A method for site-specific editing of ccr5 gene
A gene editing technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve problems such as uncontrollable, risky, fatal safety, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0038] Example 1 High-efficiency gRNA screening of CRISPR-Cas9 knockout CCR5 gene
[0039] 1. gRNA preparation
[0040] (1) Design a 20nt gRNA sequence according to the sequence of the CCR5 gene (including the exon and the intron sequence adjacent to the exon), the 3'-end of the target sequence of the gRNA on the CCR5 gene is NGG;
[0041] (2) Synthesize the sense strand and antisense strand of the target sequence with cohesive ends respectively (cacc is added to the 5'-end of the sense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, add cacc to the sense strand Add caccG at the 5'-end of the antisense strand; add aaac at the 5'-end of the antisense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, add C at the 3'-end of the antisense strand );
[0042] (3) The above-mentioned sense strand and antisense strand were mixed, treated at 90°C, cooled naturally to room temperature for annealing treatment, and do...
Embodiment 2
[0068] Example 2 Construction of a donor vector for site-specific integration of the hTRIM5 / CypA gene at the CCR5 site
[0069] (1) Acquisition of the upstream homology arm F86-F of CCR5F86
[0070] Construction of pDonor-TALEN-EGFP-hCCR5F86-F expression vector
[0071] 1. PCR amplification of the target fragment F86-F;
[0072] Primers F86-SnaB I-L / F86-Sal I-R were used to amplify the F86-F target fragment using the HepG2 cell genome as a template.
[0073] F86-SnaB I-L (SEQ ID NO: 3):
[0074] 5′-gttctagtggttggctacgtagcaccatgcttgacccagtttc-3′
[0075] F86-Sal I-R (SEQ ID NO:4):
[0076] 5′-tagcttatcgataccgtcgacGTCACCACCCCAAAGGTGAC-3′
[0077] The resulting PCR product sequence contains the DNA sequence SEQ ID NO:5 of the F86 upstream homology arm F86-F:
[0078]gcaccatgcttgacccagtttcttaaaattgttgtcaaagcttcattcactccatggtgctatagagcacaagattttatttggtgagatggtgctttcatgaattcccccaacagagccaagctctccatctagtggacagggaagctagcagcaaaccttcccttcactacaaaacttcattgcttggccaaaaagagagttaattCAA...
Embodiment 3HI
[0134] The transformation of embodiment 3 HIV-1 susceptible cell lines
[0135] 1. Culture Hela-CD4 (X4 tropism-susceptible cell line) and TZM-bl (R5 tropism-susceptible cell line) cells on the wall of 24-well plate, the plating density is 1.2×10^5cells / well, use DMEM medium (Add 10% FBS) culture;
[0136] 2. Extract the plasmid pDonor-EF1α-hTRIM5 / CypA-EGFP-hCCR5F86 ( Figure 10 ) and pX458-F86;
[0137] 3. Using Lipofectamine 3000Transfection Reagent, the above two plasmids were co-transformed into Hela-CD4 and TZM-bl cell lines;
[0138] 4. From the fifth day, add G418 with a final concentration of 800μg / ml for selection, passaging once every 2-3 days, and replace each time with fresh DMEM medium containing 800μg / mL G418;
[0139] 5. After 12 days of drug screening, cells were digested, sorted by flow cytometry and inoculated into 96-well plates;
[0140] 6. Expand the culture of cells sorted into 96-well plates, select cell lines that stably and uniformly express green ...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap