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

Lisa Simirenko; Jan-Fang Cheng; Ian Blaby

doi:10.3389/fbioe.2023.1217811

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

Front Bioeng Biotechnol. 2023 Aug 29:11:1217811. doi: 10.3389/fbioe.2023.1217811. eCollection 2023.

Authors

Lisa Simirenko¹, Jan-Fang Cheng^{1

2}, Ian Blaby^{1

2}

Affiliations

¹ US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
² Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.

Abstract

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).

Keywords: CRISPR; CRISPR screen; batch DNA extraction; design tool; gRNA.

Grants and funding

This work has been supported by the DOE Joint Genome Institute (http://jgi.doe.gov) by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through Contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.