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

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

doi:10.1007/s42994-021-00051-4

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

aBIOTECH. 2021 Jun 25;3(2):89-98. doi: 10.1007/s42994-021-00051-4. eCollection 2022 Jun.

Authors

Reqing He^#¹, Pengxiang Zhang^#¹, Yuchuan Yan¹, Chen Yu¹, Liyun Jiang¹, Youlin Zhu¹, Dong Wang¹

Affiliation

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

^# Contributed equally.

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.

Supplementary information: The online version contains supplementary material available at 10.1007/s42994-021-00051-4.

Keywords: CRISPR/Cas9; Genome editing; Protospacer adjacent motif; Soybean.