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

Abstract

Background CRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing CRISPR-Cas guide RNAs (gRNAs) for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for challenging projects with (i) non-standard Cas enzymes, (ii) non-model organisms, or (iii) projects that need to design a panel of gRNA for genome-wide screens. Findings GuideMaker can rapidly design gRNAs for gene targets across the genome using a degenerate protospacer-adjacent motif (PAM) and a genome. The tool applies hierarchical navigable small world graphs to speed up the comparison of guide RNAs and optionally provides on-target and off-target scoring. This allows the user to design effective gRNAs targeting all genes in a typical bacterial genome in ∼1–2 minutes. Conclusions GuideMaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.

CRISPR-COPIES: Anin silicoplatform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration

“…Therefore, we search and report the number of 4 nt complementarity within the gRNA and between the gRNA and a custom backbone. As SpCas9 remains the most widely used Cas endonuclease, we integrate on-target scores based on Rule Set 2 if canonical NGG PAM is used (28, 44). Recently, Rule Set 2 algorithm was modified to obtain higher prediction accuracy over the existing tool (30).…”

“…Design rules and on-target models are also added to prioritize gRNAs with high on-target activity. While bioinformatics tools like CHOPCHOP (61) and GuideMaker (28) help in designing genome-wide gRNA libraries for CRISPR-mediated activation, interference, and deletion, the primary aim of CRISPR-COPIES is to guide the selection of genome-wide intergenic sites for gene integration. We performed a thorough computational analysis for organisms across the tree of life to illustrate the speed and versatility of the software.…”

“…Genome, protein sequences, and feature table for all organisms were procured from the NCBI’s Genome (https://www.ncbi.nlm.nih.gov/genome) or RefSeq (https://www.ncbi.nlm.nih.gov/refseq/) database (27). Pre-trained models and the code to predict on-target scores were downloaded from the GitHub repository of the following software: GuideMaker (28), DeepGuide (29), CROPSR (30), and sgRNA design for E. coli (31).…”

Seedlessness Trait and Genome Editing—A Review

“…Guide RNA (gRNA) design for the particular gene and cloning the gRNAs into appropriate vector backbones are the primary tasks for genome editing vector construction. Different online tools for gRNA design (i.e., CRISPOR [ 141 ], CRISPR-P [ 142 ], CCTop [ 143 ], CHOPCHOP [ 144 ], and GuideMaker [ 145 ]) with customized features are openly accessible. High-efficiency cloning technology (MoClo) [ 146 ] and readymade cloning materials of different expression modules are readily available from addgene ( ) and other sources.…”

Generating minimum set of gRNA to cover multiple targets in multiple genomes with MINORg

“…Furthermore, the experimental burden of designing and cloning separately designed gRNA for multiple genes in multiple genomes may render large pangenomic screens tedious, which highlights the need for gRNA design tools to be able to generate a minimum gRNA set capable of covering all desired targets in the pangenome era. Recent tools such as MultiTargeter ( 11 ), which designs minimum gRNA for multiple targets, GuideMaker ( 12 ), which designs gRNA in non-reference genomes, and CRISPR-Local ( 13 ), which designs minimum gRNA for multiple targets in non-reference genomes on a per-genome basis, address some but not all of these considerations.…”