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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (7): 978-986.doi: 10.3724/SP.J.1006.2020.93064

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

Establishment of an efficient genotyping technique based on targeted DNA endonuclease in vitro activity of CRISPR/Cas9 ribonucleoprotein

WANG Nan1,QI Xian-Tao1,2,LIU Chang-Lin1,XIE Chuan-Xiao1,*(),ZHU Jin-Jie1,*()   

  1. 1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    2 Anhui Agricultural University, Hefei 230036, Anhui, China
  • Received:2019-12-18 Accepted:2020-03-24 Online:2020-07-12 Published:2020-04-10
  • Contact: Chuan-Xiao XIE,Jin-Jie ZHU E-mail:xiechuanxiao@caas.cn;zhujinjie@caas.cn
  • Supported by:
    National Major Project of Developing New GM Crops(2019ZX08010-003)

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Abstract:

Establishing a rapid, accurate, high-throughput and easily implementable genotyping method is highly desirable for functional genomics, genetic improvement and mutant screening. Here, we describe a convenient and inexpensive technique for genotyping using the targeted DNA endonuclease activity of Cas9 or Cas9NG ribonucleoproteins complex (sgRNA/Cas9-RNP or sgRNA/Cas9NG-RNP). In this study, Cas9 and Cas9NG protein purified from E. coli extract was assembled with in vitro transcribed single guide RNA (sgRNA)or enhanced sgRNA (esgRNA) as an assembled ribonucleoprotein (RNP) complex to fulfill the targeted endonuclease activity on the PCR amplicons of ZmWx exon 7. The restriction profiles can be converted into genotyping results of wildtype, homozygous or heterozygous mutant, respectively. Our data showed that ZmWx gene-edited mutants can be genotyped rapidly and efficiently by the sgRNA-optimized system esgRNA/Cas9. The reaction component optimization data suggested that 500 ng of DNA substrates could be cleavaged completely by incubating with 1 μg 1:1 molar ratio of esgRNA/Cas9 ribonucleoproteins for 30 minutes at 37℃, or by 4 μg 1:1 molar ratio of esgRNA/Cas9NG ribonucleoproteins for 4 hours at 37℃. Expanding the targeting flexibility of mutant detection via esgRNA/Cas9NG indicated that Cas9NG variant might recognize relax NG PAM (protospacer-adjacent-motif, PAM) at the expense of decreasing restriction activity, which is necessary to improve the activity of esgRNA/Cas9NG by further optimization. Therefore, the establishment and application of esgRNA/Cas9 based PCR/RNP technique provides an easy, simple and low-cost approach to genotyping in functional genomics, molecular breeding and mutant screening. In addition, our in vitro data on esgRNA/Cas9NG has certain and significant reference value for developing it into in vivo genome editing studies.

Key words: sgRNA/Cas9 ribonucleoprotein complex, Cas9NG, enhanced single guide RNA (esgRNA), genotyping, mutant screening

Table 1

Primer sequences used in this paper"

引物名称
Primer name		 序列
Sequence (5′-3′)
Cas9 F GACGACGACGACAAGGCCATGATGGACAAGAAGTACTCCA
Cas9 R TCGACGGAGCTCGCTAGCGTCGCCGCCGAGCTGGGAGAGG
Cas9NG F ACGACGACGACAAGGCCATGGACAAGAAGTACTCCATCGGC
Cas9NG R CTCGAGTGCGGCCGCAAGCTTGTCGCCGCCGAGCTGGCTCAG
sgRNA scaffold F GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG
sgRNA scaffold R AAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAA
esgRNA scaffold F GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATC
esgRNA scaffold R AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTC
ZmWx F CATACTTCTCCGGACCATACGGTAA
ZmWx R TCCCTGCTGGGGTCCCACTC
T7 F AAGCTAATACGACTCACTATAGGAGGTTCAGCTCCGGGTAGTGTTTTAGAGCTAGAAA
eT7 F AAGCTAATACGACTCACTATAGGAGGTTCAGCTCCGGGTAGTGTTTCAGAGCTATGCT
Target1 T7 F AAGCTAATACGACTCACTATAGGTTCAGCTCCGGGTAGTCGGGTTTCAGAGCTATGCT
Target2 T7 F AAGCTAATACGACTCACTATAGCTCCGGGTAGTCGGAGAAGGGTTTCAGAGCTATGCT
Target3 T7 F AAGCTAATACGACTCACTATAGCTTCTCCGACTACCCGGAGCGTTTCAGAGCTATGCT
Target4 T7 F AAGCTAATACGACTCACTATAGTTCGCCTTCTCCGACTACCCGTTTCAGAGCTATGCT
Target5 T7 F AAGCTAATACGACTCACTATAGGCCTTCTCCGACTACCCGGAGTTTCAGAGCTATGCT
Target6 T7 F AAGCTAATACGACTCACTATAGCAGCTCCGGGTAGTCGGAGAGTTTCAGAGCTATGCT
T7 R AAAAAGCACCGACTCGGTGCCACTTTTT

Fig. 1

Purification of Cas9 and Cas9NG protein A: Schematics diagram of protein expression vector; B: 10% SDS-PAGE of purified Cas9; C: 10% SDS-PAGE of purified Cas9NG. M: Protein marker. 1: Supernatant protein; 2: Ni-column crude purified protein; 3: Cation exchange column purification protein."

Fig. 2

In vitro transcription of sgRNA and esgRNA, as well as the establishment of cleavage assay via esgRNA/Cas9-RNP and esgRNA/Cas9NG-RNP A: Predicted secondary structure of sgRNA; B: Predicted secondary structure of esgRNA; C: 10% Urea-PAGE of sgRNA and esgRNA; D: PCR/RNP cleavage assay; CK: ZmWx wild PCR product without esgRNA and Cas protein."

Fig. 3

Optimization of cleavage assay via esgRNA /Cas9-RNP and esgRNA/ Cas9NG-RNP A: Effect of different gradients of esgRNA; B: Effect of different gradients of Cas9 protein; C: Effect of different gradients of digestion time; D: Digestion of PCR products of different genotypes; E: Effect of different gradients of esgRNA; F: Effect of different gradients of Cas9NG protein; G: Effect of different gradients of digestion time; H: Digestion of PCR products of different genotypes; CK: ZmWx wild PCR product without esgRNA and Cas protein."

Fig. 4

Cleavage assay of different targets for ZmWx locus via esgRNA/Cas9NG-RNP A: Targets selection of Cas9NG; B: 10% Urea-PAGE of 6 target-related esgRNA; C: PCR/RNP cleavage assay; CK: ZmWx wild PCR product without esgRNA and Cas protein."

Fig. 5

Genotyping genome-edited ZmWx mutant via esgRNA/Cas9-RNP A: Genotyping genome-edited ZmWx mutant via esgRNA/Cas9-RNP; B: Sanger sequencing peak graph of different genotypes. WT/WT is a wild type, -1 bp/-1 bp is a homozygous mutant with 1 bp deleted, and WT/-3 bp is a heterozygous mutant with 3 bp allele deleted; CK: ZmWx wild PCR product without esgRNA and Cas protein."

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