Guide RNA Design for Genome-Wide CRISPR Screens in Yarrowia lipolytica.

Abstract

Genome-wide functional genomic screens are essential to determining the genetic underpinning of a biological process. Novel and powerful tools for perturbing gene function, with the help of genetic and epigenetic information, have made it possible to systematically investigate the contribution of every gene to evolved and engineered phenotypes. Functional genomics and screening for enhanced phenotypes become ever more important when dealing with nonconventional hosts. Non-model organisms are valuable to metabolic engineering as they present a range of desirable phenotypes and can help in avoiding complex and intensive engineering of less suitable hosts that do not possess the desired phenotype(s). Domestication of such hosts however requires a suite of synthetic biology tools that allow for targeted genome engineering, regulation of gene expression, and genome-wide mutational screens. The widespread adoption of CRISPR-Cas9 and CRISPR-Cpf1 based systems has allowed for such screens in many organisms. Key considerations in any genome-wide CRISPR screen are the design of a set of unique guide RNAs targeting the required set of genes in the genome and the design of nontargeting guide RNAs that function as appropriate negative controls for the experiment. In this methods chapter, we present protocols for the design of guides for a CRISPR screen, targeting every gene in the genome of the industrially relevant oleaginous yeast Yarrowia lipolytica. The first set of protocols describes the algorithm for the design of genome targeting and nontargeting guides for a genome-wide CRISPR-Cpf1 screen. The second set of protocols describes modifications to the first for the design of guides for a CRISPR-Cas9 screen. The strategies described here should serve as an efficient guide to design a library of gRNAs for most genome-wide CRISPR screens.