Vector without frameshift mutation after recombination, method and application of site-directed gene knock-in in Xenopus frog genome

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

Inactive Publication Date: 2018-05-25
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Vector without frameshift mutation after recombination, method and application of site-directed gene knock-in in Xenopus frog genome
  • Vector without frameshift mutation after recombination, method and application of site-directed gene knock-in in Xenopus frog genome
  • Vector without frameshift mutation after recombination, method and application of site-directed gene knock-in in Xenopus frog 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), that is, 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...

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 that does not generate frameshift mutation after recombination, a method for gene-directed knock-in in Xenopus frog genome, and its application. The method comprises the following steps: 1) making the Xenopus fertilized eggs contain guide RNA, Cas9 nuclease and a donor vector with pancreatic ela-fluorescence screening label and Cas9 target fragment, 2) in the joint action of guide RNA and Cas9 nuclease Next, the target gene in the Xenopus frog genome and the double-stranded Cas9 target fragment on the donor vector are sheared, 3) Through the self-DNA repair function of the Xenopus frog cell, the targeted gene knock-in in the Xenopus frog genome is realized; 4) Embryos of the G0 generation were screened by pancreatic ela-fluorescent labeling, and F1 were identified by southern blot. The invention lays the foundation for using the clawed frog as a model animal to study genetics and human diseases.

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 Patents(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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