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ARS Home » Southeast Area » Stoneville, Mississippi » Genomics and Bioinformatics Research » Research » Publications at this Location » Publication #384526

Research Project: Development of Tn-Seq Methods for the Functional Characterization of Bacteria

Location: Genomics and Bioinformatics Research

Title: GuideMaker: software to design CRISPR-Cas guide RNA pools in non-model genomes

item POUDEL, RAVIN - University Of Florida
item TRUJILLO-RODRIGUEZ, LIDIMARIE - University Of Florida
item REISCH, CHRISTOPHER - University Of Florida
item Rivers, Adam

Submitted to: Gigascience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2022
Publication Date: 4/1/2022
Citation: Poudel, R., Trujillo-Rodriguez, L., Reisch, C., Rivers, A.R. 2022. GuideMaker: software to design CRISPR-Cas guide RNA pools in non-model genomes. Gigascience. 11. Article giac007.

Interpretive Summary: Science still does not know the function of many genes. Discovering gene function can help create new products to control or use bacteria. Traditionally scientists have created a mutation (change) in one gene to make it non-functional and observe the effect. Newer methods mutate one random gene in millions of different bacterial cells at the same time in a single test tube. That mix of mutated bacteria can then be grown together. By DNA sequencing the mix at the beginning and end of growth the genes that are required for growth can be found. A new gene-editing technology called CRISPR/Cas can be used to mutate the genes at many specific points, which is more efficient than randomly mutating them. Guidemaker is software that rapidly designs the short pieces of RNA needed to mutate genes at many specific points. Guidemaker is the first software to make this new gene discovery method more accessible.

Technical Abstract: CRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing the CRISPR-Cas guide RNA’s for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy to use design tool for atypical projects with: 1) non-standard Cas enzymes, 2) non-model organisms, or 3) projects that need to design a panel of guide RNAs (gRNA) for genome-wide screens. Given a degenerate protospacer adjacent motif (PAM) sequence and a GenBank file, GuideMaker can rapidly design gRNAs meeting specific target genes and gRNA criteria. The tool applies Hierarchical Navigable Small World (HNSW) graphs to speed up the comparison of guide RNAs enabling the user to design gRNA for all genes for a typical bacterial genome and PAM sequence in about 1-2 minutes on a laptop. Guidemaker enables the rapid design of genome-wide CRISPR/Cas gene function studies in non-model organisms with any Cas enzyme. While GuideMaker is designed with prokaryotic genomes in mind, it can process smaller eukaryotic genomes as well. GuideMaker is available as command-line software and as a web application at and in the CyCverse Discovery Environment.