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윤재영
유전체 교정 연구단
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A zero-background CRISPR binary vector system for construction of sgRNA libraries in plant functional genomics applications

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Title
A zero-background CRISPR binary vector system for construction of sgRNA libraries in plant functional genomics applications
Author(s)
Jae‑Young Yun; Sang‑Tae Kim; Kim S.-G.; Jin‑Soo Kim
Publication Date
2019-10
Journal
PLANT BIOTECHNOLOGY REPORTS, v.13, no.5, pp.543 - 551
Publisher
SPRINGER
Abstract
© 2019, Korean Society for Plant Biotechnology.Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing is a ground-breaking biotechnology for agricultural applications such as precision breeding in crop plants. Agrobacterium-mediated CRISPR delivery has been successfully adapted for gene knockout applications for basic research and agricultural technology development. However, selecting proper single-guide RNA (sgRNA) for CRISPR binary constructs to induce double-strand break in certain target genes has presented difficulties mainly due to unpredictable in vivo sgRNA activities. Therefore, more than three independent CRISPR constructs, each harboring different sgRNAs, are often applied to ensure the desired CRISPR-induced knockout alleles. Here, we report a zero-background CRISPR binary vector platform, featuring ccdB conjugation within sgRNA cloning cassette, which is later removed by AarI endonuclease, that allows positive survival selection for bona-fide sgRNA clones and effective exclusion of uncut or self-ligated ‘background’ negative clones. We demonstrate the advantage of using the zero-background CRISPR binary platform in a high-throughput pooled cloning strategy of multiple different sgRNAs which produced Agrobacteria containing multiple sgRNAs without any background. We also tested the integrity of pooled CRISPR sgRNA construct libraries during extended bacterial culture and during the transfer between Escherichia coli to Agrobacterium, and verified that the fidelity of sgRNA species representation was faithfully maintained during library generation
URI
https://pr.ibs.re.kr/handle/8788114/6857
ISSN
1863-5466
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > Journal Papers (저널논문)
Files in This Item:
1910_윤재영_Plant Biotechnology Reports.pdfDownload

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