{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T06:34:21Z","timestamp":1773297261601,"version":"3.50.1"},"reference-count":38,"publisher":"Oxford University Press (OUP)","issue":"1","license":[{"start":{"date-parts":[[2017,9,8]],"date-time":"2017-09-08T00:00:00Z","timestamp":1504828800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/about_us\/legal\/notices"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["MCB #1553250, CBET #1511881, and CBET #1360867"],"award-info":[{"award-number":["MCB #1553250, CBET #1511881, and CBET #1360867"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2018,1,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Motivation<\/jats:title>\n                    <jats:p>Genetic diversity of non-model organisms offers a repertoire of unique phenotypic features for exploration and cultivation for synthetic biology and metabolic engineering applications. To realize this enormous potential, it is critical to have an efficient genome editing tool for rapid strain engineering of these organisms to perform novel programmed functions.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>To accommodate the use of CRISPR\/Cas systems for genome editing across organisms, we have developed a novel method, named CRISPR Associated Software for Pathway Engineering and Research (CASPER), for identifying on- and off-targets with enhanced predictability coupled with an analysis of non-unique (repeated) targets to assist in editing any organism with various endonucleases. Utilizing CASPER, we demonstrated a modest 2.4% and significant 30.2% improvement (F-test, P\u2009&amp;lt;\u20090.05) over the conventional methods for predicting on- and off-target activities, respectively. Further we used CASPER to develop novel applications in genome editing: multitargeting analysis (i.e. simultaneous multiple-site modification on a target genome with a sole guide-RNA requirement) and multispecies population analysis (i.e. guide-RNA design for genome editing across a consortium of organisms). Our analysis on a selection of industrially relevant organisms revealed a number of non-unique target sites associated with genes and transposable elements that can be used as potential sites for multitargeting. The analysis also identified shared and unshared targets that enable genome editing of single or multiple genomes in a consortium of interest. We envision CASPER as a useful platform to enhance the precise CRISPR genome editing for metabolic engineering and synthetic biology applications.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Availability and implementation<\/jats:title>\n                    <jats:p>https:\/\/github.com\/TrinhLab\/CASPER.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Supplementary information<\/jats:title>\n                    <jats:p>Supplementary data are available at Bioinformatics online.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/bioinformatics\/btx564","type":"journal-article","created":{"date-parts":[[2017,9,7]],"date-time":"2017-09-07T01:56:40Z","timestamp":1504749400000},"page":"16-23","source":"Crossref","is-referenced-by-count":43,"title":["Enhanced guide-RNA design and targeting analysis for precise CRISPR genome editing of single and consortia of industrially relevant and non-model organisms"],"prefix":"10.1093","volume":"34","author":[{"given":"Brian J","family":"Mendoza","sequence":"first","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN, USA"},{"name":"Oak Ridge National Laboratory, Bioenergy Science Center (BESC), Oak Ridge, TN, USA"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8362-725X","authenticated-orcid":false,"given":"Cong T","family":"Trinh","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN, USA"},{"name":"Oak Ridge National Laboratory, Bioenergy Science Center (BESC), Oak Ridge, TN, USA"}]}],"member":"286","published-online":{"date-parts":[[2017,9,8]]},"reference":[{"key":"2023020301105611600_btx564-B1","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.jbiotec.2015.06.427","article-title":"Systematic analysis of CRISPR\u2013Cas9 mismatch tolerance reveals low levels of off-target activity","volume":"211","author":"Anderson","year":"2015","journal-title":"J. Biotechnol"},{"key":"2023020301105611600_btx564-B2","doi-asserted-by":"crossref","DOI":"10.1038\/msb4100073","article-title":"Synthetic biology: new engineering rules for an emerging discipline","volume":"2","author":"Andrianantoandro","year":"2006","journal-title":"Mol. Syst. Biol"},{"key":"2023020301105611600_btx564-B3","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1126\/science.1231143","article-title":"Multiplex genome engineering using CRISPR\/Cas systems","volume":"339","author":"Cong","year":"2013","journal-title":"Science"},{"key":"2023020301105611600_btx564-B4","author":"Connelly","year":"2015"},{"key":"2023020301105611600_btx564-B5","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/nbt.3026","article-title":"Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation","volume":"32","author":"Doench","year":"2014","journal-title":"Nat. Biotechnol"},{"key":"2023020301105611600_btx564-B6","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1038\/nbt.3437","article-title":"Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9","volume":"34","author":"Doench","year":"2016","journal-title":"Nat. Biotech"},{"key":"2023020301105611600_btx564-B7","doi-asserted-by":"crossref","first-page":"e1004724.","DOI":"10.1371\/journal.pcbi.1004724","article-title":"A biophysical model of CRISPR\/Cas9 activity for rational design of genome editing and gene regulation","volume":"12","author":"Farasat","year":"2016","journal-title":"PLoS Comput. Biol"},{"key":"2023020301105611600_btx564-B8","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.ymben.2014.10.001","article-title":"Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production","volume":"27","author":"Feng","year":"2015","journal-title":"Metab. Eng"},{"key":"2023020301105611600_btx564-B9","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1038\/nbt.3718","article-title":"Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering","volume":"35","author":"Garst","year":"2017","journal-title":"Nat. Biotechol"},{"key":"2023020301105611600_btx564-B10","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/S1525-0016(03)00099-6","article-title":"Gene transfer into genomes of human cells by the sleeping beauty transposon system","volume":"8","author":"Geurts","year":"2003","journal-title":"Mol. Ther"},{"key":"2023020301105611600_btx564-B11","doi-asserted-by":"crossref","first-page":"e00928","DOI":"10.1128\/mBio.00928-13","article-title":"Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems","volume":"5","author":"Gomaa","year":"2014","journal-title":"MBio"},{"key":"2023020301105611600_btx564-B12","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1038\/nbt.3290","article-title":"Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells","volume":"33","author":"Hendel","year":"2015","journal-title":"Nat. Biotechnol"},{"key":"2023020301105611600_btx564-B13","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1038\/nbt.2647","article-title":"DNA targeting specificity of RNA-guided Cas9 nucleases","volume":"31","author":"Hsu","year":"2013","journal-title":"Nat. Biotechnol"},{"key":"2023020301105611600_btx564-B14","doi-asserted-by":"crossref","first-page":"1262","DOI":"10.1016\/j.cell.2014.05.010","article-title":"Development and applications of CRISPR-Cas9 for genome engineering","volume":"157","author":"Hsu","year":"2014","journal-title":"Cell"},{"key":"2023020301105611600_btx564-B15","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1016\/S0092-8674(00)80436-5","article-title":"Molecular reconstruction of sleeping beauty, a Tc1-like transposon from fish, and its transposition in human cells","volume":"91","author":"Ivics","year":"1997","journal-title":"Cell"},{"key":"2023020301105611600_btx564-B16","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ymben.2015.01.008","article-title":"Multiplex metabolic pathway engineering using CRISPR\/Cas9 in Saccharomyces cerevisiae","volume":"28","author":"Jako\u010di\u016bnas","year":"2015","journal-title":"Metab. Eng"},{"key":"2023020301105611600_btx564-B17","doi-asserted-by":"crossref","first-page":"1395","DOI":"10.1016\/j.biotechadv.2015.02.011","article-title":"Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals","volume":"33","author":"Jullesson","year":"2015","journal-title":"Biotechnol. Adv"},{"key":"2023020301105611600_btx564-B18","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1038\/nature14592","article-title":"Engineered CRISPR-Cas9 nucleases with altered PAM specificities","volume":"523","author":"Kleinstiver","year":"2015","journal-title":"Nature"},{"key":"2023020301105611600_btx564-B19","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1038\/nature16526","article-title":"High-fidelity CRISPR\u2013Cas9 nucleases with no detectable genome-wide off-target effects","volume":"529","author":"Kleinstiver","year":"2016","journal-title":"Nature"},{"key":"2023020301105611600_btx564-B20","doi-asserted-by":"crossref","first-page":"W272","DOI":"10.1093\/nar\/gkw398","article-title":"CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering","volume":"44","author":"Labun","year":"2016","journal-title":"Nucleic Acids Res"},{"key":"2023020301105611600_btx564-B21","doi-asserted-by":"crossref","first-page":"536","DOI":"10.1038\/nchembio.970","article-title":"Systems metabolic engineering of microorganisms for natural and non-natural chemicals","volume":"8","author":"Lee","year":"2012","journal-title":"Nat. Chem. Biol"},{"key":"2023020301105611600_btx564-B22","doi-asserted-by":"crossref","first-page":"688","DOI":"10.1038\/nbt.2654","article-title":"Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9","volume":"31","author":"Li","year":"2013","journal-title":"Nat. Biotechnol"},{"key":"2023020301105611600_btx564-B23","doi-asserted-by":"crossref","first-page":"7473","DOI":"10.1093\/nar\/gku402","article-title":"CRISPR\/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences","volume":"42","author":"Lin","year":"2014","journal-title":"Nucleic Acids Res"},{"key":"2023020301105611600_btx564-B24","doi-asserted-by":"crossref","first-page":"10124.","DOI":"10.1038\/ncomms10124","article-title":"PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing","volume":"6","author":"Malina","year":"2015","journal-title":"Nat. Commun"},{"key":"2023020301105611600_btx564-B25","doi-asserted-by":"crossref","first-page":"982","DOI":"10.1038\/nmeth.3543","article-title":"CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo","volume":"12","author":"Moreno-Mateos","year":"2015","journal-title":"Nat. Methods"},{"key":"2023020301105611600_btx564-B26","doi-asserted-by":"crossref","first-page":"1185","DOI":"10.1016\/j.cell.2016.02.004","article-title":"Engineering cellular metabolism","volume":"164","author":"Nielsen","year":"2016","journal-title":"Cell"},{"key":"2023020301105611600_btx564-B27","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1016\/j.cell.2014.09.014","article-title":"CRISPR-Cas9 knockin mice for genome editing and cancer modeling","volume":"159","author":"Platt","year":"2014","journal-title":"Cell"},{"key":"2023020301105611600_btx564-B28","doi-asserted-by":"crossref","first-page":"e0119372.","DOI":"10.1371\/journal.pone.0119372","article-title":"CRISPR MultiTargeter: a web tool to find common and unique CRISPR single guide RNA targets in a set of similar sequences","volume":"10","author":"Prykhozhij","year":"2015","journal-title":"PLoS One"},{"key":"2023020301105611600_btx564-B29","doi-asserted-by":"crossref","first-page":"1002","DOI":"10.1038\/nbt.2355","article-title":"RNA processing enables predictable programming of gene expression","volume":"30","author":"Qi","year":"2012","journal-title":"Nat. Biotechnol"},{"key":"2023020301105611600_btx564-B30","doi-asserted-by":"crossref","first-page":"1173","DOI":"10.1016\/j.cell.2013.02.022","article-title":"Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression","volume":"152","author":"Qi","year":"2013","journal-title":"Cell"},{"key":"2023020301105611600_btx564-B31","doi-asserted-by":"crossref","first-page":"e03703.","DOI":"10.7554\/eLife.03703","article-title":"Selection of chromosomal DNA libraries using a multiplex CRISPR system","volume":"3","author":"Ryan","year":"2014","journal-title":"Elife"},{"key":"2023020301105611600_btx564-B32","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1038\/nbt.2842","article-title":"CRISPR-Cas systems for editing, regulating and targeting genomes","volume":"32","author":"Sander","year":"2014","journal-title":"Nat. Biotechnol"},{"key":"2023020301105611600_btx564-B33","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.meteno.2015.03.001","article-title":"CRISPR\u2013Cas system enables fast and simple genome editing of industrial Saccharomyces cerevisiae strains","volume":"2","author":"Stovicek","year":"2015","journal-title":"Metab. Eng. Commun"},{"key":"2023020301105611600_btx564-B34","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.coche.2016.07.005","article-title":"Modular cell design for rapid, efficient strain engineering toward industrialization of biology","volume":"14","author":"Trinh","year":"2016","journal-title":"Curr. Opin. Chem. Eng"},{"key":"2023020301105611600_btx564-B35","doi-asserted-by":"crossref","first-page":"80.","DOI":"10.1126\/science.1246981","article-title":"Genetic screens in human cells using the CRISPR-Cas9 system","volume":"343","author":"Wang","year":"2014","journal-title":"Science"},{"key":"2023020301105611600_btx564-B36","doi-asserted-by":"crossref","first-page":"949","DOI":"10.1016\/j.cell.2016.04.003","article-title":"Crystal structure of Cpf1 in complex with guide RNA and target DNA","volume":"165","author":"Yamano","year":"2016","journal-title":"Cell"},{"key":"2023020301105611600_btx564-B37","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1038\/nbt.3177","article-title":"Multiplexed tracking of combinatorial genomic mutations in engineered cell populations","volume":"33","author":"Zeitoun","year":"2015","journal-title":"Nat. Biotechnol"},{"key":"2023020301105611600_btx564-B38","doi-asserted-by":"crossref","first-page":"759","DOI":"10.1016\/j.cell.2015.09.038","article-title":"Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system","volume":"163","author":"Zetsche","year":"2015","journal-title":"Cell"}],"container-title":["Bioinformatics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/bioinformatics\/article-pdf\/34\/1\/16\/49043555\/bioinformatics_34_1_16.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/bioinformatics\/article-pdf\/34\/1\/16\/49043555\/bioinformatics_34_1_16.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,2,2]],"date-time":"2023-02-02T20:12:41Z","timestamp":1675368761000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/bioinformatics\/article\/34\/1\/16\/4107936"}},"subtitle":[],"editor":[{"given":"John","family":"Hancock","sequence":"additional","affiliation":[]}],"short-title":[],"issued":{"date-parts":[[2017,9,8]]},"references-count":38,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2018,1,1]]}},"URL":"https:\/\/doi.org\/10.1093\/bioinformatics\/btx564","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/139626","asserted-by":"object"}]},"ISSN":["1367-4803","1367-4811"],"issn-type":[{"value":"1367-4803","type":"print"},{"value":"1367-4811","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2018,1,1]]},"published":{"date-parts":[[2017,9,8]]}}}