Research on the Carrying Capacity of CLCrV-mediated VIGE System

doi:10.13560/j.cnki.biotech.bull.1985.2022-0173

Abstract

Abstract:

This work aims to explore the carrying capacity of CLCrV-mediated VIGE system. Through CLCrV-mediated sgRNA delivery system，the gene editing vector was injected into cotton overexpressing Cas9，and the genomic DNA of cotton was extracted. The effective sgRNA was screened by PCR/RE method. Then the same method was used to detect the carrying capacity of the CLCrV-mediated VIGE system. Six gene-editing vectors were successfully constructed based on the cotton GhBsr-k1 gene，and two of them achieved targeted editing of cotton GhBsr-k1 gene. The complete gene editing vector components were constructed on the CLCrV vector and no gene editing was detected in cotton cells. Two effective sgRNAs were screened for the GhBsr-k1 gene in cotton. However，CLCrV vectors carrying the entire CRISPR/Cas9 system are difficult to achieve efficient gene editing in cotton leaves.

Key words: cotton leaf crumple virus, VIGE, cotton, GhBsr-k1

ZHAO Yi, LEI Jian-feng, LIU Min, HU Zi-yao, DAI Pei-hong, LIU Chao, LI Yue, LIU Xiao-dong. Research on the Carrying Capacity of CLCrV-mediated VIGE System[J]. Biotechnology Bulletin, 2022, 38(11): 210-219.

Figures/Tables 5

Table 1 Primer and application

引物Primer	序列Sequence（5'-3'）	用途Application
GhBsr-k1-sgRNA1F GhBsr-k1-sgRNA1R	GATTGCAGCGTTTCTACCATGGAT AAACATCCATGGTAGAAACGCTGC	构建GhBsr-k1-sgRNA1 Construction of GhBsr-k1-sgRNA1
GhBsr-k1-sgRNA2F GhBsr-k1-sgRNA2R	GATTGGTGTACTTGGTAAGATCTT AAACAAGATCTTACCAAGTACACC	构建GhBsr-k1-sgRNA2 Construction of GhBsr-k1-sgRNA2
GhBsr-k1-sgRNA3F GhBsr-k1-sgRNA3R	GATTGCGCTGGCATTACCATGGGC AAACGCCCATGGTAATGCCAGCGC	构建GhBsr-k1-sgRNA3 Construction of GhBsr-k1-sgRNA3
GhBsr-k1-sgRNA4F GhBsr-k1-sgRNA4R	GATTGTTAATCAGGGGACTGCAGT AAACACTGCAGTCCCCTGATTAAC	构建GhBsr-k1-sgRNA4 Construction of GhBsr-k1-sgRNA4
GhBsr-k1-sgRNA5F GhBsr-k1-sgRNA5R	GATTGCTGGACCGGATTTAGCAAT AAACATTGCTAAATCCGGTCCAGC	构建GhBsr-k1-sgRNA5 Construction of GhBsr-k1-sgRNA5
GhBsr-k1-sgRNA6F GhBsr-k1-sgRNA6R	GATTGGTCGTGCCTACGCTGCAGA AAACTCTGCAGCGTAGGCACGACC	构建GhBsr-k1-sgRNA6 Construction of GhBsr-k1-sgRNA6
M-GhBsr-k1-F1 M-GhBsr-k1-R1	CCCTTCAGTCTTTTAATGGC ACTCCATTCTGAAACCCAAG	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
M-GhBsr-k1-F2 M-GhBsr-k1-R2	GTCACAGGATGCACGTAAAG AAAGCCACCCAGAACTCAGA	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
M-GhBsr-k1-F3 M-GhBsr-k1-R3	GAAGTGTGTGCTTAAACTTGTC ACTGCTCTGGTAGGATATGG	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
M-GhBsr-k1-F4 M-GhBsr-k1-R4	GTTTAGAATACAATGCAGAAACTTTC GACAAGTTTAAGCACACACTTC	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
M-GhBsr-k1-F5 M-GhBsr-k1-R5	GGATATCTGCAGCTGTGTCT ACTCCATTCTGAAACCCAAG	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
M-GhBsr-k1-F6 M-GhBsr-k1-R6	ACCATAATGCCCGATACTTGC ATAGAAGTGACACCTGAATCATC	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
CLCrVA-F CLCrVA-R	GCTGCTTGTATGTTTGGGTG CAAGGGAGAGTAGTTGGCA	CLCrV-A病毒检测 CLCrV-A virus detection

Table 1 Primer and application

引物Primer	序列Sequence（5'-3'）	用途Application
GhBsr-k1-sgRNA1F GhBsr-k1-sgRNA1R	GATTGCAGCGTTTCTACCATGGAT AAACATCCATGGTAGAAACGCTGC	构建GhBsr-k1-sgRNA1 Construction of GhBsr-k1-sgRNA1
GhBsr-k1-sgRNA2F GhBsr-k1-sgRNA2R	GATTGGTGTACTTGGTAAGATCTT AAACAAGATCTTACCAAGTACACC	构建GhBsr-k1-sgRNA2 Construction of GhBsr-k1-sgRNA2
GhBsr-k1-sgRNA3F GhBsr-k1-sgRNA3R	GATTGCGCTGGCATTACCATGGGC AAACGCCCATGGTAATGCCAGCGC	构建GhBsr-k1-sgRNA3 Construction of GhBsr-k1-sgRNA3
GhBsr-k1-sgRNA4F GhBsr-k1-sgRNA4R	GATTGTTAATCAGGGGACTGCAGT AAACACTGCAGTCCCCTGATTAAC	构建GhBsr-k1-sgRNA4 Construction of GhBsr-k1-sgRNA4
GhBsr-k1-sgRNA5F GhBsr-k1-sgRNA5R	GATTGCTGGACCGGATTTAGCAAT AAACATTGCTAAATCCGGTCCAGC	构建GhBsr-k1-sgRNA5 Construction of GhBsr-k1-sgRNA5
GhBsr-k1-sgRNA6F GhBsr-k1-sgRNA6R	GATTGGTCGTGCCTACGCTGCAGA AAACTCTGCAGCGTAGGCACGACC	构建GhBsr-k1-sgRNA6 Construction of GhBsr-k1-sgRNA6
M-GhBsr-k1-F1 M-GhBsr-k1-R1	CCCTTCAGTCTTTTAATGGC ACTCCATTCTGAAACCCAAG	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
M-GhBsr-k1-F2 M-GhBsr-k1-R2	GTCACAGGATGCACGTAAAG AAAGCCACCCAGAACTCAGA	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
M-GhBsr-k1-F3 M-GhBsr-k1-R3	GAAGTGTGTGCTTAAACTTGTC ACTGCTCTGGTAGGATATGG	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
M-GhBsr-k1-F4 M-GhBsr-k1-R4	GTTTAGAATACAATGCAGAAACTTTC GACAAGTTTAAGCACACACTTC	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
M-GhBsr-k1-F5 M-GhBsr-k1-R5	GGATATCTGCAGCTGTGTCT ACTCCATTCTGAAACCCAAG	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
M-GhBsr-k1-F6 M-GhBsr-k1-R6	ACCATAATGCCCGATACTTGC ATAGAAGTGACACCTGAATCATC	PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup
CLCrVA-F CLCrVA-R	GCTGCTTGTATGTTTGGGTG CAAGGGAGAGTAGTTGGCA	CLCrV-A病毒检测 CLCrV-A virus detection

Fig. 1 Construction of VIGE editing gene vector A：1-6 are B-Zero-AtU6-26∷GhBsr-k1-sgRNA（1-6）cloning vector digestion. B：1-6 are VIGE editing vector CLCrV-AtU6-26∷GhBsr-k1-sgRNA（1-6）digestion. M：Trans2K Plus II DNA marker

Fig. 2 Gene editing vector screening and mutation detection A：WT：PCR/RE assay of negative control genome before and after the Nco I enzyme digestion，1-5：PCR/RE assay of CLCrV-AtU6-26∷GhBsr-k1-sgRNA1 of 10 single plant sample after Nco I enzyme digestion. B：CLCrV-AtU6-26∷GhBsr-k1-sgRNA1 editing vector of the undigested PCR products lacked Nco I sites（due to the presence of a mutation）that were subsequently purified，cloned，and analyzed by sequencing. C：CLCrV-AtU6-26∷GhBsr-k1-sgRNA1 mutation type sequencing peak map. D：WT：PCR/RE assay of negative control genome before and after the Pst I enzyme digestion. 1：PCR/RE assay of CLCrV-AtU6-26∷GhBsr-k1-sgRNA6 of 10 single plant sample after Pst I enzyme digestion. E：CLCrV-AtU6-26∷GhBsr-k1-sgRNA6 editing vector of the undigested PCR products lacked Pst I sites（due to the presence of a mutation）that were subsequently purified，cloned，and analyzed by sequencing. F：CLCrV-AtU6-26∷GhBsr-k1-sgRNA6 mutation type sequencing peak map. “M” indicates the mutation sequence；“+” are denoted with red capital letters；“-” are shown as red dashes；yellow highlighted the differential bases in A/D subgroup of GhBsr-k1 gene. M：Trans2K Plus II DNA marker

Fig. 3 Prediction of secondary structure of sgRNA A：The secondary structure of sgRNA1. B：The secondary structure of sgRNA2. C：The secondary structure of sgRNA3. D：The secondary structure of sgRNA4. E：The secondary structure of sgRNA5. F：The secondary structure of sgRNA6

Fig. 4 CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1 editing vector construction and mutation detection A：CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1 gene editing vector diagram. B：lane 1：CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1 gene editing vector was digested with three enzymes. C：PCR/RE assay of CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1 of Xinluzao 26 and YZ-1 single plant sample after Nco I enzyme digestion. WT：PCR/RE assay of negative control genome before and after the Nco I enzyme digestion. D：PCR/RE assay of CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1 of Xinluzao26 and YZ-1 mix single plant sample after Nco I enzyme digestion. WT：PCR/RE assay of negative control genome before and after the Nco I enzyme digestion.E：Detection of CLCrV-A virus in individual samples of Xinluzao 26 and YZ-1，GhBsr-k1 is internal reference gene. N indicates negative control，and P indicates positive control. M：Trans2K Plus II DNA marker

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