We are developing a CRISPR-Cas3-based tool to programmably generate large DNA deletions.
The ability to manipulate genomes in a manner of one’s choosing enables scientific breakthroughs, new biosynthetic capacity, and new medicines. All organisms possess genes and regions of unknown function, for which removal and/or replacement would allow interrogation of function. CRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we propose to utilize the Cas3 enzyme, which naturally possesses coupled nuclease and helicase activity and is capable of deletions >250 kb, for genome engineering purposes. We will exploit Cas3 to enable the removal and experimental interrogation of entire genes, operons, islands, proviruses, and repetitive elements en masse. CRISPR-Cas3 systems are easily programmable, possesses extensive diversity in sequenced genomes, and naturally occur in many microbes. If successful, the wide-scale implementation of CRISPR-Cas3 technology has the potential to greatly enhance multiple synthetic biology applications and present a novel tool for the analysis of genomic dark matter.
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