Modulation of the CRISPR-Cas9 Immune Response

Author

Hyejin Kim

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Luciano Marraffini

Abstract

Bacteria face an onslaught of ever-changing viral (phage) invaders. Bacteria and phages are locked into an evolutionary arms race where both must continually adapt to survive. In response, bacteria have developed numerous immune systems to protect themselves, including CRISPR-Cas. CRISPR-Cas systems provide adaptive immunity through the acquisition of short DNA sequences from invading phages, known as spacers. Spacers are inserted into the CRISPR locus and serve as templates for the transcription of guides used by RNA-guided nucleases to recognize complementary sequences of the invaders and start the CRISPR immune response. CRISPR-Cas systems need to acquire enough spacers to mount an effective response against evolving threats, but not so many spacers as to risk autoimmunity through the acquisition of self-targeting spacers. In type II-A CRISPR systems, Cas9 uses the guide RNA to cleave target DNA sequences in the genome of infecting phages, and the tracrRNA to bind the promoter of cas genes and repress their transcription. We previously isolated a Cas9 mutant carrying the I473F substitution that enhanced spacer acquisition by 2-3 orders of magnitude, leading to a fitness cost due to higher levels of autoimmunity. Here we investigated the molecular basis of these findings. We found that the I473F mutation decreases the association of Cas9 to tracrRNA, limiting its repressor function, leading to high levels of expression of cas genes, which in turn strengthen the type II-A CRISPR-Cas immune response. I473 lines a conserved hydrophobic pocket that makes base-specific contacts with the nexus of tracrRNA, and substitutions within this pocket affect association of Cas9 to tracrRNA to modulate the repression of cas genes in related type II-A CRISPR-Cas systems. Our findings highlight the importance of the interaction between Cas9 and its tracrRNA cofactor in tuning the type II-A CRISPR-Cas immune response to balanced levels that enable phage defense but avoid autoimmunity.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Kim, Hyejin, "Modulation of the CRISPR-Cas9 Immune Response" (2024). Student Theses and Dissertations. 809.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/809