AUTHOR=Simirenko Lisa , Cheng Jan-Fang , Blaby Ian 

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

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1217811

DOI=10.3389/fbioe.2023.1217811

ISSN=2296-4185

ABSTRACT=<p>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 <italic>in silico</italic> 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 <italic>in silico</italic> the application of a designed gRNA library to other closely related genomes and demonstrate that for very closely related organisms Average Nucleotide Identity (ANI) &gt; 95% a large fraction of the library may be of relevance. The gRNA-SeqRET web application pipeline can be accessed at <ext-link ext-link-type="uri" xlink:href="https://grna.jgi.doe.gov/" xmlns:xlink="http://www.w3.org/1999/xlink">https://grna.jgi.doe.gov</ext-link>. The source code is comprised of freely available software tools and customized Python scripts, and is available at <ext-link ext-link-type="uri" xlink:href="https://bitbucket.org/berkeleylab/grnadesigner/src/master/" xmlns:xlink="http://www.w3.org/1999/xlink">https://bitbucket.org/berkeleylab/grnadesigner/src/master/</ext-link> under a modified BSD open-source license (<ext-link ext-link-type="uri" xlink:href="https://bitbucket.org/berkeleylab/grnadesigner" xmlns:xlink="http://www.w3.org/1999/xlink">https://bitbucket.org/berkeleylab/grnadesigner</ext-link>).</p>