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Biol."],"published-print":{"date-parts":[[2020,3]]},"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Derived from an adaptive bacterial immune system, the clustered regularly interspaced palindromic repeats (CRISPR)\/CRISPR\u2010associated 9 (Cas9) system has shown great potential in high\u2010throughput functional genomic screening, especially for protein\u2010coding genes. However, it is still challenging to apply the similar strategy to study non\u2010coding genomic elements such as long non\u2010coding RNAs (lncRNAs) or clusters of microRNAs, because short insertions or deletions may not be sufficient to generate loss\u2010of\u2010function phenotypes.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Here, we presented a systematic strategy for designing a CRISPR\u2010based paired\u2010sgRNA library for high\u2010throughput screening in non\u2010coding regions. Due to the abundance of lncRNAs and their diverse regulatory roles <jats:italic>in vivo<\/jats:italic>, we repurposed microarray datasets to select 600 highly expressed lncRNAs in non\u2010small\u2010cell lung cancer and designed two schemes for lncRNA deletion with ~20 paired\u2010sgRNAs for each lncRNA. Through Golden\u2010Gate assembly, we generated a pooled CRISPR\u2010based library with a total of 12,878 sgRNA pairs.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Over 80% of paired\u2010sgRNAs were recovered from final pooled library with a relatively even distribution. Cleavage efficiency of sgRNA pairs was validated through experiments of transient transfection and viral infection. Moreover, randomly selected paired\u2010sgRNAs showed that efficient deletion of genomic DNA could be achieved with a deletion size within the range of 500 to 3000 bp.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>In summary, we have demonstrated a strategy to design and construct a pooled paired\u2010sgRNA library to generate genomic deletion in the lncRNA regions, validated their deletion efficiency and explored the relationship of deletion efficiency with respect to deletion size. This method would be also suitable for investigation of other uncharacterized non\u2010coding genomic regions in mammalian cells in an efficient and cost\u2010effective manner.<\/jats:p><\/jats:sec>","DOI":"10.1007\/s40484-020-0194-5","type":"journal-article","created":{"date-parts":[[2020,3,6]],"date-time":"2020-03-06T04:51:26Z","timestamp":1583470286000},"page":"31-42","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Construction of a CRISPR\u2010based paired\u2010sgRNA library for chromosomal deletion of long non\u2010coding RNAs"],"prefix":"10.1002","volume":"8","author":[{"given":"Minzhen","family":"Tao","sequence":"first","affiliation":[{"name":"MOE Key Laboratory of Bioinformatics and Bioinformatics Division Center for Synthetic and System Biology Department of Automation Beijing National Research Center for Information Science and Technology Tsinghua University Beijing 100084 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