CRISPys: Optimal sgRNA Design for Editing Multiple Members of a Gene Family Using the CRISPR System

Abstract

The development of the CRISPR-Cas9 system in recent years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simple and effective task. The system is directed to a genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with it, subsequently leading to site-specific modifications. 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 modification of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes. 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 efficiency to edit the induced targets is maximized. We suggest several approaches for designing the optimal individual sgRNA and an approach to compute the optimal set of sgRNAs for cases when the experimental platform allows for more than one. The latter may optionally account for the homologous relationships among gene-family members. We further show that CRISPys outperforms simpler alignment-based techniques by in silico examination over all gene families in the Solanum lycopersicum genome.

PINCER: improved CRISPR/Cas9 screening by efficient cleavage at conserved residues

“…‘transcriptional adaptation’ mechanistically linked to NMD), and some groups have proposed simultaneous targeting of multiple paralogs (e.g. CRISPys) ( 96 , 97 ). However, as this approach requires either prior knowledge of paralog sets and/or expanding library size, it remains a limiting factor for single-guide libraries like PINCER.…”

Genome editing reveals reproductive and developmental dependencies on specific types of vitellogenin in zebrafish (Danio rerio)

“…In this context, KO refers to the process of rendering a gene incapable of producing messenger RNA (mRNA) transcripts or their respective functional proteins. The high sequence identity among zebrafish type‐I vtgs precluded single‐gene loss of function studies and compelled us to employ a gene family editing design, which is now common practice in CRISPR/Cas9 experiments (cf Hyams et al ). This approach is based upon utilization of pooled sets of guide RNAs expected to target most or all gene family members.…”

CAFRI‐Rice: CRISPR applicable functional redundancy inspector to accelerate functional genomics in rice

“…Using the CAFRI‐Rice tool, researchers can easily obtain information concerning the functional redundancy of an interesting rice gene using an informative graphical user interface, without high computing power or other substantial labor. This tool makes it possible to design multi‐ or optimal multi‐targeting guide RNA for studying gene function and interest traits (Hyams et al ., 2018).…”