Method for obtaining homozygous mutation of seamless modification

A homozygous, seamless technology, applied in the field of molecular biology, can solve the problems of low efficiency of precise and seamless gene modification, troublesome construction of plasmids, lack of screening system, etc., to save manpower, material resources and time, facilitate disease models, and select the range wide range of effects

Inactive Publication Date: 2018-03-23
UNIVERSITY OF MACAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Double-strand breaks are necessary for homologous recombination to obtain genetic mutations. Of course, this also stimulates another, more common endogenous DNA repair pathway - non-homologous end joining (NHEJ), which will be precise and seamless. Genetic Modification Creates Barriers
Synthetic ssODNs can be conveniently ordered from companies, but it lacks a screening system, making precise and seamless gene modification quite inefficient
The piggyBac transposon may be relatively troublesome to construct a plasmid, but it can carry a screening system, and it is relatively easier to obtain the desired seamless heterozygous mutation
Of course, for homozygous mutations, the efficiency of using CRISPR / Cas9 combined with piggyBac is very low, resulting in a huge waste of manpower, material resources and time

Method used

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  • Method for obtaining homozygous mutation of seamless modification
  • Method for obtaining homozygous mutation of seamless modification
  • Method for obtaining homozygous mutation of seamless modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 CRISPR / Cas9 combined with piggyBac transposon carrying puro / TK resistance screening to efficiently obtain heterozygous mutations

[0034] experimental method

[0035] 1. Cell culture and transfection

[0036] Seed iPS cells on cell culture plates pre-coated with Matrigel (BD Biosciences), and use mTeSR TM 1 (Stemcell Technologies) at 37°C, 5% CO 2 cultured in a cell culture incubator. Before Lipofectamine transfection, iPS cells were divided into 4×10 per well 5 ~5×10 5 The density was passaged on six-well plates coated with Matrigel, and mTeSR containing 10 μM ROCK inhibitor (Sigma) TM 1 was incubated overnight. On the second day after passaging, iPS cells were co-transfected with 6 μg pCas9_NeoR, 3 μg sgRNAplasmid and 50 nM ssODN (90 bpssODN), and the cells in the other well were transfected with 2 μg pmaxGFP alone as a control for transfection efficiency. The transfection process was strictly in accordance with Lipofectamine TM 3000's official opera...

Embodiment 2

[0056] Example 2 CRISPR / Cas9 combines piggyBac transposons containing puro / TK and hygR / TK resistance screening respectively to obtain homozygous mutations efficiently

[0057] experimental method

[0058] The experimental method is the same as that used in Example 1.

[0059] 1. Cell culture and transfection

[0060] With embodiment 1.

[0061] 2. iPS single cell clone culture

[0062] With embodiment 1.

[0063] 3. Construction of Cas9 and PB-based plasmids

[0064] With embodiment 1.

[0065] 4. Obtain homozygous mutation

[0066] The following still takes PSEN1ΔI83M84 as an example to illustrate the process of using CRISPR / Cas9 and the new piggyBac transposon system to obtain seamless homozygous mutations, such as Figure 2A Shown:

[0067] (a) First, determine that the site to be mutated is on exon 4 of PSEN1;

[0068] (b) using Cas9 to cut at the target site to generate DSB;

[0069] (c) Construction of piggyBac transposons (SEQ ID No: 7 and SEQ ID No: 8) carryi...

Embodiment 3

[0076] Example 3 Cells carrying AD-related gene mutations acquire gene mutation-dependent phenotypic changes experimental methods

[0077] 1. Differentiation of cortical neurons

[0078] Human iPS cells are differentiated toward neural stem cells, and the cultured iPS cells are scraped into small clonal clusters with a cell scraper at 2.5-3.0×10 4 cells / cm 2 The density of PSC Neural Induction Medium (Life Technologies) plus Y23632 (sigma) was used to culture on a 6-well plate pre-coated with matrigel (BD Biosciences), the medium was changed every two days, and the culture was continued for 7 days. After 7 days, single cells were digested with Accutase (Sigma) at 1.0-1.2×10 5 cells / cm 2 density for expansion.

[0079] For cortical neuron differentiation, neural stem cells were plated at 2 × 10 5 cells / cm 2 The density of N2B27 medium (DMEM-F12: Neural Basal medium 1:1, 2% B27, 1% N2, 1% non-essential amino acids (non-essential aminoacids), 2mM Glutamax, 100U penicillin p...

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Abstract

The invention relates to a method for obtaining homozygous mutation of seamless modification. The method comprises the following steps: (1) cutting a locus needing mutation in a cell genome to generate double-chain breaking by using a gene editing system; (2) respectively integrating two kinds of transposons, which contain a target mutation sequence and different resistance, to enter a pair of alleles of the cell genome through homologous recombination; (3) screening a cell clone of the alleles which integrate the transposons, and then cutting off the transposons; and (4) causing death to thecell clone in which the transposons are residually left to obtain the cell clone which contains the homozygous mutation of the seamless modification. The method can be used for obviously improving theprobability of obtaining the homozygous mutation, manpower, material resources and time are saved, and the application range is wide.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for obtaining seamlessly modified homozygous mutations. Background technique [0002] Compared with animal models, cell models derived from human iPS have a series of advantages, such as being able to be purified into a specific cell type for research, facilitating high-throughput screening of compounds and gene targets, and obtaining a variety of gene mutations Cells and use it to study cell phenotypes and pathogenic mechanisms, and there is no species difference with humans. Cells from patients are reprogrammed to obtain iPS, which can be used to study many genetic diseases. Of course, the acquisition of patient cells, the confirmation of pathogenic mutation genes, and the common analysis of cells with different genetic backgrounds all pose obstacles to the study of genetic diseases. However, these obstacles can be resolved by introducing disease-related ...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N5/10
CPCC12N5/0696C12N9/22C12N15/907C12N2510/00
Inventor 苏焕兴谭渊黄志健
Owner UNIVERSITY OF MACAU
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