CN118703607A - A high-throughput single-cell exogenous vector integration site detection method based on microfluidics technology - Google Patents

Abstract

The present invention relates to the field of biotechnology, and in particular to a high-throughput single-cell exogenous vector integration site detection method based on microfluidics technology, which can accurately detect vector integration sites in single cells and analyze the interaction information between exogenous vectors and host genomes at corresponding integration sites, thereby evaluating the impact of genetic modification on the host. The advantages of the method of the present application include: 1. High-throughput processing at the single-cell level is achieved by using microfluidics technology, which improves detection efficiency and accuracy. 2. Combined with the modified Tn5 enzyme system for targeted DNA cleavage, the specificity and resolution of detection are improved. 3. Through unique droplet encapsulation technology and microsphere capture system, accurate capture and labeling of exogenous vector integration sites in single cells are achieved. 4. After single cells are labeled, the gold standard technology for seamless integration site detection, Ligation-mediated target amplification (LTA-PCR), is used for library construction and sequencing, and the integration sites of single cells are quantitatively detected with high sensitivity and specificity.

Description

A high-throughput single-cell exogenous vector integration site detection method based on microfluidics technology

Technical Field

The present invention relates to the field of biotechnology, and in particular to a high-throughput single-cell exogenous vector integration site detection method based on microfluidics technology.

Background Art

Integration site detection of integrative vectors is a key step in gene therapy research. It helps to reveal how exogenous vector DNA interacts with the host cell genome, which is crucial for evaluating the safety of gene therapy and understanding the pathological mechanisms that may be caused by integrative vectors. Current detection methods mainly include: ligation-mediated PCR (LAM-PCR), inverse PCR, high-throughput sequencing (NGS), and emerging technologies such as target site amplification (TLA) and Alu-PCR. At present, these technologies are mainly suitable for integration site detection of mixed samples, that is, they analyze the average level of mixed cells or tissues and cannot reveal the heterogeneity of integration sites between cells in the sample. In particular, although LAM-PCR provides high sensitivity and detailed positioning, its operation complexity and potential amplification bias limit its widespread application; although inverse PCR is direct and low-cost, it is limited to integration sites near known sequences; NGS technology can detect a large number of integration sites in a single experiment, but data processing is complex and costly. As emerging technologies, target site amplification (TLA) and Alu-PCR can effectively reveal integration events in complex genomic regions, but they also require special optimization and analysis skills. Therefore, a set of mature and stable technologies is needed to reveal the heterogeneity between cells at the single-cell level and provide more accurate cell origin and clonality data, which is of great significance for further gene therapy research and clinical applications.

Summary of the invention

The present invention firstly provides a high-throughput single-cell exogenous vector integration site detection method based on microfluidics technology, and the method comprises the steps of microfluidics and high-throughput sequencing.

In certain embodiments, the method comprises the following steps:

(1) preparing a single cell suspension and pretreating the cells to increase cell membrane permeability;

(2) using Tn5 enzyme coated with a specific sequence to cut cell DNA fragments;

(3) Using microfluidics technology to form single-cell droplets to encapsulate cells and amplification reagents;

(4) Capturing DNA fragments and labeling DNA molecules using microspheres with cell tags;

(5) Mixing single-cell droplets to capture target fragments;

(6) Perform two rounds of PCR amplification on the target fragment and connect the adapter sequence. After the library is constructed, perform NGS sequencing;

(7) Use bioinformatics analysis software to process the raw sequencing data to obtain the integration site matrix of each cell and the regional analysis of its related sites in the genome.

In certain embodiments, the single cell comprises a stem cell.

In certain embodiments, the single cell is separated from a cultured cell population based on a green fluorescent signal by flow cytometry.

In certain embodiments, the pretreatment comprises centrifugation and lysis using proteinase K.

In certain embodiments, the NGS sequencing uses a MiSeq sequencer (Illumina).

In certain embodiments, the method further comprises the step of distinguishing cells from different sources using cell identity markers.

The present invention also provides an application of the above method, wherein the application is one or more of the following:

a) Analyze the interaction information between the exogenous vector and the host genome at the corresponding integration site;

b) Evaluate the impact of genetic modification on the host;

c) The differentiation of cells can be tracked through the integration site;

d) Uncover the molecular mechanisms that control cell differentiation and tissue development;

e) Evaluate the safety of gene therapy;

Optionally, the method is used for non-diagnostic purposes or non-therapeutic purposes.

The present invention also provides a single cell exogenous vector integration site detection kit, which contains all detection reagents for implementing the above method.

The present invention preferably provides an application of the above-mentioned kit, wherein the application is one or more of the following:

a) Analyze the interaction information between the exogenous vector and the host genome at the corresponding integration site;

b) Evaluate the impact of genetic modification on the host;

c) The differentiation of cells can be tracked through the integration site;

d) Uncover the molecular mechanisms that control cell differentiation and tissue development;

e) Evaluate the safety of gene therapy;

Optionally, the method is used for non-diagnostic purposes or non-therapeutic purposes.

Compared with the prior art, the present invention has at least the following beneficial effects:

The present invention provides a high-throughput single-cell exogenous vector integration site detection method based on microfluidics technology, which can accurately detect the vector integration site in a single cell and analyze the interaction information between the exogenous vector and the host genome at the corresponding integration site, thereby evaluating the impact of genetic modification on the host.

Benefits include:

1. Microfluidics technology is used to achieve high-throughput processing at the single-cell level, improving detection efficiency and accuracy.

2. Combined with the modified Tn5 enzyme system, targeted DNA cutting improves the specificity and resolution of detection.

3. Through the unique droplet encapsulation technology and microsphere capture system, the precise capture and labeling of exogenous vector integration sites in a single cell is achieved.

4. After labeling individual cells, the gold standard technology for seamless integration site detection, Ligation-mediated target amplification (LTA-PCR), is used to build a library for sequencing, and the integration sites of individual cells are quantitatively detected with high sensitivity and specificity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Technical flow chart of single cell integration site detection;

Figure 2 Bioinformatics analysis flow chart;

Figure 3 Single-cell integration site detection technology is used to detect and analyze different types of blood stem cells.

DETAILED DESCRIPTION

In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

Example 1 Method for detecting single cell integration sites

See Figure 1 for the technical flow chart of single-cell integration site detection in the present application, and see Figure 2 for the flow chart of bioinformatics analysis;

The method of the present invention comprises the following steps:

(1) preparing a single cell suspension and pretreating the cells to increase cell membrane permeability;

(2) using Tn5 enzyme coated with a specific sequence to cut cell DNA fragments;

(3) Using microfluidics technology to form single-cell droplets to encapsulate cells and amplification reagents;

(4) Capturing DNA fragments and labeling DNA molecules using microspheres with cell tags;

(5) Mixing single-cell droplets to capture target fragments;

(6) Perform two rounds of PCR amplification on the target fragment and connect the adapter sequence. After the library is constructed, perform NGS sequencing;

(7) Use bioinformatics analysis software to process the raw sequencing data to obtain the integration site matrix of each cell and the regional analysis of its related sites in the genome.

Example 2 Verification of the performance of the method of the present application by mixing cell standards with known integration sites

1.1 Preparation of monoclonal single/multi-site standards:

The purified lentivirus was used to infect the target cells at a certain MOI. After 48 hours of infection, the infection effect was observed under a fluorescence microscope. The cells were cultured for a certain period of time until the cell fusion reached more than 90%. Single cells were separated from the above cultured cell populations according to the green fluorescence signal by flow cytometry and cultured until monoclones were formed. The isolated monoclonal cells were amplified under appropriate culture conditions until sufficient cell quantity was obtained for the next experiment.

1.2 Confirmation of monoclonal single-site standard:

DNA was extracted from the monoclonal cells, and then the sample concentration was quantitatively detected by Qubit, followed by quality control of the lentiviral LTR sequence, that is, primers were designed according to the LTR sequence, and then the LTR sequence in the sample to be tested was amplified by PCR, and the presence of the LTR sequence in the sample was determined by 1% gel electrophoresis. Then, the sample to be tested was subjected to LTA-PCR combined with second-generation sequencing for integration site detection, and the specific steps were as follows: First, 1.5 μg of genomic DNA was sheared to an average length of 500 bp using a Covaris M220 gene disruptor. The broken DNA was purified and primers were extended using specific biotin-modified primers. The extension product was purified by Ampure XP magnetic beads, and then the target DNA fragment was captured using biotinylated magnetic beads and washed with sterile deionized water. The ligation product was subjected to the first nested PCR using biotinylated target fragment primers and ligation adapter primers. The biotinylated PCR product was captured by magnetic beads, the product was enriched and washed, and 1/2 of the eluate was used as a template for the second exponential PCR, and the second segment Barcode and sequencing-specific adapter were connected at the same time. The PCR product was qualitatively controlled by gel electrophoresis. The remaining product was purified and the concentration of the product was controlled by Qubit 4.0, and the sample was mixed according to 50ng sample. The mixed product was controlled again by TapeStation 4150 and Qubit 4.0, and the product was diluted to 10pM with TE buffer and sequenced by MiSeq sequencer (Illumina). After the machine was off, the integration site was analyzed according to the flanking sequence of the lentiviral LTR to determine whether the sample to be tested was a monoclonal single/multi-site sample. If it did not meet expectations, the virus MOI value was adjusted to prepare the standard again.

1.3 Mixed Standards:

The cells of the above monoclonal unit sites and background cells were digested separately, and then mixed with the background cells (total number of cells was 20,000) in the following ratios (ratio of standard): 0.1%, 0.5%, 1%, 5%, 10%, 20%, and then single-cell integration site library sequencing was performed after mixing.

1.4 Analysis of single cell integration sites in mixed samples:

First, according to the target cell samples collected above, centrifuge at 12,000 rpm for 5 minutes, discard the supernatant and resuspend the cells to 2,0000 per microliter. The resuspended cell samples are mixed with the lysis solution such as proteinase K used in the microfluidic system for 10 minutes. The lysis solution can destroy the cell membrane and release the components of the genomic DNA in the cell. The Tn5 transposase system is incorporated into the droplet for 15 minutes. The enzyme can randomly interrupt the cell DNA and add a known sequence to the genome. Then, according to the known sequence of the Tn5 enzyme and the specific primers designed for the vector sequence, targeted PCR amplification is performed in the microsphere (denaturation temperature 96°C for 20 seconds, annealing temperature about 5°C lower than the melting point of the primer for 15 seconds, extension temperature 70°C for 1 minute/kb, total number of cycles 30 times), and the amplification product is mixed with the microspheres with specific cell identity markers for subsequent cell identity identification. After the reaction is completed, the oil-water emulsion is destroyed, and the encapsulated cell microspheres are released to perform the next step of sequencing adapter PCR amplification. After sequencing is completed, the obtained sequence data is subjected to bioinformatics analysis, and cell identity markers are used to distinguish cells from different sources, and the integration site is determined based on the sequence of the viral vector.

In order to verify the detection limit of the technology of the present invention, we mixed the monoclonal single site (ratio 100%) standard of known integration sites into cells not treated with any vector according to a certain ratio, and the final detection results are shown in Table 1:

Table 1 Detection results

It can be seen that when the proportion of standard cells in mixed cells is 1%, the method of the present invention can detect standard cells in the mixed sample, and the integration sites and their proportions in the cells are consistent with expectations.

In view of the above detection limit, the present invention further verifies that the technology is used to evaluate the detection of samples with mixed multiple monoclonal multiple integration sites (the specific process is the same as above) (standard A cells: monoclonal contains 4 integration sites, the ratio is 25%; standard B cells: monoclonal contains 10 integration sites, the ratio is 10%), and the final results are shown in Table 2:

Table 2 Detection results

Standard product ratio Detection Is the integration site correct? Is the site ratio correct? 1% (Standard A) yes yes yes 1% (Standard B) yes yes yes 1% (Standard A, B) yes yes yes

Through the above-mentioned mixed monoclonal standard products with multiple integration sites, it can be seen that the technology of the present invention can stably detect standard products with multiple integration sites at a ratio of 1%, which is more in line with the actual sample situation.

Example 3 Method for cell differentiation or clonal evolution via single cell integration site

Judging cell differentiation or clonal evolution based on the integration sites within the cell is an important application scenario of integration site analysis. Its theoretical basis is as follows: When a virus (such as HIV-1) or other types of vectors (such as plasmids) are integrated into the host cell genome, they leave "footprints" at specific genomic locations, namely integration sites. These sites can serve as genetic markers of cell origin and clonal history. Therefore, integration site analysis at the single-cell level can identify and quantify the same integration sites that appear in multiple cells. If an integration site appears at the same frequency in multiple cells, this may indicate that these cells are derived from the same clone. In contrast, the distribution of integration sites in different cells is random, indicating that the cell population is polyclonal.

We performed single-cell integration site analysis on the lentivirus-modified hematopoietic stem cells and the peripheral blood after they were reinfused into the animals at different time points (Day 0, Day 10, Day 60), so that we can dynamically track the differentiation of blood stem cells (see Figure 3).

The specific steps are as follows:

2.1 Lentiviral transformation of hematopoietic stem cells

Healthy C57BL/6 mice of about 3 months old were selected, and the mice were sacrificed under sterile conditions to extract bone marrow cells. Hematopoietic stem cells HSCs were sorted using magnetic beads with CD117 (c-Kit) antibodies. HSCs were pre-cultured with a cytokine-containing culture medium (StemSpan culture medium plus SCF, TPO, IL-3, etc.) for about 24 hours. The lentivirus solution was added to the HSCs culture, and the infection was observed under a fluorescence microscope after continuing to culture at 37°C and 5% _CO2 for 48 hours. The culture was continued for a certain period of time until the cell confluence reached more than 90%. Cells were separated from the above-cultured cell population according to the green fluorescence signal by flow cytometry, and cultured until the confluence reached more than 90%.

2.2 Hematopoietic stem cell transfusion and blood collection

The transduced HSCs were transplanted into immunodeficient mice. Blood was collected from the mice at different time points (Day 0, Day 10, Day 60), and then blood cell subsets were sorted based on flow cytometry markers.

2.3 Analysis of single-cell integration sites in samples

First, according to the target cell samples collected above, centrifuge at 12,000 rpm for 5 minutes, discard the supernatant and resuspend the cells to 2,0000 per microliter. The resuspended cell samples are mixed with the lysis solution such as proteinase K used in the microfluidic system for 10 minutes. The lysis solution can destroy the cell membrane and release the components of the genomic DNA in the cell. The Tn5 transposase system is incorporated into the droplet for 15 minutes. The enzyme can randomly interrupt the cell DNA and add a known sequence to the genome. Then, according to the known sequence of the Tn5 enzyme and the specific primers designed for the vector sequence, targeted PCR amplification is performed in the microsphere (denaturation temperature 96°C for 20 seconds, annealing temperature about 5°C lower than the melting point of the primer for 15 seconds, extension temperature 70°C for 1 minute/kb, total number of cycles 30 times), and the amplification product is mixed with the microspheres with specific cell identity markers for subsequent cell identity identification. After the reaction is completed, the oil-water emulsion is destroyed, and the encapsulated cell microspheres are released to perform the next step of sequencing adapter PCR amplification. After sequencing is completed, the obtained sequence data is subjected to bioinformatics analysis. At the same time, cell identity markers are used to distinguish cells from different sources, the integration site is determined based on the sequence of the viral vector, and the differentiation trend between cells is determined based on the differentiation of the cells.

Referring to Figure 3, different types of blood stem cells are detected and analyzed based on the single-cell integration site detection technology, and cell differentiation is combed based on the integration site. Through this embodiment, it can be clearly seen that the differentiation of cells can be tracked through the integration site, which is particularly important for understanding how pluripotent stem cells differentiate into specific cell types during development, and helps to reveal the molecular mechanisms that control cell differentiation and tissue development. In gene therapy, exogenous genes introduced by viral vectors or other methods may be randomly integrated into the genome of host cells. Tracking these integration sites can evaluate the behavior of cells after gene editing, including whether they maintain the expected differentiation potential or whether unexpected genetic changes, such as carcinogenic transformation, have occurred. This is crucial to ensuring the safety of gene therapy.

The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (10)

1. A high-flux single-cell exogenous vector integration site detection method based on a microfluidic technology, which is characterized by comprising the steps of microfluidic control and high-flux sequencing.

2. The method according to claim 1, characterized in that it comprises the steps of:

(1) Preparing single cell suspension, and pretreating cells to increase cell membrane permeability;

(2) Cleaving a cellular DNA fragment using a Tn5 enzyme coated with a specific sequence;

(3) Forming single-cell droplets by adopting a microfluidic technology, and wrapping cells and an amplification reagent;

(4) Capturing DNA fragments and labeled DNA molecules by means of microspheres bearing cellular tags;

(5) Mixing single-cell droplets to capture target fragments;

(6) Performing two rounds of PCR amplification on the target fragment, connecting the adaptor sequences, and performing NGS sequencing after constructing a library;

(7) Sequencing raw data is processed by using a bioinformatic analysis software to obtain an integration site matrix of each cell and the regional analysis of related sites in a genome.

3. The method of claim 1, wherein the single cell comprises a stem cell.

4. The method of claim 1, wherein the single cells are separated from the cultured cell population by flow cytometry based on green fluorescent signal.

5. The method of claim 1, wherein the pretreatment comprises centrifugation of the supernatant and cleavage with proteinase K used.

6. The method of claim 1, wherein the NGS sequencing uses a MiSeq sequencer (Illumina).

7. The method of claim 1, further comprising the step of distinguishing between cells of different origins using a cell identity tag.

8. Use of the method according to claim 1 or 2, characterized in that the use is one or more of the following;

a) Analyzing interaction information between the exogenous vector and the host genome at the corresponding integration site;

b) Assessing the effect of the genetic modification on the host;

c) The differentiation of cells can be followed by integration sites;

d) Molecular mechanisms controlling cell differentiation and tissue development are disclosed;

e) Evaluating the safety of gene therapy;

optionally, the method is for non-diagnostic or non-therapeutic purposes.

9. A single cell exogenous vector integration site detection kit comprising all detection reagents for performing the method of any one of claims 1-7.

10. Use of a kit according to claim 9, wherein the use is one or more of the following;

a) Analyzing interaction information between the exogenous vector and the host genome at the corresponding integration site;

b) Assessing the effect of the genetic modification on the host;

c) The differentiation of cells can be followed by integration sites;

d) Molecular mechanisms controlling cell differentiation and tissue development are disclosed;

e) Evaluating the safety of gene therapy;

optionally, the method is for non-diagnostic or non-therapeutic purposes.