TYPE III-A CRISPR/CAS SYSTEMS AND COMPARISON OF CAS1, CAS2, AND CAS10 PROTEINS OF LACTOBACILLI
OZGE KAHRAMAN ILIKKAN
*
Food Quality Control and Analysis Program, Kahramankazan Vocational School, Başkent University, Ankara, Turkey.
*Author to whom correspondence should be addressed.
Abstract
The most abundant CRISPR systems are type I and type II in lactic acid bacteria, however, type III also exists in a few genera. In this study, lactobacilli that possess a type III-A CRISPR system were detected and Cas10 signature nucleases, Cas1, Cas2 proteins, and repeat sequences were compared. 39 strains belonging to 9 species had Type III-A CRISPR system within all lactobacilli. Ligilactobacillus salivarius strains had the most abundant type III-A system among all species. “agtaagaacatatctccgaatataggagacgaaaac” was the most frequent repeat sequence and the secondary structure was predicted using the RNAfold analysis tool. Cas1, Cas2, and Cas10 amino acid sequence alignment and the phylogenetic tree revealed that the strains tended to split into branches according to species. L. ruminis ATCC 27782 and ATCC 27780 had IS66 family insertion sequence element accessory protein TnpB between cas6 and CRISPR array, and also, DEDDh genes. The endogenous Cas10 enzyme of type III-A CRISPR systems has recently started being utilized in genetic engineering studies, and reprogrammed type III-A systems have been evaluated for endogenous genome editing. Therefore, this study retraced type III-A CRISPR systems of lactobacilli to evaluate clustering of repeat sequences, Cas1, Cas2, Cas10 proteins among strain, and also intended to discover lactobacilli as a candidate for utilization in reprogrammed endogenous genome editing in the future.
Keywords: Type III-A, CRISPR/Cas, Cas10, repeat sequence, lactobacilli