Shedding Light on Cas9 Target Search

Abstract

The CRISPR/Cas adaptive immune system provides prokaryotes with a defensive mechanism against invading viruses and plasmids. The invading viral DNA fragments are incorporated into the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) locus in the bacterial genome and are later used to recognize and destroy the invader when it returns. In the last stage of CRISPR immunity, called the interference stage, Cas (CRISPR-associated) proteins assemble with short guide RNA molecules which are transcribed from the CRISPR locus. These guide RNA molecules can be programmed to recognize any DNA sequence. In recent years the CRISPR/Cas adaptive immune system has seen an immense growth in interest with the type II CRISPR-Cas9 system being in the center of attention. In this system, the DNA of the invading virus is recognized and cleaved by a single protein Cas9 which is guided by an RNA duplex. Due to its simplicity, CRISPR-Cas9 system is a promising tool in gene engineering as its guide RNA can be programmed to recognize virtually any sequence in the genome. The CRISPR-Cas9 has been demonstrated to work in a variety of organisms, however, despite the large interest in this system, the precise mechanism by which Cas9 finds and cleaves its target remains ambiguous. We utilize biophysical single-molecule techniques, namely total internal reflection fluorescence microscopy (TIRFM) together with Forster resonance energy transfer (FRET) to investigate the mechanics of Cas9 target search with nanometer sensitivity. We are probing one-dimensional diffusion of the protein along the DNA strand and investigating the effects different DNA sequences have on the mechanics of Cas9 target search.