gRNA-SeqRET: a universal tool for targeted and genome-scale gRNA design and sequence extraction for prokaryotes and eukaryotes

Frontiers in Bioengineering and Biotechnology

Bioengineering and Biotechnology

Lisa Simirenko¹  Jan-Fang Cheng²  Ian Blaby² 

[1] US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, United States;US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, United States;Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States;

关键词: CRISPR;    gRNA;    design tool;    batch DNA extraction;    CRISPR screen;   

DOI  :  10.3389/fbioe.2023.1217811

 received in 2023-05-05, accepted in 2023-08-21,  发布年份 2023

来源: Frontiers

【 摘 要 】

High-throughput genetic screening is frequently employed to rapidly associate gene with phenotype and establish sequence-function relationships. With the advent of CRISPR technology, and the ability to functionally interrogate previously genetically recalcitrant organisms, non-model organisms can be investigated using pooled guide RNA (gRNA) libraries and sequencing-based assays to quantitatively assess fitness of every targeted locus in parallel. To aid the construction of pooled gRNA assemblies, we have developed an in silico design workflow for gRNA selection using the gRNA Sequence Region Extraction Tool (gRNA-SeqRET). Built upon the previously developed CCTop, gRNA-SeqRET enables automated, scalable design of gRNA libraries that target user-specified regions or whole genomes of any prokaryote or eukaryote. Additionally, gRNA-SeqRET automates the bulk extraction of any regions of sequence relative to genes or other features, aiding in the design of homology arms for insertion or deletion constructs. We also assess in silico the application of a designed gRNA library to other closely related genomes and demonstrate that for very closely related organisms Average Nucleotide Identity (ANI) > 95% a large fraction of the library may be of relevance. The gRNA-SeqRET web application pipeline can be accessed at https://grna.jgi.doe.gov. The source code is comprised of freely available software tools and customized Python scripts, and is available at https://bitbucket.org/berkeleylab/grnadesigner/src/master/ under a modified BSD open-source license (https://bitbucket.org/berkeleylab/grnadesigner).

【 授权许可】

Unknown   
Copyright © 2023 Simirenko, Cheng and Blaby.