利用单转录本表达Cas9和sgRNA条件性编辑果蝇基因组

doi:10.16288/j.yczz.23-099

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Hereditas(Beijing) ›› 2023, Vol. 45 ›› Issue (7): 593-601.doi: 10.16288/j.yczz.23-099

• Research Article • Previous Articles Next Articles

Conditional editing of the Drosophila melanogaster genome using single transcripts expressing Cas9 and sgRNA

Bingzheng Wang¹(), Chao Zhang², Jiali Zhang², Jin Sun¹()

1. School of Laboratory Animal & Shandong Laboratory Animal Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250024, China
2. School of Clinical and Basic Medical Sciences, Shandong First Medical University, Jinan 250024, China

Received:2023-04-15 Revised:2023-06-09 Online:2023-07-20 Published:2023-07-03
Contact: Jin Sun E-mail:370756420@qq.com;sunjin@sdfmu.edu.cn
Supported by:
National Natural Science Foundation of China(31801079)

Abstract

Abstract:

The CRISPR/Cas9(clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR- associated protein 9) system, a highly efficient, simple, and easy genome editing technology, offers significant potential for genetic engineering and has been commonly applied in gene function studies in Drosophila melanogaster. However, when using CRISPR/Cas9 system to edit Drosophila melanogaster gene, Cas9 and sgRNA expression elements exist in different Drosophila melanogaster individuals, and Cas9 and sgRNA must be integrated into an individual through a complex genetic hybridization process, which has a long and complex operation cycle In this study, on the basis of the CRISPR/Cas9 system, we introduced the tRNA-sgRNA system and triplex elements, used triplex elements to link Cas9 and tRNA-sgRNA genes, stabilized the end of Cas9 mRNA after single transcript cutting, and made the expression of both Cas9 protein and sgRNA with a single transcript a reality. And as we obtained the corresponding phenotypic progeny in one hybridization, genetic manipulation was simplified. We found that conditional knockout of the white(w) gene in the Drosophila melanogaster eye and the broad(br) gene in the adult wing disc resulted in corresponding phenotypes that matched expectations using our new conditional gene editing system. So the significant advances in this new conditional gene editing system over the existing CRISPR/Cas9 system are that it is more efficient, extendable, and easy to use.

Key words: CRISPR/Cas9, tRNA-sgRNA, triplex elements, conditional gene editing

Bingzheng Wang, Chao Zhang, Jiali Zhang, Jin Sun. Conditional editing of the Drosophila melanogaster genome using single transcripts expressing Cas9 and sgRNA[J]. Hereditas(Beijing), 2023, 45(7): 593-601.

Figures/Tables 8

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引物名称	引物序列(5′→3′)
cas9-F	AGACGCATACCAAAGTCTAGAATGGCCCCAAAGAAGAAGC
cas9-R	cctagGTGCTAGATTATTTCTTTTTCTTAGC
Triplex-F	AAGAAATAATCTAGCACctaggaggatccactcgAGGTTTAGAG
tRNA-sgRNA-R	CCTACCTGATGCCAACAATTctagTTTGCTCTAGTCCTAGtgg
White-F	tgcaCAGGAGCTATTAATTCGCGG
White-R	aaacCCGCGAATTAATAGCTCCTG
br-sg-F	CCCGGGTTCGATTCCCGGCCGATGCACACAGCACTTCTGCCTGCGCGTTTTAGAGCTAGAAATAGCAAG
br-sg-R	TAACTTGCTATTTCTAGCTCTAAAACCGGCGTTCGAGAATCTGCGCTGCACCAGCCGGGAATCGAA
Dmt-Fs	AAGCATCGGTGGTTCAGTGG
ftz	GATTGCAAAGAACTAGAGCCG

Conditional editing of the Drosophila melanogaster genome using single transcripts expressing Cas9 and sgRNA

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