Abstract
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.
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Acknowledgements
We thank all members of Center for Genome Engineering for support. We thank J. Suh for comments on the manuscript. This work was supported by the research fund from Institute for Basic Science, Republic of Korea (IBS-R021-D1).
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J-YY and J-SK conceived and supervised the project; J-YY designed the experiments; J-YY generated all the constructs; J-YY and S-TK performed the library construction and analyzed NGS data; S-GK contributed to the cloning of a main construct; J-YY wrote the main part of manuscript; All the authors analyzed the results and commented on the manuscript.
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Yun, JY., Kim, ST., Kim, SG. et al. A zero-background CRISPR binary vector system for construction of sgRNA libraries in plant functional genomics applications. Plant Biotechnol Rep 13, 543–551 (2019). https://doi.org/10.1007/s11816-019-00567-8
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DOI: https://doi.org/10.1007/s11816-019-00567-8