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Expanding the range of CRISPR/Cas9-directed genome editing in soybean

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Abstract

The CRISPR/Cas9 system has been widely applied for plant genome editing. The commonly used SpCas9 has been shown to rely on the protospacer adjacent motif (PAM) sequences in the canonical form NGG and non-canonical NAG. Although these PAM sequences are extensively distributed across plant genomes, a broader scope of PAM sequence is required to expand the range of genome editing. Here we report the adoption of three variant enzymes, xCas9, SpCas9-NG and XNG-Cas9, to produce targeted mutation in soybean. Sequencing results determined that xCas9 with the NGG and KGA (contains TGA and GGA) PAMs successfully induces genome editing in soybean genome. SpCas9-NG could recognize NGD (contains NGG, NGA and NGT), RGC (contains AGC and GGC), GAA and GAT PAM sites. In addition, XNG-Cas9 was observed to cleave soybean genomic regions with NGG, GAA and AGY (contains AGC and AGT) PAM. Moreover, off-target analyses on soybean editing events induced by SpCas9 and xCas9 indicated that two high-fidelity Cas9 variants including eSpCas9 (enhanced specificity SpCas9) and exCas9 (enhanced specificity xCas9) could improve the specificity of the GGA PAM sequence without reducing on-target editing efficiency. These findings significantly expand the scope of Cas9-mediated genome editing in soybean.

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Data availability

Deep sequencing data produced in this article have been submitted to NCBI under accession number PRJNA685266.

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Funding

This work was supported by the grants from Natural Science Foundation of Jiangxi Province (20171ACB20001) and National Natural Science Foundation of China (31800224, 31960138 and 31960433).

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Author notes

  1. Reqing He and Pengxiang Zhang have contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Molecular Biology and Gene Engineering in Jiangxi Province, College of Life Science, Nanchang University, Nanchang, 330031, Jiangxi, China

    Reqing He, Pengxiang Zhang, Yuchuan Yan, Chen Yu, Liyun Jiang, Youlin Zhu & Dong Wang

Authors
  1. Reqing He
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  2. Pengxiang Zhang
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  3. Yuchuan Yan
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  4. Chen Yu
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  5. Liyun Jiang
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  6. Youlin Zhu
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  7. Dong Wang
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Contributions

DW and RH designed the experiments. PZ, YY and CY performed experiments. LJ, YZ, RH and DW analyzed the data. DW and RH wrote the manuscript.

Corresponding author

Correspondence to Dong Wang.

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He, R., Zhang, P., Yan, Y. et al. Expanding the range of CRISPR/Cas9-directed genome editing in soybean. aBIOTECH 3, 89–98 (2022). https://doi.org/10.1007/s42994-021-00051-4

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