Novel clustered regularly interspaced short palindromic repeats-associated 9 platform with divergent targeting capabilities

Abstract

Thesis: S.M., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2018.Cataloged from PDF version of thesis.Includes bibliographical references (pages 47-49).RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. The range of sequences that CRISPR endonucleases can recognize, however, is constrained by the need for a specific protospacer adjacent motif (PAM) flanking the target site. In this thesis, we demonstrate the natural PAM plasticity of a highly-similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report a divergent affinity to 5'-NNGT-3' PAM sequences, as well as preferences for expanded 5'-NNG- 3' motifs, and demonstrate the editing capabilities of the ortholog in both bacterial and human cells. We subsequently build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise-overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be utilized both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities. Finally, we develop original machine learning-based tools to both predict the efficacy of single guide RNA (sgRNA) sequences targeting specific loci, as well as to classify and characterize the recently-discovered anti-CRISPR proteins.by Pranam Chatterjee.S.M