Determining the PAM sequence for the Streptococcus thermophilus CRISPR4 Cas1-Cas2 complex

Tate Bothner

Mentor: Dr. Scott Bailey

Supervisor: Elvar Bjarkason

Johns Hopkins Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated) systems are adaptive bacterial immune systems found in bacteria and archaea, used to fight against invaders. In S. thermophilus, type I-E systems utilize the Cas1-Cas2 complex to identify foreign DNA elements for adaptation. This is the first step in CRISPR immunity in which fragments of foreign DNA, known as protospacers, are integrated into the CRISPR array within the bacterial genome as spacers. A key component of the identification of foreign DNA is the protospacer adjacent motif (PAM) sequence, which is used by Cas1-Cas2 to identify a potential protospacer before further investigation of the downstream target sequence. While previous studies have outlined the general functions and applications of the S. thermophilus CRISPR-Cas system, little is known about the PAM. The aim of this study was to determine the optimal PAM sequence of S. thermophilus. Based on a bioinformatics analysis of spacer sequences, the PAM was identified as AA or GA in the S. thermophilus CRISPR4 system. While next steps are required to validate this study, the PAM identification helps take a first step towards a greater understanding of the CRISPR-Cas system in this bacterium.