Direct Repeats and Spacer Diversities as Found in Lactobacillus pentosus KCA1 CRISPR loci and Cas9 Structural Signature

Aim: To provide an insight on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) direct repeats and spacer organization present in Lactobacillus pentosus KCA1. Little or no information exist on CRISPR and CRISPR-associated (Cas) genes in Lactobacilli as a vista of new technique for potential genome editing.

Methodology: The genome sequence of Lactobacillus pentosus KCA1 was obtained from gene bank and bioinformatic tools such as CRISPR finder was used to analyze direct repeats and spacer diversities. CRISPR Target tool was used for spacer target prediction. The iterative threading assembly refinement (I-TASSER) algorithm was used for the prediction of the Cas9 3D structure, and the binding site by this integrated algorithm. CueMol2 version 2.2.2.366 was used to visualize the binding sites.

Results: The genome of Lactobacillus pentosus KCA1 encodes six CRISPR arrays with direct repeat (DR) length consensus exhibiting repeat polymorphisms ending with adenine-guanine (AG) nucleotide except CRISPR1 that has mainstream DR length ending with adenine-cytosine (AC) and terminal repeats ending with adenine-thymine (AT). The domain of KCA1_Cas9 has 153 amino acid residues belonging to HNH Cas9-type, located between positions 786-938. KCA1_0112 Cas9 has one chain A architecture and three putative ligand binding sites residues located at 10 (Asp), 777 (Glu) and 992 (Asp) positions of the 4086 base pairs.

Conclusion: Spacer analysis revealed that L. pentosus KCA1 may have been exposed to several mobile genetic elements as the spacers matched sequences from bacteriophages and plasmids. Further studies are needed to explore the structural architecture of KCA1 Cas9 as a potential part of CRISPR genome editing functionality.