Identification of Novel Cas Proteins in Metagenomic Datasets of Microbiomes in Puerto Rican Ecosystems

CRISPR-Cas9 technology has become one of the most groundbreaking discoveries of the 21st century. CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, along with CRISPR-associated (Cas) proteins, function as an adaptive immune system for many prokaryotic organisms. As a gene editing tool, CRISPR-Cas9 allows for sequence-specific targeting, which scientists have exploited in favor of biomedicine, as a way to alter and remove genes and genomic regions altogether in almost all eukaryotic cells. In spite of its great implications in biomedical research, and as a means of studying gene function, CRISPR-Cas9 has important limitations. Perhaps, most notably, Cas9 interacts with a specific DNA recognition site, known as the protospacer adjacent motif (PAM). In the absence of the correct PAM sequence, Cas9 will be unable to bind DNA and, hence, it will be unable to perform its endonuclease function. While the PAM sequences for the commonly used S. pyogenes Cas9 (SpCas9) is commonly found throughout the human genome, they are not always efficiently positioned to target a particular gene. Our current understanding of Cas9 orthologs is deficient, and the characterization of novel PAM sequences will continue to improve the gene-editing efficiency of CRISPR technology as we know it. Moreover, the isolation of novel Cas9 and other CRISPR-associated proteins has and will continue to increase the number of CRISPR applications. Throughout this project, we aim to use functional metagenomics as a vehicle for bioprospecting novel CRISPR-Cas systems within different environmental samples of Puerto Rico. We analyzed two metagenomic datasets from the microbial and functional diversity of distinct water systems: the Guajataca water reservoir, and the Clara Cave and Empalme Sinkhole waters. Sequencing data was analyzed regarding CRISPR-Cas analogy through CRISPR databases via CRISPRCasFinder and NCBI-BLAST. Multiple sequence alignments were conducted to describe conserved regions in Cas genes using the ConSurf server. Several CRISPR-Cas-like regions were identified within the metagenomic datasets. Candidate prospects will be analyzed concerning Cas protein identity and function with in vivo and in vitro approaches. Throughout this approach, we expect to unravel previously unexplored CRISPR-Cas systems, as well as characterizing and describing Cas protein orthologs within these environments respecting PAM sequence specificity and other functional and structural characteristics.