Using of bioinformatic software methods for determining PAM sequences by analyzing of CRISPR sites

Abstract

Abstract Currently, CRISPR/Cas technology is one of the most actively developing in molecular biology as an instrument for genomic DNA editing and controlling gene activity at various levels. For today this technology has a number of limitations that makes it difficult to use. One of the main limitation is the conditions under which the Cas type proteins or their analogues cut DNA: 1) the complementary between sequence of protospacer and guiding RNA; 2 )the special sequences flanking this protospacer on one or both ends. This special sequences usually unique for each enzyme and named PAM sites. However, if the ability to change guiding RNA leads to the emergence of an accurate genomic editing tool, the presence of an immutable PAM site significantly limits the areas of the genome in which editing can be performed. At the moment, more than 3.5 thousand of Cas proteins using different PAM sites have been identified in various bacteria with the help of bioinfomatic tools, but in practice less than ten PAM are used. To be able to use new proteins, it is necessary to obtain their characteristics. In particular the optimal physical and chemical conditions for enzyme, the structure of guiding RNA and PAM-sequence, characteristic for new protein. Currently, several programs have been created to determine PAM sequences for previously unexplored Cas protein analogues, but all of them prone to mistakes and require data confirmation from classical molecular biology. The purpose of this work was to create a new efficient algorithm and software for idetification of PAM sequences characteristic of any microorganism. To test the program, we used CRISPR bacterial cassettes, formed by Cas9 enzyme, as well as current homology search programs for the bacterial genomes. As a result of the study, a modular program was created that allows identifying the sequence of the PAM site based on the analysis of CRISPR cassettes with an efficiency not inferior to world analogues.