CRISPys: Optimal sgRNA design for editing multiple members of a gene family using the CRISPR system

Abstract

The discovery and development of the CRISPR-Cas9 system in the past few years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simpler, efficient, and effective task. The system is directed to the genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with the DNA target, subsequently leading to site-specific double-strand breaks. However, many gene families in eukaryotic genomes exhibit partially overlapping functions and, thus, the knockout of one gene might be concealed by the function of the other. In such cases, the reduced specificity of the CRISPR-Cas9 system, which may lead to the cleavage of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes. Here, we introduce CRISPys, an algorithm for the optimal design of sgRNAs that would potentially target multiple members of a given gene family. CRISPys first clusters all the potential targets in the input sequences into a hierarchical tree structure that specifies the similarity among them. Then, sgRNAs are proposed in the internal nodes of the tree by embedding mismatches where needed, such that the cleavage efficiencies of the induced targets are maximized. We suggest several approaches for designing the optimal individual sgRNA, and an approach that provides a set of sgRNAs that also accounts for the homologous relationships among gene-family members. We further show by in-silico examination over all gene families in the Solanum lycopersicum genome that our suggested approach outperforms simpler alignment-based techniques.\n\nGraphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY\n\nHighlightsO_LIMany genes in eukaryotic genomes exhibit partially overlapping functions. This imposes difficulties on reverse-genetics, as the knockout of one gene might be concealed by the function of the other.\nC_LIO_LIWe present CRISPys, a graph-based algorithm for the optimal design of CRISPR systems given a set of redundant genes.\nC_LIO_LICRISPys harnesses the lack of specificity of the CRISPR-Cas9 genome editing technique, providing researchers the ability to simultaneously mutate multiple genes.\nC_LIO_LIWe show that CRISPys outperforms existing approaches that are based on simple alignment of the input gene family.\nC_LI