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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (3): 427-437.doi: 10.3724/SP.J.1006.2021.04178

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Efficient screening system of effective sgRNA for cotton CRISPR/Cas9 gene editing

ZHOU Guan-Tong(), LEI Jian-Feng, DAI Pei-Hong, LIU Chao, LI Yue, LIU Xiao-Dong*()   

  1. College of Agriculture, Xinjiang Agricultural University, Engineering Research Centre of Cotton of Ministry of Education, Urumqi 830052, Xinjiang, China
  • Received:2020-08-05 Accepted:2020-10-14 Online:2021-03-12 Published:2020-10-28
  • Contact: LIU Xiao-Dong E-mail:601930485@qq.com;xiaodongliu75@aliyun.com
  • Supported by:
    National Natural Science Foundation of China(31660433);Xinjiang Agricultural University Postgraduate Research and Innovation Project(XJAUGRI2017003)

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Abstract:

Single guide RNA (sgRNA) is one of the important elements of the CRISPR/Cas9 genome editing technology system. However, studies have shown that many sgRNAs cannot work effectively. It is worth screening to verify the effectiveness of multiple design candidate sgRNAs. Instantaneous transformation of protoplasts or leaves with complete editing vectors were used to verification of the effectiveness of sgRNA in the early stage. These methods are time-consuming and laborious, and the success rate is not high, especially for cotton with low efficiency of the protoplasmic system. In this study, target sequences were designed for GhMAPKKK2 and GhAE genes, and two vectors of GhU6-5P::MAPKKK2-sgRNA-1300, GhU6-5P::AE-sgRNA-1300 which transcibed only sgRNA were constructed and injected YZ-1 Cas9 transgenic cotton plant leaves through Agrobacterium; meanwhile, two corresponding complete CRISPR/Cas9 genome editing vectors of GhU6-5P::MAPKKK2-sgRNA-Cas9 and GhU6-5P::AE-sgRNA-Cas9 were constructed and injected YZ-1 wild-type cotton leaves with Agrobacterium. In addition, target sequences were designed for GhPDS, GhCLA1, GhMAPKKK2, and GhAE genes, respectively, and GhU6-5P-2::PDS-sgRNA- CLCrVA, GhU6-5P-2::CLA1-sgRNA-CLCrVA, GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA and GhU6-5P-2::AE-sgRNA-CLCrVA virus delivery vectors were constructed and injected YZ-1 Cas9 transgenic cotton plant leaves through Agrobacterium. In the above experiments, the plants transformed with the empty vector were used as controls. The genomic DNA of the transformed cotton leaves was subjected to PCR and enzyme digestion, and the PCR products which were not completely digested were cloned and sequenced. The results showed that no mutation in target gene was detected in the cotton plants transformed with the GhU6-5P::AE-sgRNA-1300, GhU6-5P::MAPKKK2-sgRNA-Cas9 and GhU6-5P::AE-sgRNA-Cas9, and the target genes mutation in the Cas9 transgenic plants transformed with GhU6-5P::MAPKKK2-sgRNA-1300, GhU6-5P-2::PDS-sgRNA-CLCrVA, GhU6-5P-2::CLA1-sgRNA-CLCrVA, GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA and GhU6-5P-2::AE-sgRNA-CLCrVA vector was uncovered. The types of mutations included base substitution, base deletion and base insertion. The results indicated that the strategy of using Cas9 transgenic plants as transformation recipients can efficiently and truly verify the effectiveness of sgRNA, which eliminated false negative results due to low transformation efficiency, and the strategy of using virus as vectors to deliver sgRNA was more efficient and accurate. The establishment of this sgRNA high-efficiency verification system provides an important technical basis for cotton functional genomics research.

Key words: cotton, transient transformation, CRISPR/Cas9, genome editing

Table 1

Primer sequences used in this study"

引物名称
Primer name		 上游引物序列
Forward sequence (5'-3')		 下游引物序列
Reverse sequence (5'-3')
GhU6-5P-MAPKKK2-sg AAGGGTTCCCAGCTGACATA TATGTCAGCTGGGAACCCTT
GhU6-5P-AE-sg GAGTTTGGAGGGCTTACAAT ATTGTAAGCCCTCCAAACTC
GhU6-5P-2-PDS-sg GAAGCGAGAGATGTTCTAGG CCTAGAACATCTCTCGCTTC
GhU6-5P-2-CLA1-sg TATGCTCGCGGAATGATCAG CTGATCATTCCGCGAGCATA
Test MAPKKK2-sg CCATGTCGTAGCTTATAAAGG TCATTTACCTTCTCTTCCCAG
Test AE-sg AGACTTGTTTCAATGGACTC AATAAGCTGACAGCAGTTGG
Test PDS-sg TGCATGATCCATCACTCAAGTTT GAACGAAAGGCCCTTTCTTTC
Test CLA1-sg GGATCTGAAAGGTGAAAGGAATC TACCGTGATACTTGTCAGCAGCT

Fig. 1

Identification of gene editing vector by restriction enzyme digestion A: 1, 2 are GhU6-5P::MAPKKK2-sgRNA-1300 and GhU6-5P::MAPKKK2-sgRNA-Cas9. B: 1, 2 are GhU6-5P::AE-sgRNA-1300 and GhU6-5P:: AE-sgRNA-Cas9. C: 1-4 are GhU6-5P-2::PDS-sgRNA-ClCrVA , GhU6-5P-2::CLA1-sgRNA-ClCrVA, GhU6-5P-2::MAPKKK2-sgRNA- ClCrVA and GhU6-5P-2::AE-sgRNA-ClCrVA. M: 2K plus II DNA marker."

Fig. 2

GhU6-5P::MAPKKK2-sgRNA targeted mutations A: M: 2K plus II DNA marker; 1, 2: PCR/RE assay of negative control genome before and after the enzyme digestion; 3-6: PCR/RE assay of GhU6-5P::MAPKKK2-sgRNA-Cas9 genome of single plant sample after enzyme digestion. B: M: 2K plus II DNA marker; 1, 2: PCR/RE assay of negative control genome before and of single plant sample after the enzyme digestion; 3-6: PCR/RE assay of GhU6-5P::MAPKKK2-sgRNA-1300 genome after enzyme digestion. C: sequencing of GhMAPKKK2-sgRNA target sequence and mutant PCR products. M1 occurred at GhMAPKKK2 from D subgenome, M2, M3 occurred at GhMAPKKK2 from A subgenome. D: sequencing peak diagram of the mutant PCR product clone, the red box indicates that the base has a mutation region compared to the control. PCR/RE indicates PCR/restriction enzyme assays."

Fig. 3

GhU6-5P::AE-sgRNA targeted mutations A: M: 2K plus II DNA marker; 12, 11: PCR/RE assay of negative control genome before and after the enzyme digestion; 3-12: PCR/RE assay of GhU6-5P::AE-sgRNA-Cas9 of single plant sample after enzyme digestion. B: M: 2K plus II DNA marker; 1, 2: PCR/RE assay of negative control genome before and after the enzyme digestion; 3-12: PCR/RE assay of GhU6-5P::AE-sgRNA-1300 of single plant sample genome after enzyme digestion. PCR/RE indicates PCR/restriction enzyme assays."

Fig. 4

Results of GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA partial editing effect sequencing A: M: 2K plus II DNA marker; 1, 2: PCR/RE assay of negative control genome before and after the enzyme digestion. B: M: 2K plus II DNA marker; 3-6: PCR/RE assay of GhU6-5P::MAPKKK2-sgRNA-CLCrVA genome of single plant sample after enzyme digestion. C: M: 2K plus II DNA marker, 1-10: clony PCR product after enzyme digestion. PCR/RE indicates PCR/restriction enzyme assays."

Fig. 5

GhU6-5P-2::AE-sgRNA-CLCrVA partial editing effect sequencing results A: M: 2K plus II DNA marker; 1, 2: PCR/RE assay of negative control genome before and after the enzyme digestion. B: M: 2K plus II DNA marker, 3-7: PCR/RE assay of GhU6-5P::AE-sgRNA-CLCrVA genome of single plant sample after enzyme digestion. C: M: 2K plus II DNA marker; 1-10: clony PCR product after enzyme digestion. PCR/RE indicates PCR/restriction enzyme assays."

Fig. 6

Results of GhU6-5P-2::PDS-sgRNA-CLCrVA partial editing effect sequencing A: M: 2K plus II DNA marker; 1, 2: PCR/RE assay of negative control genome after and before the enzyme digestion. B: M: 2K plus II DNA marker; 3-5: PCR/RE assay of GhU6-5P-2::PDS-sgRNA-CLCrVA of single plant sample genome after enzyme digestion. C: sequencing of GhPDS-sgRNA target sequence and mutant PCR products. M1, M2 occurred at GhPDS from A subgenome; M3 occurred at GhPDS from D subgenome. D: sequencing peak diagram of the mutant PCR product; the red box indicates that the base has a mutation region compared to the control. PCR/RE indicates PCR/restriction enzyme assays."

Fig. 7

Results of GhU6-5P-2::CLA1-sgRNA-CLCrVA partial editing effect sequencing A: M: 2K plus II DNA marker; 1, 2: PCR/RE assay of negative control genome before and after the enzyme digestion. B: M: 2K plus II DNA marker, 3-5: PCR/RE assay of GhU6-5P::CLA1-sgRNA-CLCrVA genome of single plant sample after enzyme digestion. C: sequencing of GhCLA1-sgRNA target sequence and mutant PCR products. M1, M3 occurred at GhCLA1 from D subgenome; M2 occurred at GhCLA1 from A subgenome. D: sequencing peak diagram of the mutant PCR product; the red box indicates that the base has a mutation region compared to the control. PCR/RE indicates PCR/restriction enzyme assays."

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