Carrier incapable of generating frameshift mutation after recombination as well as method and application for gene fixe-point knock-in in Xenopus laevis genome

A genome and clawed frog technology, applied in the field of genetic engineering, can solve the problems of inability to do long-term genetic research, tediousness, and low efficiency

Inactive Publication Date: 2015-05-13
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

However, both methods are time-sensitive and cannot be used for long-term genetic research
[0005] At the end of the 20th century, scientists used gene homologous recombination technology to complete gene knockout and site-specific modification. Due to the extremely low efficiency of this method (Thomas et al., 1987), the research assistance to life science is limited to those with embryonic stem cells and body Species with mature nuclear transfer technology
[0009] Its disadvantages are: high requirements on homology arms, low efficiency, and cumbersome
However, the data in this article show that this modification has no information transmitted through the gonad and is limited to genes with high expression intensity
Insertion screening is not possible for weakly expressed genes or lncRNAs

Method used

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  • Carrier incapable of generating frameshift mutation after recombination as well as method and application for gene fixe-point knock-in in Xenopus laevis genome
  • Carrier incapable of generating frameshift mutation after recombination as well as method and application for gene fixe-point knock-in in Xenopus laevis genome
  • Carrier incapable of generating frameshift mutation after recombination as well as method and application for gene fixe-point knock-in in Xenopus laevis genome

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0093] Example 1 Gene site-specific insertion in exon 5 of the ets1 gene in Xenopus tropicalis

[0094] 1. Cas9 target site

[0095] The ets1 target is the target site corresponding to ets1-T2 in Table 1 in the literature (Guo et al., 2014) (the sequence is shown in SEQUENCE NO.15, that is, 5-GGTTCAGAGAATTCAGAGGGCGG-3)

[0096] 2. Preparation of Cas9mRNA and gRNA

[0097] Synthesize Cas9 mRNA and gRNA according to the following scheme:

[0098] 1) Preparation of Cas9 mRNA

[0099] Endonuclease NotI will 10ug pCS2-3×FLAG-NLS-SpCas9-NLS (addgene ID: 51307; vector structure as image 3 Shown in a) The plasmid was linearized, and Cas9 mRNA was prepared by in vitro transcription using SP6.

[0100] 2) gRNA preparation

[0101] a. Vector preparation

[0102] Referring to the method of the literature (Guo et al., 2014), the Cas9 target site shown in SEQUENCE NO.15 was cloned into the backbone vector by using the enzyme digestion effect of BbsI, and the backbone vector was pUC57...

example 2

[0131] Example 2 Carrying out site-directed gene insertion in exon 3 of the ets2 gene in Xenopus tropicalis

[0132] 1. Cas9 target site

[0133] The ets2 target is the target site corresponding to ets2 in Table 1 in the literature (Guo et al., 2014) (the sequence is shown in SEQUENCE NO.25), ie, ggtctggact cttactctca tgg.

[0134] 2. Preparation of Cas9mRNA and gRNA

[0135] 1. Preparation of Cas9 mRNA, see Example 1

[0136] 2. Preparation of gRNA, see Example 1

[0137] a. Vector preparation

[0138] Referring to the method of the literature (Guo et al., 2014), the Cas9 target site as shown in SEQUENCE NO.25 was cloned into the backbone vector by using the enzyme digestion effect of BbsI, and the backbone vector was pUC57-T7-gRNA plasmid (vector structured as image 3 b) to obtain ets2-specific gRNA.

[0139] b. gRNA transcription synthesis, see Example 1

[0140] 3.donor preparation

[0141] 1) PCR amplification

[0142] The genome sequence (611bp) containing the ...

example 3

[0157] Example 3 Carrying out gene-directed insertion at intron 1 of the tropical clawed frog tyrosinase gene to achieve the effect of no frame shift

[0158] 1. Preparation of Cas9 mRNA and gRNA

[0159] 1) Cas9mRNA synthesis method is the same as in Example 1

[0160] 2) Preparation of gRNA

[0161] The target site is within intron 1, about 600bp away from exon 2, named tyr-int1.

[0162] The target site recognition sequence is 5-GGGGTCCCTAACTTCCTCTATGG-3 (SEQUENCE NO.31).

[0163] The two annealed single-stranded sequences are as follows:

[0164] tyr-int1-S: 5-TAGGGGTCCCTAACTTCCTCTA-3 (SEQUENCE NO.32)

[0165] tyr-int1-A: 5-AAACTAGAGGAAGTTAGGGACC-3 (SEQUENCE NO.33)

[0166] The gRNA synthesis method is the same as Example 1.

[0167] 2.donor preparation

[0168] 1) PCR amplification

[0169] Using PCR amplification (template is the genome of Xenopus tropicalis) to obtain the partial sequence of the intron containing the tyr-int1 target site and the exon adjacent to...

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Abstract

The invention provides a carrier incapable of generating frameshift mutation after recombination as well as a method and an application for gene fixe-point knock-in in a Xenopus laevis genome. The method comprises steps as follows: (1), guide RNA (ribonucleic acid), Cas9 nuclease and a donor carrier with a pancreas ela-fluorescent screening label and a Cas9 target fragment are contained in a fertilized egg of Xenopus laevis; (2) under the joint action of guide RNA and Cas9 nuclease, a target gene in the Xenopus laevis genome and the double-chain Cas9 target fragment on the donor carrier are shorn; (3), gene fixed-point knock-in of the Xenopus laevis genome is realized through a DNA (deoxyribonucleic acid) recovery function of Xenopus laevis cells; (4), G0-generation embryos are screened through the pancreas ela-fluorescent screening label, and F1 is subjected to southern blot identification. The carrier incapable of generating frameshift mutation after recombination as well as the method and the application for gene fixe-point knock-in in the Xenopus laevis genome lay a foundation for research of genetics and human diseases with Xenopus laevis as a model animal.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a carrier that does not produce frameshift mutations after recombination, a method for gene-directed knock-in in the Xenopus genome, and an application thereof. Background technique [0002] The African clawed frog (Xenopus laevis) is a classic model animal for the study of early embryology. It has the advantages of large embryos and large egg production. The tropical clawed frog (Xenopus tropicalis) has all the advantages of the African clawed frog. In addition, this species is small, has a short reproductive cycle (4-5 months), and is a diploid species, which is suitable for genetic research. [0003] The entire genome of the tropical clawed frog has been sequenced. Its genome has about 1.7 billion base pairs and contains 20,000 to 21,000 genes, of which about 1,700 genes are very similar to the corresponding genes of humans, and nearly 80% of all human genes Genes related ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/033
Inventor 陈永龙石照应
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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