(498c) Computational Design of CRISPR Guide RNAs to Enable Strain-Specific Control of Microbial Consortia

Microbes naturally coexist in complex, multi-strain communities. However, extracting individual microbes from and specifically manipulating the composition of these consortia remains challenging. The sequence-specific nature of CRISPR guide RNAs can be leveraged to accurately differentiate microorganisms and facilitate the creation of tools that can achieve these tasks. We developed a computational program, ssCRISPR, which designs strain-specific CRISPR guide RNA spacer sequences with user-specified target strains, protected strains, and guide RNA properties. We experimentally verify the accuracy of the strain-specificity predictions in both Escherichia coli and Pseudomonas spp. and show that up to three nucleotide mismatches are required to ensure perfect specificity. To demonstrate the functionality of ssCRISPR, we apply computationally designed CRISPR-Cas9 guide RNAs to two applications: the purification and engineering of specific microbes through one- and two-plasmid transformation workflows and the targeted removal of specific microbes using DNA-loaded liposomes. ssCRISPR will be of use in diverse microbiota engineering applications. In this talk, we will discuss this promising and impactful technology to engineer diverse microbiota.

Austin Rottinghaus and Tae Seok Moon, US Provisional Patent Application

Austin G. Rottinghaus, Steven Vo, and Tae Seok Moon, Paper under review.