Preparation method of cell line with high homologous recombination rate

Abstract

The invention relates to a preparation method of a cell line with a high homologous recombination rate, which comprises the following steps: stably inserting a donor vector into an AAVS1 position by using a siRNA technology and a gene editing technology, knocking down important genes LIG4 and Ku70 in an NHEJ pathway, and simultaneously overexpressing important proteins SWIT5 and SFR1 in a homologous recombination pathway, so that the cell line with the high homologous recombination rate is obtained. And screening by a monoclonal sorting method to obtain the improved 293T cell line with high homologous recombination efficiency. The proliferation effect and transfection effect of the cell line obtained by the method are similar to those of a wild type, and the homologous recombination efficiency is 20 times that of the wild type, so that the subsequent accurate gene editing operation is facilitated, and the scientific research can be more efficiently carried out at low cost.

Description

technical field

[0001] The present invention relates to a preparation method of a cell line with high homologous recombination rate. Background technique

[0002] HEK293 cell, also known as human embryonic kidney cell 293, is a cell line derived from human embryonic kidney cells. It has the characteristics of high transfection efficiency and easy culture. It is a very commonly used cell line for expressing foreign genes.

[0003] CRISPR-Cas gene editing technology is a DNA double-strand break (DSB) generated by RNA-guided Cas nuclease cleavage at a specific site in the genome, and then the DSB is repaired by the endogenous repair machine of the cell. edit. These breaks are primarily repaired through one of two major repair pathways, classical non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter of which is restricted to the S / G2 phase of the cell cycle and occurs with significant frequency lower. Precise genome editing applications rely on HD...

Examples

preparation example Construction

[0051] The preparation method of the donor vector is as follows:

[0052] (1) Determine the siRNA sequence of Ku70.

[0053] SEQ ID No. 4 (Ku70i): GGAAGAGATAGTTTGATTT.

[0054] (2) Determine the effective siRNA sequence of LIG4.

[0055] SEQ ID No. 5 (LIG4i): GCTAGATGGTGAACGTATG.

[0056] The gene fragment H1-LIG4i-spacer-U6-Ku70i was synthesized by an outsourcing company.

[0057] (3) Determine the CDS sequences of functional proteins expressed by SWI5 and SFR1

[0058] The CDS sequences of the important proteins SWI5 and SFR1 in the homologous recombination pathway were queried by NCBI, and the corresponding primers were designed to amplify the two genes of SWI5 and SFR1 respectively, and then bridge PCR to fuse SWI5-P2A-SFR1 together, using Nhe1 and Apa1 two genes Each restriction site was linked to the pcDNA3.1 vector.

[0059] 1) Using cDNA as a template, the SWI5 gene was amplified by amplification.

[0060] The amplification primers are:

[0061] SWI5 F (SEQ ID N...

experiment example 1

[0113] The vectors Lam-Donor-gn and NLS-cas9-gk-H1-lamin sg were constructed, and 293THDR cells and wild-type 293T cells were co-transfected. After transfection, they were cultured for 72 hours. Flow cytometric analysis showed that 293THDR cells were homologously recombined. The ratio is about 20 times of the homologous recombination efficiency of wild-type 293T cells. The result is as figure 1 shown.

experiment example 2

[0115] Constructed the vector tubB-Donor-gn and NLS-cas9-gk-H1-TUB SG1, co-transfected 293THDR cells and wild-type 293T cells, and cultured for 72 hours after transfection. Flow cytometry analysis showed that 293THDR cells were homologously recombined. The ratio is about 20 times of the homologous recombination efficiency of wild-type 293T cells. The result is as figure 2 and image 3 shown.

technical field

[0001] The present invention relates to a preparation method of a cell line with high homologous recombination rate. Background technique

[0002] HEK293 cell, also known as human embryonic kidney cell 293, is a cell line derived from human embryonic kidney cells. It has the characteristics of high transfection efficiency and easy culture. It is a very commonly used cell line for expressing foreign genes.

[0003] CRISPR-Cas gene editing technology is a DNA double-strand break (DSB) generated by RNA-guided Cas nuclease cleavage at a specific site in the genome, and then the DSB is repaired by the endogenous repair machine of the cell. edit. These breaks are primarily repaired through one of two major repair pathways, classical non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter of which is restricted to the S / G2 phase of the cell cycle and occurs with significant frequency lower. Precise genome editing applications rely on HD...

Examples

preparation example Construction

[0051] The preparation method of the donor vector is as follows:

[0052] (1) Determine the siRNA sequence of Ku70.

[0053] SEQ ID No. 4 (Ku70i): GGAAGAGATAGTTTGATTT.

[0054] (2) Determine the effective siRNA sequence of LIG4.

[0055] SEQ ID No. 5 (LIG4i): GCTAGATGGTGAACGTATG.

[0056] The gene fragment H1-LIG4i-spacer-U6-Ku70i was synthesized by an outsourcing company.

[0057] (3) Determine the CDS sequences of functional proteins expressed by SWI5 and SFR1

[0058] The CDS sequences of the important proteins SWI5 and SFR1 in the homologous recombination pathway were queried by NCBI, and the corresponding primers were designed to amplify the two genes of SWI5 and SFR1 respectively, and then bridge PCR to fuse SWI5-P2A-SFR1 together, using Nhe1 and Apa1 two genes Each restriction site was linked to the pcDNA3.1 vector.

[0059] 1) Using cDNA as a template, the SWI5 gene was amplified by amplification.

[0060] The amplification primers are:

[0061] SWI5 F (SEQ ID N...

experiment example 1

[0113] The vectors Lam-Donor-gn and NLS-cas9-gk-H1-lamin sg were constructed, and 293THDR cells and wild-type 293T cells were co-transfected. After transfection, they were cultured for 72 hours. Flow cytometric analysis showed that 293THDR cells were homologously recombined. The ratio is about 20 times of the homologous recombination efficiency of wild-type 293T cells. The result is as figure 1 shown.

experiment example 2

[0115] Constructed the vector tubB-Donor-gn and NLS-cas9-gk-H1-TUB SG1, co-transfected 293THDR cells and wild-type 293T cells, and cultured for 72 hours after transfection. Flow cytometry analysis showed that 293THDR cells were homologously recombined. The ratio is about 20 times of the homologous recombination efficiency of wild-type 293T cells. The result is as figure 2 and image 3 shown.

Images