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Current popular sgRNA design tools only consider the sgRNA perfectly matched to the target site and provide the results without any on-target mismatch. We suppose taking on-target gRNA-DNA mismatches into consideration might provide better sgRNA with similar binding activity and reduced off-target sites. Here, we trained a seq2seq-attention model with feedback-loop architecture, to automatically generate sgRNAs with on-target mismatches. Dual-luciferase reporter experiment showed that multiple sgRNAs with three mismatches could achieve the 80% of the relative activity of the perfect matched sgRNA. Meanwhile, it could reduce the number of off-target sites using sgRNAs with on-target mismatches. Finally, we provided a freely accessible web server sgRNA design tool named ExsgRNA. Users could submit their target sequence to this server and get optimal sgRNAs with less off-targets and similar on-target activity compared with the perfect-matched sgRNA.<\/jats:p>","DOI":"10.1093\/bib\/bbac183","type":"journal-article","created":{"date-parts":[[2022,4,26]],"date-time":"2022-04-26T11:57:13Z","timestamp":1650974233000},"source":"Crossref","is-referenced-by-count":6,"title":["ExsgRNA: reduce off-target efficiency by on-target mismatched sgRNA"],"prefix":"10.1093","volume":"23","author":[{"given":"Wei-Xin","family":"Hu","sequence":"first","affiliation":[{"name":"Biomedical Informatics & Genomics Center, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi\u2019an Jiaotong University , Xi\u2019an 710049, P.R. China"}]},{"given":"Yu","family":"Rong","sequence":"additional","affiliation":[{"name":"Biomedical Informatics & Genomics Center, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi\u2019an Jiaotong University , Xi\u2019an 710049, P.R. China"}]},{"given":"Yan","family":"Guo","sequence":"additional","affiliation":[{"name":"Biomedical Informatics & Genomics Center, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi\u2019an Jiaotong University , Xi\u2019an 710049, P.R. China"}]},{"given":"Feng","family":"Jiang","sequence":"additional","affiliation":[{"name":"Biomedical Informatics & Genomics Center, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi\u2019an Jiaotong University , Xi\u2019an 710049, P.R. China"}]},{"given":"Wen","family":"Tian","sequence":"additional","affiliation":[{"name":"Biomedical Informatics & Genomics Center, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi\u2019an Jiaotong University , Xi\u2019an 710049, P.R. China"}]},{"given":"Hao","family":"Chen","sequence":"additional","affiliation":[{"name":"Biomedical Informatics & Genomics Center, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi\u2019an Jiaotong University , Xi\u2019an 710049, P.R. China"}]},{"given":"Shan-Shan","family":"Dong","sequence":"additional","affiliation":[{"name":"Biomedical Informatics & Genomics Center, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi\u2019an Jiaotong University , Xi\u2019an 710049, P.R. China"},{"name":"Research Institute of Xi\u2019an Jiaotong University , Zhejiang, China"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7062-3025","authenticated-orcid":false,"given":"Tie-Lin","family":"Yang","sequence":"additional","affiliation":[{"name":"Biomedical Informatics & Genomics Center, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi\u2019an Jiaotong University , Xi\u2019an 710049, P.R. China"}]}],"member":"286","published-online":{"date-parts":[[2022,5,18]]},"reference":[{"issue":"12","key":"2022092013201755400_ref1","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"},{"issue":"2","key":"2022092013201755400_ref2","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1038\/nmeth.2812","article-title":"E-CRISP: fast CRISPR target site identification","volume":"11","author":"Heigwer","year":"2014","journal-title":"Nat Methods"},{"issue":"9","key":"2022092013201755400_ref3","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1038\/nmeth.3473","article-title":"Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach","volume":"12","author":"Chari","year":"2015","journal-title":"Nat 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