{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,24]],"date-time":"2026-04-24T21:27:53Z","timestamp":1777066073976,"version":"3.51.4"},"reference-count":28,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2025,4,27]],"date-time":"2025-04-27T00:00:00Z","timestamp":1745712000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>CRISPR-Cas9 has revolutionized genetic research with bioinformatics tools essential for tasks like guide RNA design, off-target prediction, and data analysis. This systematic review summarizes the functionality and key features of such tools. Studies published after 2012 were selected through searches in PubMed, Google Scholar, and other sources, with the final search conducted on 9 November 2024. Seven studies met the criteria, describing around 45 tools, including databases and functional programs. Tools like CRISPResso, CHOPCHOP, and Cas-OFFinder were commonly highlighted, with a major focus on single-guide RNA (sgRNA) design and optimization. Some tools provided specific solutions, while others offered broader functionality, but most lacked experimental validation. Several tools were developed by the authors of the studies, introducing potential bias. Findings highlight a need for integrated platforms that combine functionalities, reducing reliance on fragmented workflows. Current tools often address narrow tasks, complicating their practical application. Future development should focus on comprehensive, multitasking tools to improve accessibility and streamline research processes. Limitations include the descriptive nature of most studies, potential author bias, and challenges in comparing tools objectively. Nonetheless, this review underscores bioinformatics\u2019 critical role in CRISPR research and emphasizes the need for innovative, standardized platforms. This study received no funding and was not registered.<\/jats:p>","DOI":"10.3390\/cimb47050307","type":"journal-article","created":{"date-parts":[[2025,4,28]],"date-time":"2025-04-28T09:39:47Z","timestamp":1745833187000},"page":"307","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["CRISPR-Cas9 and Its Bioinformatics Tools: A Systematic Review"],"prefix":"10.3390","volume":"47","author":[{"given":"Alicja","family":"Jasieniecka","sequence":"first","affiliation":[{"name":"Polytechnic University of Coimbra, Rua da Miseric\u00f3rdia, Lagar dos Corti\u00e7os, S. Martinho do Bispo, 3045-093 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2334-7280","authenticated-orcid":false,"given":"In\u00eas","family":"Domingues","sequence":"additional","affiliation":[{"name":"Polytechnic University of Coimbra, Rua da Miseric\u00f3rdia, Lagar dos Corti\u00e7os, S. Martinho do Bispo, 3045-093 Coimbra, Portugal"},{"name":"Medical Physics, Radiobiology and Radiological Protection Group, Research Centre of the Portuguese Institute of Oncology of Porto (CI-IPOP), 4200-072 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2025,4,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"816","DOI":"10.1126\/science.1225829","article-title":"A Programmable Dual-RNA\u2013Guided DNA Endonuclease in Adaptive Bacterial Immunity","volume":"337","author":"Jinek","year":"2012","journal-title":"Science"},{"key":"ref_2","unstructured":"(2024, October 04). The Nobel Prize in Chemistry 2020. Available online: https:\/\/www.nobelprize.org\/prizes\/chemistry\/2020\/popular-information\/."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1136\/archdischild-2016-310459","article-title":"What is CRISPR\/Cas9?","volume":"101","author":"Redman","year":"2016","journal-title":"Arch. Dis. Child. Educ. Pract."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.ymeth.2019.07.013","article-title":"CRISPR-Cas bioinformatics","volume":"172","author":"Alkhnbashi","year":"2020","journal-title":"Methods"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Wadas, I., and Domingues, I. (2025). Systematic Review of Phylogenetic Analysis Techniques for RNA Viruses Using Bioinformatics. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26052180"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Naeem, M., and Alkhnbashi, O.S. (2023). Current Bioinformatics Tools to Optimize CRISPR\/Cas9 Experiments to Reduce Off-Target Effects. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24076261"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Li, W., Xu, H., Xiao, T., Cong, L., Love, M.I., Zhang, F., Irizarry, R.A., Liu, J.S., Liu, M.B., and Liu, X.S. (2024). MAGeCK enables robust identification of essential genes from genome-scale CRISPR\/Cas9 knockout screens. Genome Biol., 15.","DOI":"10.1186\/s13059-014-0554-4"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1147","DOI":"10.1101\/gr.191452.115","article-title":"Sequence determinants of improved CRISPR sgRNA design","volume":"25","author":"Xu","year":"2015","journal-title":"Genome Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1779","DOI":"10.1038\/ng.3984","article-title":"Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells","volume":"49","author":"Meyers","year":"2017","journal-title":"Nat. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1262","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":"ref_11","doi-asserted-by":"crossref","first-page":"828","DOI":"10.1038\/nprot.2017.016","article-title":"Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening","volume":"12","author":"Joung","year":"2017","journal-title":"Nat. Protoc."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Zhang, Q., and Ye, Y. (2017). Not all predicted crispr-cas systems are equal: Isolated cas genes and classes of crispr like elements. BMC Bioinform., 18.","DOI":"10.1186\/s12859-017-1512-4"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Grissa, I., Vergnaud, G., and Pourcel, C. (2007). The crisprdb database and tools to display crisprs and to generate dictionaries of spacers and repeats. BMC Bioinform., 8.","DOI":"10.1186\/1471-2105-8-172"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"D393","DOI":"10.1093\/nar\/gkx835","article-title":"Anti-crisprdb: A comprehensive online resource for anti-crispr proteins","volume":"46","author":"Dong","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"W52","DOI":"10.1093\/nar\/gkm360","article-title":"Crisprfinder: A web tool to identify clustered regularly interspaced short palindromic repeats","volume":"35","author":"Grissa","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"W246","DOI":"10.1093\/nar\/gky425","article-title":"CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins","volume":"46","author":"Couvin","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1089\/crispr.2017.0022","article-title":"CRISPRdisco: An automated pipeline for the discovery and analysis of CRISPR-Cas systems","volume":"1","author":"Crawley","year":"2018","journal-title":"CRISPR J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"i489","DOI":"10.1093\/bioinformatics\/btu459","article-title":"CRISPRstrand: Predicting repeat orientations to determine the crRNA-encoding strand at CRISPR loci","volume":"30","author":"Alkhnbashi","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e105","DOI":"10.1093\/nar\/gkt183","article-title":"Crass: Identification and reconstruction of CRISPR from unassembled metagenomic data","volume":"41","author":"Skennerton","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"i520","DOI":"10.1093\/bioinformatics\/btw456","article-title":"Assemble CRISPRs from metagenomic sequencing data","volume":"32","author":"Lei","year":"2016","journal-title":"Bioinformatics"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"i576","DOI":"10.1093\/bioinformatics\/btw454","article-title":"Characterizing leader sequences of crispr loci","volume":"32","author":"Alkhnbashi","year":"2016","journal-title":"Bioinformatics"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"i145","DOI":"10.1093\/nar\/gkn228","article-title":"CRISPRcompar: A website to compare clustered regularly interspaced short palindromic repeats","volume":"36","author":"Grissa","year":"2008","journal-title":"Nucleic Acids Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"7975","DOI":"10.1038\/s41467-022-35743-y","article-title":"TAPE-seq is a cell-based method for predicting genome-wide off-target effects of prime editor","volume":"13","author":"Kwon","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"W401","DOI":"10.1093\/nar\/gku410","article-title":"CHOPCHOP: A CRISPR\/Cas9 and TALEN web tool for genome editing","volume":"42","author":"Montague","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1126\/science.1247005","article-title":"Genome-scale CRISPR-Cas9 knockout screening in human cells","volume":"343","author":"Shalem","year":"2014","journal-title":"Science"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1096","DOI":"10.1126\/science.aac7041","article-title":"Identification and characterization of essential genes in the human genome","volume":"350","author":"Wang","year":"2015","journal-title":"Science"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1186\/s12967-024-06013-w","article-title":"Transitioning from wet lab to artificial intelligence: A systematic review of AI predictors in CRISPR","volume":"23","author":"Abbasi","year":"2025","journal-title":"J. Transl. Med."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1186\/s12967-022-03765-1","article-title":"Integration of CRISPR\/Cas9 with artificial intelligence for improved cancer therapeutics","volume":"20","author":"Bhat","year":"2022","journal-title":"J. Transl. Med."}],"container-title":["Current Issues in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1467-3045\/47\/5\/307\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:22:26Z","timestamp":1760030546000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1467-3045\/47\/5\/307"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,27]]},"references-count":28,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2025,5]]}},"alternative-id":["cimb47050307"],"URL":"https:\/\/doi.org\/10.3390\/cimb47050307","relation":{},"ISSN":["1467-3045"],"issn-type":[{"value":"1467-3045","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,27]]}}}