRISPR leader region
Author(s) -
Rajan Rakhi,
Van Orden Mason,
Klein Peter,
Kesavan Babu,
Najar Fares
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.909.7
Subject(s) - crispr , biology , plasmid , genetics , gene , locus (genetics) , dna , rna , trans activating crrna , streptococcus thermophilus , conserved sequence , crispr interference , computational biology , cas9 , bacteria , peptide sequence , lactobacillus
The C lustered R egularly I nterspaced S hort Pa lindromic Re peats (CRISPR) and CR ISPR‐ ass ociated (Cas) proteins consist of RNA‐protein complexes that provide bacteria and archaea sequence‐specific immunity against bacteriophages and conjugative plasmids. The bacteria or archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA site‐specifically into the leader‐repeat (repetitive units of CRISPR RNA gene) junction, in a process called adaptation. The hypothesis of the present work is that sequence/structural motifs present in the leader‐repeat junction recruits type‐specific Cas proteins for successful adaptation. To test this, we analyzed 167 type IIA CRISPR loci belonging to different bacterial genera. Our dataset consisted of 150 nucleotides of the CRISPR leader, the first repeat, and the protein sequences of Cas1, Cas2, Cas9, and Csn2 proteins. After performing multiple sequence alignments using MUSCLE, phylogenetic trees were generated in UGENE. Our results indicate two highly conserved DNA motifs at the 3′ leader end; 5′‐ATTTGAG‐3′ (indicated as group 1) and 5′‐CTRCGAG‐3′ (indicated as group 2). A third group with a very short 5′‐CG‐3′ DNA conservation at the 3′ leader end is observed mostly in Lactobacilli . Interestingly, the 3′‐leader end of group 1 is associated with the CRISPR‐1 locus of Streptococcus thermophilus DGCC7710, while that of group 2 is associated with the CRISPR‐3 locus of S. thermophilus DGCC7710. Since the CRISPR loci 1 and 3 of S. thermophilus DGCC7710 have been established as two distinct type IIA locus, our results demonstrate the association of these two loci with distinct 3′‐leader end sequences. A phylogenetic analysis of the repeat and Cas proteins revealed segregation reflecting the 3′ leader end conservation and pointed to the coevolution of the leader region and the CRISPR components. Previous studies have shown that the 3′‐leader end sequences are important for adaptation in S. thermophilus (group 1) and S. pyogenes (group 2) type IIA systems. Our results expanded this and demonstrated that the 3′‐leader end sequence conservation is more universally conserved covering several (~50 in this study) bacterial genera. Studies are ongoing to experimentally prove the importance of the 3′ leader end sequences and its co‐relation to locus‐specific Cas proteins. Similar studies can be used to identify conserved leader sequences in other CRISPR types and sub‐types. Support or Funding Information Research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under the grant number P20GM103640.