Goat-friendly site SETD5-IN, sgRNA specifically targeting site, and coding DNA and application thereof

A SETD5-IN and friendly site technology, applied in the field of genetic engineering, can solve problems such as system off-target, reduced efficiency of gene-directed editing, and functional impact of off-target sites

Active Publication Date: 2021-09-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the CRISPR / Cas9 system also has the problem of off-target
Some studies have found that some sequences that do not completely match the sgRNA recognition region also have DSBs. This situation is called the off-target effect of Cas9. functionality is affected

Method used

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  • Goat-friendly site SETD5-IN, sgRNA specifically targeting site, and coding DNA and application thereof
  • Goat-friendly site SETD5-IN, sgRNA specifically targeting site, and coding DNA and application thereof
  • Goat-friendly site SETD5-IN, sgRNA specifically targeting site, and coding DNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1. Determination of goat-friendly site SETD5-IN

[0078] The goat-friendly site SETD5-IN is 2kb in length, located on goat chromosome 22, and its position on the goat genome (GCA_001704415.1) is Chr22:17026455-17028455, which is located on the first intron of the SETD5 gene , the sequence is shown as SEQ ID No.1, and its position on the genome is as follows figure 1 shown. The prediction based on the AnimalTFDB database showed that there was no transcription factor binding site in this friendly site; based on the gene position information recorded in the *.gff annotation file of the NCBI existing goat genome, it showed that there was no alternative splicing site in this friendly site. Gene editing within this site will not affect the expression of SETD5 gene and adjacent genes.

Embodiment 2

[0079] Example 2, Construction of CRISPR / Cas9 Targeting Vector

[0080] The structure of the CRISPR / Cas9 targeting vector (sgRNA expression vector) is as follows: image 3 shown.

[0081] 1) According to the sgRNA screening principle of the CRISPR / Cas9 system, screen the PAM sequence (5'-NGG) in the goat SETD5-IN site region and the corresponding sgRNA target, and predict the off-target efficiency: select the 13nt at the 5' end of the PAM sequence The sequence was used as the seed sequence and compared with the NCBI online database. Use the sgRNA structure online prediction website (http: / / unafold.rna.albany.edu / q=mfold / RNA-Folding-Form / ) to evaluate the structure and free energy of the sgRNA corresponding to each target, and confirm that the recognition sequence on the sgRNA is not pair with itself.

[0082]The sequence correspondence between the sgRNA target sequence (excluding the PAM region, the sequence in the table below is the positive strand sequence of the genomic ...

Embodiment 3

[0094] Example 3. Verification of gene editing efficiency at the cellular level

[0095] Step 1) Prepare goat fibroblasts. After 40 days of goat pregnancy, the fetus was taken out by cesarean section, and the head, tail, limbs and internal organs were removed, and the tissue was cut into 1mm in size 3 of small pieces. The shredded tissue blocks were evenly placed in the culture dish, and the culture dish was inverted in a cell culture incubator at 38.5°C, 5% CO2, and saturated humidity for 2 hours to make the tissue block adhere to the surface of the culture dish. Add a small amount of DMEM / F12 culture solution containing 10% FBS, continue to culture in the incubator for 24 hours, and add 3 mL of medium to continue the culture. Afterwards, the medium was changed every 3 days, observed every day, and the growth status of the cells was recorded.

[0096] Step 2) The sgRNA expression vector (CRISPR / Cas9 targeting vector) is detoxified and extracted.

[0097] According to the ...

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PUM

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Abstract

The invention discloses a goat-friendly site SETD5-IN, sgRNA specifically targeting the site and a coding DNA and application thereof. The invention specifically discloses sgRNA for editing a goat-friendly site SETD5-IN (SEQ ID No.1), a DNA molecule for coding the sgRNA, a CRISPR / Cas9 targeting vector for editing the friendly site SETD5-IN, and a gene editing method. The goat-friendly site SETD5-IN of the invention can be transferred into an exogenous gene or a site-specific overexpression endogenous gene and the like, the expression of the SETD5 gene and an adjacent gene cannot be influenced, a new safe site is provided for gene site-specific integration, gene editing can be efficiently and specifically carried out by utilizing the sgRNA with high targeting efficiency and low off-target rate of the invention, and a foundation is laid for researching functions of important economic character related genes of livestock and producing transgenic animals.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a goat-friendly site SETD5-IN, a sgRNA specifically targeting the site, its coding DNA and applications. Background technique [0002] In the process of producing transgenic animals, integrating the target gene into a specific site (target site) in the animal genome is a key step. When selecting the target, on the one hand, the transferred segment should be avoided to affect the normal physiological activities of the animal. Therefore, the target is usually located in the non-coding region of the genome. Rosa26 (Reverse orientation splice acceptorβ-geo 26) gene is a common insertion site for exogenous genes, and its transcription product is long non-coding RNA (long none coding RNA, lncRNA), which can be widely expressed in various tissues of the body, so many studies have included Rosa26 Genes are considered as friendly transgenic sites. However, with th...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/85C12N5/10C12N15/90
CPCC12N15/113C12N15/85C12N15/907C12N2310/20
Inventor 韩红兵齐钰王梦瑶
Owner CHINA AGRI UNIV
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