RU2827872C1 - Method of creating cell models of translocations using genome editing tools - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a method for creating a cell model of leukaemia for modelling and studying processes occurring in leukaemia cells. Method is aimed at detecting those single cells in the population in which translocation has occurred, and their selection. Primary selection is carried out by fluorescence of the reporter protein, and ensuring that all elements (Cas9 and RNA guide genes) required for translocation induction have entered the cell is ensured by the fact that all these elements together with the fluorescent protein gene are assembled into one plasmid. Such a plasmid for inducing a new translocation is created on the basis of a universal vector in two cloning steps. To detect translocation, PCR is used, and without a separate step of DNA extraction (so-called direct PCR), combining this approach with embedded PCR, which increases sensitivity of the method. For reliable production of a cell line with a translocation with a probability of not less than 0.95, a preliminary check of the effectiveness of RNA guides and counting the frequency of translocations can be carried out. Based on the frequency of translocations, an optimal cloning strategy into subpopulations and a strategy of PCR analysis with combining several samples into one reaction are selected. Cloning is carried out using a cell sorter. Obtained monoclonal lines are characterized for the presence of translocation, the rearrangement region is sequenced, the expression of the fused gene product is shown and can be further used for the declared purposes.

EFFECT: invention enables to obtain monoclonal lines with translocations in the shortest possible time and with minimal time and laboratory resources.

21 cl, 5 dwg, 1 ex

Description

Field of technology to which the invention relates

The present invention relates to biotechnology, namely, to technologies for creating cell lines with translocations using nuclease-genetic editors; to medical and scientific technologies, namely, to technologies for modeling events occurring in leukemia on cells for the purpose of studying and selecting the most effective drugs. Creating cell lines with translocations makes it possible to obtain a convenient model of leukemia for studying them at the cellular level. Thus, it is possible to conduct a study of differential gene expression on the obtained cells in comparison with cells without translocation or to compare initially homogeneous cells with different translocations. The results of such studies will allow a better understanding of the characteristics of leukemia cells and identify potential targets for therapy. It is possible to test various chemotherapeutic agents on the obtained cells. For this purpose, cells with translocation and cells without translocation are treated with various therapeutic compounds, and then cytotoxic tests are carried out to identify compounds that act most effectively on cells with translocation.

State of the art

Obtaining cell lines with translocations is an important task for both research purposes and practical applications. Studying cell models with different translocations allows us to identify the mechanisms of translocation formation and the mechanisms of cancer development associated with these translocations. And testing drugs on cell models with translocations will allow us to better select different drugs for different types of cancer, depending on the specific translocation.

The creation of cells with translocations began with the use of Cre-Lox systems. LoxP sequences are inserted into the target loci of the cell genome, and when a transgene encoding Cre recombinase is expressed in these cells, recombination occurs between the LoxP sites, which leads to the formation of chromosomal translocations between the target loci. An example of such an approach is the work Inducible chromosomal translocation of AML1 and ETO genes through Cre/loxP-mediated recombination in the mouse (doi: 10.1093/embo-reports/kvd027). This approach is very time-consuming - it is necessary to select cells first with a LoxP site in one chromosome, then with a LoxP site in another, check the correctness of the insertion, then start the work of Cre recombinase and then select cells with translocation. Moreover, the frequency of occurrence of translocations in cells is not very high, for example, in the mentioned work it did not exceed 1 × 10 ^-4 , in another similar work, Cre-Mediated Site-Specific Translocation between Nonhomologous Mouse Chromosomes (http://www.jstor.org/stable/2368247), the frequency of translocations in cells containing LoxP sites in target loci and expressing Cre is estimated as 1 in 1.2-2.4 × 10 ^-3 .

A technological revolution in obtaining cells with translocations has been the use of programmable nucleases. Expression of nucleases in a cell directed at two target sites results in the occurrence of a translocation between these sites in some of the cells. The number of cells in which a translocation occurs can vary, but on average ranges from 1 in 300 cells to 1 in 10,000 cells.

To obtain a cell culture in which all cells contain a translocation, it is necessary to select individual cells with a translocation from a mixed population and obtain a so-called monoclonal culture from them. Various approaches are used to select cells containing a translocation. The simplest is culture cloning. Cells transfected with nucleases are distributed one by one into the wells of a plate. Clones grow from single cells. When the clones grow, DNA is isolated from them and PCR analysis is performed to determine the presence of the desired translocation. Using this algorithm with CRISPR/Cas, human eHAP cell lines with CD74-ROS1 rearrangement were obtained (Efficient generation and reversion of chromosomal translocations using CRISPR/Cas technology, https://doi.org/10.1186/s12864-016-3084-5). Plasmids encoding the guide RNA genes, as well as a plasmid encoding Cas9 and a plasmid with resistance to the antibiotic blasticidin, were transfected together into cells using Turbofectin transfection reagent (Origene). The cell population was then enriched for transfected cells by treatment with the antibiotic blasticidin for 2=3 days. Cells were diluted by serial dilutions to a concentration of 15 cells/ml, then distributed 50 μl into the wells of a 384-well plate and allowed to grow into clones for further analysis by PCR. A total of 192 clones were analyzed, of which 2 were positive. This method demonstrated the possibility of obtaining translocations, but it is quite time- and resource-intensive: for example, to obtain 2 clones, the authors performed serial dilutions manually, monitored the growing clones using specialized equipment that is not available in all laboratories = a robot for working with cells Microlab STAR Line robotic system, and performed multiple PCR reactions (at least one with each of the 192 clones). To analyze the cells using PCR, they were lysed with the DirectPCR Lysis reagent kit (PeqLab), which requires the use of ≈ 40,000 cells or more and their lysis overnight.

Another example is the generation of a mouse stem cell line (mESC) with a Cdx2-Gsk3α rearrangement and a HEK293 cell line with a BCR-ABL1 translocation (Induction of site-specific chromosomal translocations in embryonic stem cells by CRISPR/Cas9, https://doi.org/10.1038/srep21918). The authors first generated a mESC cell line expressing Cas9 and then delivered guide RNA genes using lentiviral transduction. These cells were initially distributed at 5 per well, and 3 positive wells out of 33 were detected using PCR analysis. Then the cells from the positive wells were cloned into single cells. Three out of 16 monoclones had a rearrangement. For HEK293 cells with a BCR-ABL1 translocation, the authors do not indicate the number of clones analyzed, but note that this analysis is time-consuming. The disadvantage of this strategy is that in order to obtain the same translocation in several different cell lines, it is necessary to first integrate the Cas9 gene into the genome in each of them, and also that the use of lentiviral transduction will lead to the constant expression of the CRISPR/Cas system, which has off-target activity and will constantly introduce mutations at off-target sites in the resulting cell line.

Both approaches described use cloning and PCR analysis of numerous clones to detect those containing the translocation. However, another method has been proposed that involves the possibility of selecting cells in which the event of formation of the target translocation has occurred, which allows one to do without cloning. This approach involves the integration of a selection cassette into the rearrangement point. To do this, in addition to plasmids encoding nucleases, a plasmid containing regions of homology to two rearranged loci, between which the selection cassette is located, is introduced into the cells. The cassette encodes a fluorescent protein or an antibiotic resistance protein. In this case, cells with the rearrangement can be selected from the population by fluorescence or antibiotic resistance. Using this approach, cells with the EWSR1-WT1 translocation were obtained based on human mesenchymal stem cells hMSC (Generation of chromosomal translocations that lead to conditional fusion protein expression using CRISPR-Cas9 and homology-directed repair, doi: 10.1016/j.ymeth.2017.05.006). However, this approach is unreliable, since in many cases cells are selected that do not carry translocations, but only carry a cassette integrated at a non-homologous DNA break end joining. Also, after obtaining a translocation, it is necessary to cut out this cassette using Cre recombinase expression. An additional problem is the complexity of translocation diagnostics using PCR, since the primers flanking the integrated construct and the homology arms are located far from each other, and such PCR requires additional optimization for reliable detection. The use of such an approach to obtain lines with translocations on LCL cells was not reliable and did not provide a gain in time compared to the claimed method (data from our laboratory). Thus, in an attempt to model various translocations, it was possible to obtain only one line out of 4, and this took 16 weeks.

The technical problem solved by the claimed invention is the need to overcome the shortcomings inherent in analogues by developing a method for obtaining cell cultures for modeling and studying the processes occurring in leukemic cells with a certain chromosomal translocation, and for testing drugs, as well as developing a universal and rapid method for obtaining such lines.

Disclosure of invention

The technical result of the invention is the possibility of obtaining model cultures in a shorter period of time (8 weeks) from transplanted (immortalized) cells with a reproduction probability of at least 0.95.

The following advantages of the claimed method can be highlighted. Firstly, the reliability of the method of obtaining - the developed method allows to estimate the probability of translocation and, based on it, modify the protocol in order to obtain a cell line with a target rearrangement with a probability of 95% as quickly as possible. Secondly, the method requires minimal expenditure of working time and laboratory resources - when analyzing subpopulations and clones, the minimum possible number of PCR reactions with the minimum possible number of cells is used, and the subcloning procedure is carried out using a cell sorter, and not by the limiting dilution method.

The technical result is achieved by a method of creating a cellular model of leukemia for modeling and studying the processes occurring in leukemic cells with a certain chromosomal translocation, and for testing drugs on them, including taking a sample/cells from a patient, identifying target loci that are associated with the pathology being studied, i.e. determining the rearrangement loci in the genome, then synthesizing at least one plasmid construct encoding genome editing tools, guide RNAs directed to the target loci (rearrangement loci), and including a fluorescent protein, transfecting the cells with the resulting plasmid or plasmids, after transfection, the cells expressing the fluorescent protein are selected using a cell sorter and distributed in groups of 20±10 into the wells of a plate with a medium, increasing the cell subpopulation to 20,000±10,000 cells, then analyzing the resulting subpopulations for the presence of cells with translocation using direct nested PCR and cloning the subpopulations with confirmed translocation into monoclonal lines, among which lines with translocation are identified, confirming the expression of the fusion gene, while for the analysis of subpopulations, the content of cells with translocation is first assessed in a group of wells and One PCR is performed with mixed material, and if a translocation is detected, the contents in individual wells are checked. In this case, cells from several wells are combined into groups of 2 to 12 wells; if there is no translocation in a group, cells from all wells of such a group are excluded from further work. A subpopulation of cells with a confirmed rearrangement is cloned using a cell sorter and monoclones are grown to 10,000 - 100,000 cells. Nucleases that specifically recognize target loci are used as genome editing tools, and systems based on CRISPR/Cas, zinc finger nucleases, TALE nucleases, endonucleases for inserting mobile genetic elements into the genome, as well as other nucleases that have the property of recognizing and cutting target loci are used as editing tools. The green fluorescent protein from Aequorea victoria and its homologues, biliverdin-binding bacteriophytochromes and cyanobacterichromes, phycocyanobilin-binding cyanobacterial phytochromes, small far-red fluorescent proteins, small green oxygen-independent fluorescent proteins, and fluorescence-activated labels are used as fluorescent proteins. The plasmid construct for transfection contains an origin and a gene for resistance to a selective antibiotic and encodes genome editing tools, includes a region containing a type IIS restriction enzyme recognition site, into which a sequence responsible for recognition of the target locus by the nuclease is inserted, and also encodes genes for elements of the genome editing system and a fluorescent protein gene. The cells used are those capable of undergoing more than 30-40 divisions, such as immortalized cells or tumor cell cultures. Cell transfection is performed using chemical transfection, electroporation, and transduction using viral particles. Cells with translocations are assessed using the PCR method. To obtain monoclones, cells are distributed from populations containing translocation, one cell per well.

To study leukemogenesis (the formation of hemoblastoses), a cell line is used, which is modified immortalized lymphoblasts of the LCL line, containing the MLL-ARHGAP26 translocation, obtained by the claimed method. To obtain a cellular model, immortalized lymphoblasts (LCL line) are used, the loci of rearrangement within the MLL-ARHGAP26 genes are determined and the genetic construct px330_gRNA_Cas9 Santaka and plasmid 53188-PcisI are synthesized, the gene for the second guide RNA from plasmid 53188-PcisI is inserted into px330_gRNA_Cas9_Santaka, at the sites of restriction enzymes HindIII and BstAFI, the recognition region of one guide RNA to MLL is introduced into the resulting plasmid px330_gRNA_Cas9_Santaka_gRNA using the restriction enzyme BstV21, and the recognition region of another guide RNA to ARHGAP26 is introduced using the restriction enzyme PcisI, the resulting plasmid px330_gMLL_Santaka_gARH is carried out transfection of LCL cells, after 2 days the cells are sorted by the Santaka transfection marker at 20±10 cells per well of a 96-well plate, the subpopulation of cells is increased to 20,000±10,000 cells, the content of cells with translocation is assessed using direct nested PCR first in a group of wells, then in individual wells, the subpopulation of cells with confirmed rearrangement is cloned using a cell sorter and monoclones are increased to 10,000 - 100,000 cells; then the presence of translocation is determined using direct nested PCR and the monoclone of cells with translocation is used for further studies. The px330_gRNA_Cas9 Santaka construct includes elements of the CRISPR/Cas system that introduce breaks into loci in the 11th intron of the MLL gene and in the 11th intron of the ARHGAP26 gene, and contains the Santaka fluorescent protein gene, the SV40 promoter sequence, the BmtI restriction enzyme site obtained on the basis of commercial plasmids px330 and phU6-gRNA, as well as a set of primers with the sequences SEQ ID No. 1 - SEQ ID No. 4:

To synthesize the genetic constructs of plasmid 53188-PcisI, a commercial plasmid phU6-gRNA with oligonucleotides that form an insert with PciSI sites when annealed to each other and a set of primers with the sequences SEQ ID No. 5 - SEQ ID No. 8 are used:

To check for the presence of translocation using the nested PCR method, primers with sequences SEQ ID No. 9 - SEQ ID No. 12 are used:

The presence of translocation is tested by nested PCR in two stages, where in the first stage of PCR, amplification is performed using the following temperature conditions: 95°C 5 min, (95°C 20 sec, 58°C 1 min, 72°C 1 min) × 20, 72°C 5 min, after PCR, 4 μl of the reaction mixture is added to 396 μl of deionized water, then 4 μl of the resulting solution is used as a template in the second stage of PCR. In the second stage of PCR, amplification is performed using the following temperature conditions: 95°C 5 min, (95°C 20°s, 58°C 1 min, 72°C 40 sec) × 30, 72°C 5 min, the results are analyzed by electrophoresis in agarose gel.

Brief description of the drawings

The invention is illustrated by the following illustrative material.

Fig. 1 shows a map of the pX330_gRNA_dCas9 plasmid, which is used to obtain plasmid constructs that induce translocation. The diagram shows: the Cas9 nuclease gene containing the SV40 NLS and the elements required for its expression (CMV enhancer, beta-actin promoter, hybrid intron, Kozak sequence), elements of the guide RNA gene (U6 promoter, region encoding the guide RNA scaffold), and other plasmid elements required for cloning.

Fig. 2 shows a map of the plasmid from which the plasmid inducing the rearrangement is obtained by cloning at the BstV2I and PciSI sites. The diagram shows the genes for the Cas9 nuclease and the Santaka fluorescent protein containing the SV40 NLS and the elements necessary for its expression (CMV enhancer, beta-actin promoter, hybrid intron, Kozak sequence), elements of the guide RNA genes (U6 promoter, region encoding the guide RNA scaffold), and other elements of the plasmid necessary for cloning.

Fig. 3 shows a diagram of the claimed method for obtaining cell models, where A. Transfection of cells with a plasmid to induce translocation. B. Sorting by Santaka fluorescence, 20 cells per well of the plate. C. Analysis of groups of 6 wells with cells for the presence of translocation using direct nested PCR. D. Analysis of individual wells of the groups that showed the presence of translocation in the previous step for the presence of translocation. D. Sorting cells one by one into wells of the plate to obtain monoclones. E. Analysis of monoclones for the presence of translocation using direct PCR.

Fig. 4 shows: A. a diagram of the location of primer annealing points on the intact MLL gene and on the fused MLL-ARHGAP26 gene. B. an electropherogram demonstrating the results of PCR for the MLL-ARHGAP26 translocation.

Fig. 5 shows an electropherogram demonstrating the result of checking the expression of the fusion gene. A. Scheme of the arrangement of primers when checking the obtained cells for the expression of mRNA of the fusion gene. B. Electrophoresis of PCR products with reverse transcription. GAPDH is used as a reaction control.

Implementation of the invention

To model a leukemia-associated translocation, the points between which the rearrangement occurs in patients are determined (PMID: 37019990). There are many ways to determine the loci of the rearrangement, both with accuracy to the intron of the rearranged gene and with accuracy to the nucleotide (see the review of methods: PMID: 33205680). To model the rearrangement as in patients, it is sufficient to introduce a break in each of the partner genes at any point within the intron in which the break occurred in the patient (except for splice sites), since the intron does not contain a protein-coding sequence, and the oncogenic effect of the rearrangement is caused by the chimeric protein. The choice of the target locus is carried out based on the data on the breakpoints, as well as the possibility of introducing a break using a programmable nuclease. Thus, when using the Cas9 nuclease from Streptococcus pyogenes, its work requires the GG sequence following 1 nucleotide from the 20-nucleotide region recognized by the guide RNA - the so-called PAM (protospacer-adjacent motif), and Cas9 orthologs from other bacteria use the following PAMs: SaCas9 from Staphylococcus aureus - NNGRRT (R = A, G) (PMID: 25830891); CjCas9 from Campylobacter jejuni - N4RYAC (Y = C, T) (PMID: 28220790); St1Cas9 from Streptococcus thermophilus - NNRGAA (PMID: 31900288). For each Cas nuclease described in the scientific literature, its PAM is always defined.

The claimed method for obtaining cell lines with translocations is as follows.

First, a plasmid construct encoding genome editing tools directed at 2 target loci (between which the rearrangement occurs) and a fluorescent protein are obtained. Plasmids must contain all the elements necessary for replication and selection. Any methods of assembling plasmid constructs are used. The elements from which the plasmid (or plasmids) are obtained can be obtained from other plasmids or synthesized (gene synthesis is performed). The necessary plasmid elements and assembly methods are described in the Plasmids 101 e-book, Addgene. It is permissible to split these components into several plasmids, but this reduces the transfection efficiency. It is also permissible to deliver genome editing tools to cells directly as proteins or nucleoprotein complexes by any method, or by transfecting cells with mRNA encoding these components.

Any nucleases that specifically recognize target loci between which translocation is obtained can act as genome editing tools. Those that are more effective are selected from those available. To compare the effectiveness, the methods described in the article PMID: 31876277 are used. Systems based on CRISPR/Cas, zinc finger nucleases, TALE nucleases, mobile genetic element homing endonucleases and other programmable nucleases or meganucleases act as genome editing tools.

Fluorescent proteins can be any avGFP (green fluorescent protein from Aequorea victoria ) homologues - GFP, RFP, CFP, YFP, etc., as well as other fluorescent proteins, such as biliverdin-binding bacteriophytochromes and cyanobacterichromes, as well as phycocyanobilin-binding cyanobacterial phytochromes, small far-red fluorescent proteins - smURFP and small green oxygen-independent fluorescent proteins, such as those based on eel fluorescent proteins (UnaG). It is also possible to use fluorescence-activating and absorption-shifting tags, which require the addition of a fluorogen to the cells for fluorescence. Any fluorescent protein that enables selection of cells carrying the plasmid construct(s) by cell sorting will do. To reduce the time required to obtain several cell lines with different translocations, translocation-inducing plasmids are obtained based on a universal construct. The universal plasmid construct contains regions that include type IIS restriction enzyme recognition sites for rapid cloning of any sequences responsible for target locus recognition into these sites. In the case of the CRISPR/Cas system, 20 nucleotides of the guide RNA gene encoding the target-recognizing part of the guide RNA are responsible for target locus recognition. Thus, based on one plasmid, a plasmid aimed at inducing one target translocation is obtained in just 2 cloning stages.

Secondly, cells are transfected to deliver elements of the genome editing system to them. Any cells that are kept in the form of a culture and are capable of undergoing more than 30-40 divisions can act as cells. These can be immortalized cells or a tumor cell culture, for example, HeLa, HCT116, LCL, Jurkat, HL60, etc. It is preferable to use cells that are close in type to the cells of the model tumor. Cell transfection is carried out by any of the following methods: chemical transfection, electroporation, transduction using viral particles.

Third, after transfection, the cells expressing the fluorescent protein are sorted using a cell sorter and distributed into wells of a plate with a medium, 20±10 at a time. The medium used is one that ensures the existence, growth, and division of these cells. The number of wells in a plate (or several plates) is calculated based on the expected translocation frequency in the cell population. This value can be determined in preliminary experiments by any method that allows for the calculation of translocations (PCR, sequencing, etc.). To estimate the expected translocation frequency, preliminary experiments are performed by transfecting cells with genetic constructs that induce translocations. After 24-72 hours, an analysis is performed: dilutions are made (e.g., 10,000 cells, 1,000 cells, 100 cells) and PCR analysis is performed for each dilution to detect translocation. The preferred PCR analysis technique is described below. If the proportion of cells with translocations is below 1 per 1000, it is advisable to select other target sites within the same intron of the gene involved in the translocation. For example, in the case of using the CRISPR/Cas system, other guide RNAs are synthesized in order to find more effective ones. More effective methods of cell transfection can also be used - various conditions of electroporation, chemical transfection or viral transduction. You can also focus on literary data: for example, in human hematopoietic cells transfected with Cas9 ribonucleoprotein complexes with guide RNAs, a translocation frequency of approximately 1 per 200 cells is shown (DOI 10.1182/bloodadvances.2019000450). As a result, the number of wells in the plate is selected so that at least several wells contain a cell with a translocation with a probability of at least 0.95. It is also acceptable to distribute not 20 cells per well, but another quantity, however, it is less convenient. The number 20 is due to the fact that in the future the subpopulation obtained on the basis of these 20 cells will need to be cloned again to obtain monoclones - populations grown from single cells. And in order to obtain at least one monoclone with translocation with a probability of more than 0.95, it is necessary to grow and test 60 monoclones. Considering that a population does not always grow from a single cell (some cells die after sorting), take with a reserve - 96 monoclones (distributed among the wells of one 96-well plate).

The presence of translocations in a population is checked by the PCR method, as the simplest and fastest among the methods for detecting translocations. When checking for cells with translocation in subpopulations obtained from 20 cells, then to speed up the check, several wells are combined into groups, in the range of 2-12 wells, and one PCR is performed with mixed material from each group. Then the group of wells in which the translocation was detected is checked in individual wells. This reduces the total number of reactions, since approximately only single wells with translocation are found in one plate.

To reduce the time and material costs, so-called direct PCR is used, when DNA is not isolated, but simply a cell lysate is added as a matrix. DNA isolation is allowed using columns, beads and other methods of DNA isolation and purification from cells, but this is much more time-consuming and requires a large expenditure of kits or reagents for isolation. DNA isolation by such methods requires several hundred thousand cells, while for direct PCR several thousand cells are taken, the cell population reaches this size a week faster.

To increase the sensitivity of PCR (PCR sensitivity is how few matrix molecules are needed for the reaction to occur), a nested PCR approach is used (PMID: 33134100). This approach involves performing PCR in 2 stages: at the first stage, a reaction is carried out with the first pair of primers, at the second stage, PCR is carried out with another pair of primers that lie inside the first pair. Semi-nested PCR is allowed, when one of the primers of the second stage coincides with one of the primers of the first stage.

Fourthly, cells from populations containing translocation are distributed one cell per well to obtain monoclones. A cell sorter is used for this procedure, allowing distribution of one cell per well of the plate. Other cloning methods are allowed, such as the limiting dilution method, but this increases the labor costs of the claimed method.

The obtained monoclones are tested for translocation using direct PCR. To confirm the rearrangement, the PCR product is sequenced using Sanger. To confirm the expression of the fusion gene product, PCR with reverse transcription is performed using primers located on the exons of the chimeric mRNA.

The resulting cell lines are used to study the mechanisms of leukemia development, to identify new markers for patient stratification, and to study the effects of various drugs on cells with this translocation.

Example

As an example of the claimed method, we give an example of obtaining a cell line containing the MLL-ARHGAP26 translocation . This is one of the translocations involving the MLL gene, occurring in hemoblastoses (blood cancer). Immortalized lymphoblasts (LCL line) were used to obtain this line. A genetic construct was created containing elements of the CRISPR/Cas system, introducing breaks into loci in the 11th intron of the MLL gene and in the 11th intron of the ARHGAP26 gene, and containing the Santaka fluorescent protein gene. The px330_gRNA_Cas9 plasmid was used to create the genetic construct (Fig. 1).

1) inserted the SV40 promoter sequence into the CciNI and SbfI sites. At the same time, a restriction enzyme site was added The phU6-gRNA plasmid (#53188 Addgene) and the following primers were used as a template:

2) the Kozak-Santaka-SV40polyA sequence was developed for its insertion into the BmtI and SbfI sites in the resulting plasmid. At the same time, sites for the BstAF I and HindIII restriction enzymes were inserted into the primer on the polyA side. Primer sequences:

The resulting construct was named px330_gRNA_Cas9_Santaka.

3) the gene for the second guide RNA from plasmid 53188-PcisI (it already contains sites not for BstV21, but for PciSI, another type IIS restriction enzyme) was inserted into px330_gRNA_Cas9_Santaka at the sites of HindIII and BstAFI restriction enzymes. To obtain plasmid 53188-PcisI, plasmid phU6-gRNA (#53188 Addgene) and the following oligonucleotides were used, which form an insert with PciSI sites when annealed to each other:

To insert the guide RNA gene into the px330_gRNA_Cas9_Santaka plasmid at the BstAFI and Hind III (W) sites, it was amplified using the plasmid 53188-PcisI and the following primers:

The final construct was called px330_gRNA_Cas9_Santaka_gRNA (Fig. 2) and was used to obtain a plasmid inducing the target rearrangement of MLL-ARHGAP26 . For this purpose, sequences recognized by guide RNAs were selected within the introns of the target genes between which the translocation occurs. The 20-nucleotide fragments recognized by guide RNAs were inserted into the plasmid px330_gRNA_Cas9_Santaka_gRNA into the guide RNA genes in two stages using the restriction enzymes BstV21 and PciSI. For this cloning, single-stranded oligonucleotides were synthesized that, when annealed with each other, yield the desired insert with protruding sticky ends corresponding to the sticky ends of the plasmid treated with BstV21 or PciSI. The correctness of the plasmid assembly was confirmed by Sanger sequencing. The resulting plasmid was named px330_gMLL_Santaka_gARH.

Before obtaining cells with translocation, the detection limit of target translocations was determined. For this, the cells were transfected with a plasmid, sorted for fluorescence after 1-2 days, and 16 identical direct nested PCR reactions were performed from the selected cells with a certain number of cells to assess the proportion of cells with translocation in the culture. Translocation can be expected approximately in 1 cell per several hundred to thousands of cells without translocation. Based on this, 16 reactions were performed with lysates of 100 cells as a matrix. Having assessed the detectable proportion of cells with translocations, a protocol was developed that allows obtaining monoclones of cells with translocation with the smallest number of sortings and analytical PCRs.

To obtain monoclonal cultures with the MLL-ARHGAP26 translocation, the following steps were performed (scheme in Fig. 3). The initial cell culture was transfected with the px330_gMLL_Santaka_gARH plasmid, which induces the target translocation. After 2 days, the cells were sorted by the Santaka transfection marker, 20 cells per well of a 96-well plate. When populations of approximately 20,000 cells grew from 20 cells in the wells, wells containing cells with the translocation were identified among the resulting populations using direct nested PCR. For direct PCR, cell lysates were obtained as follows: up to 10,000 cells were pelleted in PCR tubes by centrifugation at 1000 g for 2 min. The supernatant was removed and 10 μl of the lysis mixture containing 0.2 mg/ml proteinase K and 0.2 mM of each dNTP in 1x PCR buffer were added. The cells were lysed for 15 min at 65°C, then the proteinase was inactivated at 95°C for 5 min. Then 10 μl of a mixture of HS TaqPol polymerase (Eurogen, Russia), primers (synthesized on request by Evrogen, Russia) and dNTPs (Eurogen, Russia) in PCR buffer (from the HS TaqPol kit, Evrogen, Russia) were added so that the final concentrations corresponded to those recommended by the polymerase manufacturer, and semi-nested PCR was performed. Samples from 6 wells were combined into one reaction.

At the first stage the following primers were used:

The program used was: 95°C 5 min, (95°C 20 sec, 58°C 1 min, 72°C 1 min) × 20, 72°C 5 min. After PCR, 4 μl of the reaction mixture was added to 396 μl of deionized water. 4 μl of the resulting solution was used as a template for the second round of PCR. The following primers were used in the second stage of PCR:

The program used was: 95°C 5 min, (95°C 20 sec, 58°C 1 min, 72°C 40 sec) × 30, 72°C 5 min. The results were analyzed by electrophoresis in agarose gel (Fig. 4. - electrophoresis of PCR products).

If the material from a group of wells gave a positive PCR result, then the wells of this group were then analyzed separately using the same PCR.

Subpopulations of cells from positive wells (with rearrangement) were then cloned one cell per well of the plate using a cell sorter. Clones from single cells were grown to approximately 20 thousand cells and these monoclonal lines were analyzed using direct PCR in one step using primers MLLi11_F and ARH_R_in, according to the amplification program: 95°C 5 min, (95°C 20 sec, 58°C 20 sec min, 72°C 40 sec) × 35, 72°C 5 min. The PCR product was sequenced using Sanger for additional confirmation of rearrangement.

Clones with the rearrangement were analyzed for expression of the fusion gene. For this purpose, RNA was isolated from the cells, reverse transcription was performed, and PCR was performed with the resulting cDNA. The following primers were used:

PCR was performed using SYBR Green according to the protocol: 95°C 2 min, (95°C 15 sec, 60°C 20 sec, 72°C 30 sec) × 35, 72°C 5 min. The results were analyzed by electrophoresis in agarose gel. The result of the fusion gene expression test is shown in Fig. 5. The product was also sequenced by Sanger to confirm the result, and karyotyping of the cells was also performed to confirm the chromosomal translocation.

Thus, in 8 weeks, a cell model for studying leukemia with MLL-ARHGAP26 rearrangement at the cellular level was obtained from transplanted (immortalized) cells.

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<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..31</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q2">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aataagcggccgcctgaggcggaaagaacca</INSDSeq_sequence

>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>45</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..45</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q4">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aataacctgcaggatatgctagcctgtctcttgatcgatctttgc</IN

SDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="3">

<INSDSeq>

<INSDSeq_length>36</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..36</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q6">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aataagctagcgccaccatggtgtcgaagggagaag</INSDSeq_seq

uence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="4">

<INSDSeq>

<INSDSeq_length>48</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..48</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q8">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aataacctgcaggaagcttaataacttaagatacattgatgagtttgg<

/INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="5">

<INSDSeq>

<INSDSeq_length>25</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..25</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q10">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>caccggaagagcaataagctcttca</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="6">

<INSDSeq>

<INSDSeq_length>25</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..25</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q12">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aaactgaagagcttattgctcttcc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="7">

<INSDSeq>

<INSDSeq_length>26</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..26</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q14">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aataacttaaggtcgggcaggaagag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="8">

<INSDSeq>

<INSDSeq_length>29</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..29</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q16">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aatttaagcttctcagtacgctcagcgga</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="9">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q18">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctgcactcctaaagcatgacca</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="10">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q20">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>catccaccaggtgacaacga</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="11">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q22">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctgcactcctaaagcatgacca</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="12">

<INSDSeq>

<INSDSeq_length>21</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q24">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gaggcatctcaagactaggca</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="13">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q26">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ccacctccggtcaataagca</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="14">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q28">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ttcgatacagcccttgctcg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (21)

1. A method for creating a cell model of leukemia for modeling and studying the processes occurring in leukemia cells, including taking a sample/cells from a patient, identifying target loci that are associated with the pathology under study, then synthesizing at least one plasmid construct encoding genome editing tools, RNA guides directed to the target loci and including a fluorescent protein, transfecting the cells with the resulting plasmid or plasmids, after transfection, cells expressing the fluorescent protein are selected using a cell sorter and distributed in 20±10 pieces into the wells of a plate with a medium, increasing the cell subpopulation to 20,000±10,000 cells, then analyzing the resulting subpopulations for the presence of cells with translocation using direct nested PCR and cloning subpopulations with confirmed translocation into monoclonal lines, among which lines with translocation, confirm the expression of the fusion gene; in this case, to analyze subpopulations, the content of cells with translocation is first assessed in a group of wells and one PCR is performed with mixed material; if translocation is detected, the content in individual wells is checked. 2. The method according to item 1, characterized in that cells from several wells are combined into groups of 2 to 12 wells; in the absence of translocation in a group, cells from all wells of such a group are excluded from further work. 3. The method according to item 1, characterized in that a subpopulation of cells with a confirmed rearrangement is cloned using a cell sorter and monoclones are grown to 10,000-100,000 cells. 4. The method according to claim 1, characterized in that nucleases that specifically recognize target loci are used as genome editing tools. 5. The method according to item 4, characterized in that the editing tools used are systems based on CRISPR/Cas, zinc finger nucleases, TALE nucleases, endonucleases for inserting mobile genetic elements into the genome, as well as other nucleases that have the property of recognizing and cutting target loci. 6. The method according to claim 1, characterized in that the green fluorescent protein from Aequorea victoria and its homologues, biliverdin-binding bacteriophytochromes and cyanobacterichromes, phycocyanobilin-binding cyanobacterial phytochromes, small far-red fluorescent proteins, small green oxygen-independent fluorescent proteins, as well as fluorescence-activated labels are used as the fluorescent protein. 7. The method according to claim 1, characterized in that the plasmid construct contains an origin and a gene of resistance to a selective antibiotic and encodes genome editing tools, includes a region containing a type IIS restriction enzyme recognition site, into which a sequence responsible for recognition of the target locus by the nuclease is inserted, and also encodes genes of elements of the genome editing system and a fluorescent protein gene. 8. The method according to item 1, characterized in that the cells used are those capable of undergoing more than 30-40 divisions. 9. The method according to item 6, characterized in that immortalized cells or a culture of tumor cells are used as cells. 10. The method according to item 1, characterized in that the transfection of cells is carried out using chemical transfection, electroporation, transduction using viral particles. 11. The method according to item 1, characterized in that the assessment of cells with translocations is carried out using the PCR method. 12. The method according to item 1, characterized in that in order to obtain monoclones, cells from populations containing a translocation are distributed, one cell per well. 13. The method according to claim 1, characterized in that for studying leukemogenesis, a cell line is used that is modified immortalized lymphoblasts of the LCL line containing the MLL-ARHGAP26 translocation. 14. The method according to claim 13, characterized in that immortalized lymphoblasts (LCL line) are used to obtain the cell model, the rearrangement loci within the MLL-ARHGAP26 genes are determined and the genetic construct px330_gRNA_Cas9 Santaka and plasmid 53188-PcisI are synthesized, the gene for the second guide RNA from plasmid 53188-PcisI is inserted into px330_gRNA_Cas9_Santaka, at the sites of restriction enzymes HindIII and BstAFI, the recognition region of one guide RNA to MLL is introduced into the obtained plasmid px330_gRNA_Cas9_Santaka_gRNA using restriction enzyme BstV21, and the recognition region of another guide RNA to ARHGAP26 is introduced using restriction enzyme PcisI, obtained by plasmid px330_gMLL_Santaka_gARH transfect LCL cells, after 2 days sort cells by Santaka transfection marker, expand cell subpopulation, assess content of cells with translocation using direct nested PCR, clone subpopulation of cells with confirmed rearrangement using cell sorter and expand monoclones; then determine presence of translocation using direct nested PCR and use monoclone of cells with translocation for further studies. 15. The method according to claim 14, characterized in that the px330_gRNA_Cas9 Santaka construct includes elements of the CRISPR/Cas system that introduce breaks into the loci in the 11th intron of the MLL gene and in the 11th intron of the ARHGAP26 gene, and contains the Santaka fluorescent protein gene, the SV40 promoter sequence, and the BmtI restriction enzyme site. 16. The method according to item 15, characterized in that commercial plasmids px330, phU6-gRNA and a set of primers with sequences SEQ ID No. 1 - SEQ ID No. 4 are used to synthesize the genetic construct px330_gRNA_Cas9 Santaka. 17. The method according to claim 14, characterized in that for the synthesis of the genetic construct of plasmid 53188-PcisI, the plasmid phU6-gRNA is used with oligonucleotides that, when annealed to each other, form an insert with PciSI sites and a set of primers with the sequences SEQ ID No. 5 - SEQ ID No. 8. 18. The method according to item 14, characterized in that primers with sequences SEQ ID No. 9 - SEQ ID No. 12 are used to check for the presence of translocation using the nested PCR method. 19. The method according to item 14, characterized in that the verification of the presence of translocation is carried out using the nested PCR method in two stages. 20. The method according to claim 19, characterized in that at the first stage of PCR, amplification is carried out using the following temperature conditions: 95°C 5 min, (95°C 20 sec, 58°C 1 min, 72°C 1 min) × 20, 72°C 5 min, after completing the PCR, 4 μl of the reaction mixture is added to 396 μl of deionized water, then 4 μl of the resulting solution is used as a matrix at the second stage of PCR. 21. The method according to item 19, characterized in that at the second stage of PCR, amplification is carried out using the following temperature conditions: 95°C 5 min, (95°C 20 sec, 58°C 1 min, 72°C 40 sec) × 30, 72°C 5 min, the results are analyzed by electrophoresis in agarose gel.