CN114686522A

CN114686522A - Efficient organoid editing method

Info

Publication number: CN114686522A
Application number: CN202111669152.7A
Authority: CN
Inventors: 金鸣; 袁鹏飞; 刘娜; 申红艳; 苏美华; 李玉兰; 郑雅茹
Original assignee: Edigene Biotechnology Inc
Current assignee: Beijing Jiyin Medical Technology Co ltd
Priority date: 2020-12-31
Filing date: 2021-12-31
Publication date: 2022-07-01

Abstract

The present application relates to an optimized gene editing organoid method comprising: mixing the lentivirus packaged with the Cas9 protein coding sequence and the gRNA coding sequence with organoids to form a mixture; and co-culturing the lentivirus with the organoid mixture. By the method, the efficiency of the CRISPR system in editing organoids can be improved. The application also relates to organoids edited by the optimized gene editing organoid method, organoid high-throughput screening libraries constructed by the method, and uses of organoids edited by the method or organoid high-throughput screening libraries constructed.

Description

Efficient organoid editing method

Technical Field

The application relates to a gene editing technology, in particular to a technology for editing a gene of an organoid by using a lentivirus infection method.

Background

Organoids are three-dimensional cell cultures cultured in vitro, mainly from tissues, embryonic stem cells or induced pluripotent stem cells, which can be differentiated into a plurality of organoid-specific cell types due to their self-renewal and differentiation ability, possess spatial tissues similar to the corresponding organs and can reproduce partial functions of the corresponding organs, thereby providing a highly physiologically relevant system. For organoid research, most of the previous researches are related researches by establishing models on PDX mice, the method is time-consuming and high in cost, and since 3D models appear, more and more people select the 3D models to research diseases and treatments, so that the time cost can be saved. At present, there are two methods for organoid editing, one is electrotransformation, the number of organoids is limited during electrotransformation, and only small volume electrotransfection is needed, that is, when electrotransfection is performed, the total volume of organoids subjected to electrotransfection simultaneously is small. This is not satisfactory for later applications such as library construction; secondly, for larger plasmids, the single electrotransformation efficiency is lower, and if the two-step electrotransformation is adopted, the organoid survival rate is reduced; the other is lentivirus infection, the method is not limited by the initial amount of total cells of organoids transfected simultaneously when the organoids are transfected by lentiviruses of the organoids, but the problem of low infection efficiency and editing efficiency when the organoids are infected by the lentiviruses in the prior art can occur; the organoid sources are basically normal tissues or tumor tissues, and few primary tumor cells are isolated from PDX mouse tumors to obtain organoids for editing.

Disclosure of Invention

In order to solve the problems that in the prior art, organoids derived from primary tumor cells are difficult to culture, and the infection efficiency and the editing efficiency are low, the application provides a method for gene editing of organoids, which comprises the following steps: cas9 enzyme and gRNA were simultaneously introduced into organoids and expressed by lentiviral transfection.

1. A method of gene editing an organoid, comprising:

mixing the lentivirus packaged with the Cas9 protein coding sequence and the gRNA coding sequence with organoids to form a mixture;

co-culturing the lentivirus with the organoid mixture.

2. The method of item 1, wherein the organoid is cultured from primary tumor cells isolated from a human tumor xenograft model (PDX) animal tumor,

wherein, matrigel is used in the process of obtaining organoid by culturing primary tumor cells.

3. The method of item 2, wherein prior to mixing the lentivirus with the organoids, the organoids are treated with mild proteolytic enzyme, preferably Tryple, to separate from the matrigel and break into small organoids of uniform size. In some embodiments, the time for the mild protein digesting enzyme treatment is from 3 to 15 minutes. In some embodiments, the time for the mild protein digesting enzyme treatment is 10 minutes.

4. The method according to any one of items 1-3, wherein a staining assisting agent is further added to the mixture when the lentivirus is mixed with the organoid, preferably the staining assisting agent is TransDux Max transfection agent or a transfection agent identical to the active ingredient thereof.

5. The method of any one of items 1-4, wherein the Cas9 protein is a spCas9 protein.

6. The method according to any one of items 1-5, wherein the Cas9 protein coding sequence and the gRNA coding sequence are packaged into a lentivirus by the same lentivirus expression vector.

7. The method of item 6, wherein the lentiviral expression vector further comprises a selectable marker gene, preferably the selectable marker is a fluorescent protein.

8. The method of clause 6 or 7, wherein the lentiviral expression vector is greater than 9KB, e.g., 9.5KB, 10KB, 11-30 KB, 12-25 KB, 13-20 KB, 14KB, etc.

9. The method of any one of items 1-8, wherein the co-culturing comprises:

a centrifugal incubation step and a matrigel culture step,

wherein the centrifugation incubation step is to centrifuge the mixture, and matrigel is not added in the centrifugation incubation step;

the matrigel culturing step is to mix the organoids in the mixture after centrifugation with matrigel and culture in a culture medium.

10. The method according to item 9, wherein the centrifugation is carried out at 100 to 800g for 0.5 to 3 hours; preferably 500-800 g, 1-2 h; most preferably 600g, 1 h.

11. The method of clause 9 or 10, wherein the co-culturing further comprises:

a standing incubation step between the centrifugation incubation step and the matrigel incubation step,

the step of standing incubation is to perform standing incubation on the mixture for 1-6 hours, such as 3-6 hours, 4.5-5 hours and the like; preferably 4 h.

12. The method of any one of claims 9-11, wherein the centrifugation incubation step is performed at ambient temperature.

13. A method of transfecting an organoid with a lentivirus, comprising:

mixing lentivirus with organoids to form a mixture;

co-culturing the lentivirus with the organoid mixture.

In some embodiments, the organoids are cultured from primary tumor cells isolated from a human tumor xenograft model (PDX) animal tumor.

In some embodiments, the organoids are small organoids formed by protease digestion of a larger organoid.

In some embodiments, the time for the mild protein digesting enzyme treatment is from 3 to 15 minutes. In some embodiments, the time for the mild protein digesting enzyme treatment is 10 minutes.

14. The method of item 13, wherein in mixing the lentivirus with the organoid, a staining assisting agent is also added to the mixture, preferably the staining assisting agent is TransDux Max transfection agent or a transfection agent identical to the active ingredient thereof.

15. The method according to item 13 or 14, wherein the co-culturing comprises:

a centrifugal incubation step and a matrigel culture step,

16. The method as set forth in item 15, wherein the centrifugation is carried out under 100 to 800g for 0.5 to 3 hours; preferably 500-800 g, 1-2 h; most preferably 600g, 1 h.

17. The method of clause 15 or 16, wherein the co-culturing further comprises:

the standing incubation step is to perform standing incubation on the mixture for 1-6 hours; preferably 3-6 h; most preferably 4 h.

In some embodiments, the centrifugation incubation step is performed at ambient temperature.

18. An organoid edited using the method of any one of items 1 to 12. In some embodiments, the organoid is an organoid edited by the method of any of claims 1 to 12 for a period of 7 to 10 days, for example, an organoid edited by the method of any of claims 1 to 12 for a period of 8 days or 9 days, or an organoid edited by the method of any of claims 1 to 12 for a period of 11, 12, 13, 14, 15, 16, 17, 18, or 19 days.

19. An organoid gene library consisting of organoids described in item 18.

20. Use of the organoid as described in item 18 for disease mechanism and therapeutic studies.

21. A method of high throughput screening using the organoid gene library as described in item 19, said high throughput screening being selected from the group consisting of: functional gene screening, drug target screening, drug sensitive gene screening, and drug resistant gene screening.

In the above embodiment, it is preferred that the primary tumor cell is a primary intestinal cancer cell.

According to the technical scheme, on one hand, the problem of obtaining a large number of organoid sources is solved, on the other hand, the organoid editing efficiency is improved, the screening process is simplified, and meanwhile, the problem of low organoid infection efficiency caused by large plasmids is solved. By using the technical scheme of the application, the accurately edited organoids can be obtained in a large quantity more easily, and further support is provided for organoid-based gene function research, drug screening, research and development.

Drawings

FIG. 1 is an organoid derived from primary tumor cells;

FIG. 2 is organoids after passage;

FIG. 3 white light (left panel) and green fluorescence pictures (right panel) of organoids 3 days after infection;

FIG. 4 is a white light (left) and green fluorescence image (right) of day 10 of one-step infestation of organoid sample 1;

FIG. 5 is a white light (left panel) and fluorescence image (right panel) of day 10 of one-step invasion organoid sample 2;

fig. 6 is a 20-day infected organoid in two steps for displaying editing efficiency, where 6A is a white light picture, 6B is a GFP fluorescence picture, and 6C is a mCherry fluorescence picture;

FIG. 7 white (left) and fluorescence (right) pictures 48 hours after organoid electrotransformation;

FIG. 8pCRISPR-GFP plasmid map.

Detailed Description

Cancer is a health problem worldwide and we need more innovative targeted therapies. In the development of new drugs and therapies, the tissue complexity and genetic heterogeneity of tumors can be well replicated in good tumor models, which often affects the final success or failure of development. Compared with a plurality of preclinical models, the organoids have good potential in success rate, maintenance difficulty and screening difficulty. The gene editing technology is helpful to generate the organoid with the objective mutation in a targeted manner, and provides a more flexible and abundant organoid model. However, in the prior art, the gene editing efficiency of the organoid is low, the editing process takes a long time, and it is difficult to enlarge the editing scale.

To solve the above technical problems, the present application provides an improved method for gene editing organoids; a method of transfecting an organoid; organoids edited by the method of gene editing and methods of using the method to construct organoid libraries, organoid gene libraries constructed by the method of constructing organoid libraries; and the use of the gene-edited organoids and the organoid library.

Definition of

As used herein, the term "primary intestinal cancer cells" refers to intestinal tumor cells isolated from malignant tissue of the intestine of patients with intestinal cancer or mouse tumor of PDX tumor model, and cells obtained by subjecting the intestinal cancer cells to amplification culture in vitro without any modification.

As used herein, the term "primary tumor cells" refers to tumor cells isolated from a tumor of a tumor patient or a tumor of a PDX tumor model animal, as well as cells obtained by subjecting said tumor cells to expansion culture in vitro without any modification. The primary tumor cells described herein include, for example, primary intestinal cancer cells, primary melanoma cells, primary thyroid cancer cells, primary renal cancer cells, primary skin cancer cells, primary bone cancer cells.

As used herein, the term "organoid" refers to a class of microscopic three-dimensional structures capable of self-assembly formed by stem cells, which may be pluripotent stem cells or adult stem cells, in vitro culture. Unless otherwise indicated in this application, it is used interchangeably with "tumor organoids", i.e., cell masses cultured from tumor primary tumor cells that have a three-dimensional structure and a tumor microenvironment.

As used herein, the term "PDX animal model" refers to a tumor model constructed by transplanting tumor tissue from a tumor patient into a critically deficient animal and allowing the tumor tissue to grow in the critically deficient animal. In some embodiments, the severely immunodeficient animal comprises a severely immunodeficient rat, mouse, or the like. In some embodiments, the severe immunodeficient animal includes an FPG rat, F334RG rat, Rag2 rat, SCID rat, Nude mouse, Beige mouse, Xid mouse, SCID mouse, NOD/SCID mouse, BNX mouse, and the like. When the critically-immunodeficient animal is a critically-immunodeficient mouse, then the "PDX animal model" is a "PDX mouse model". "PDX mouse tumor" refers to a tumor formed by transplantation of patient tumor tissue "in a PDX mouse model.

As used herein, the term "staining assisting agent" refers to an agent that assists a lentivirus in infecting a cell, increases the contact of the virus with the cell, and maximizes viral infection of the cell, and includes Polybrene (Polybrene), TransDux, and like agents, preferably Polybrene, TransDux, and more preferably TransDux. It will be appreciated by those skilled in the art that other transfection reagents having the same effective components as the TransDux et al transfection reagent may be used in the protocol of the present application to perform the same function.

As used herein, the term "matrigel" is a basement membrane matrix that polymerizes during cell or organoid culture to form a bioactive three-dimensional matrix that mimics the structure, composition, physical characteristics and function of the basement membrane of cells in vivo. Matrigel is mainly composed of polysaccharides and proteins, or proteoglycans, which form a complex network structure, support and connect tissue structures, regulate tissue generation and physiological activities of cells. In some embodiments, the matrigel may be extracted from animal tissue. In some embodiments, the matrigel is extracted from an EHS mouse tumor that is enriched for extracellular matrix proteins.

As used herein, the term "spCas 9" refers to Cas9 protein derived from streptococcus pyogenes. Such as Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F.Multiplex Genome Engineering using CRISPR/Cas systems.science.2013.15; 339(6121) 819-23. published documents. As described in the publications.

As used herein, the term "lentiviral expression vector" is an HIV genome-based viral vector that contains the genetic information required for packaging, transfection, stable integration, and can carry foreign nucleic acid sequences. When the lentiviral expression vector is transfected into cells with a lentiviral packaging plasmid containing all of the helper proteins necessary to provide all of the transcription and packaging of the RNA into the recombinant pseudoviral vector, a packaged lentivirus is obtained. Herein, when the exogenous nucleic acid sequence carried by the lentiviral expression vector comprises a Cas9 protein coding sequence and a sgRNA coding sequence, then the packaged lentivirus is a lentivirus packaged with a Cas9 protein coding sequence and a sgRNA coding sequence. When a cell is infected with the lentivirus, the lentivirus can insert the exogenous nucleic acid sequence into the genome of the cell. Herein, an exemplary lentiviral expression vector is provided: pCRISPR-GFP, the main structure of which is shown in FIG. 8. It will be appreciated by those skilled in the art that the replacement of GFP in the present plasmid with other fluorescent protein genes may still function in the same manner as described in the present application.

As used herein, a "mild protease" is a protease used for digesting cell adhesion factors, extracellular matrix, etc. to detach cells from cell walls or cells from each other, and is called a "mild protease" because it does not require serum to stop digestion, and digestion is weaker than pancreatin. Exemplary mild proteases include Tryple, and the like.

As used herein, the "non-adherent state" refers to a state in which cells are not bound to the bottom or wall of a culture vessel by an adhesion factor, extracellular matrix, or matrigel. In the present application, the state of the cells when the virus is added is the "non-adherent state". The cells in the non-adherent state are not required to be digested by pancreatin, a dissociation agent, shaking and/or the like during passage or taking.

As used herein, the term "stationary incubation" is mixing lentivirus with primary tumor cells or organoids and placing the mixture in a liquid medium (preferably a liquid medium suitable for growth of the primary tumor cells or organoids) without centrifugation or shaking of the container containing the lentivirus and primary tumor cells or organoids. During the "resting incubation" process, the primary tumor cells are in a non-adherent state, and the organoids are not in contact with matrigel.

In this context, the term "centrifugation incubation" step is the centrifugation of a mixture of lentivirus and primary tumor cells or organoids for a certain period of time. In the step of centrifugal incubation, neither feeder cells nor matrigel are added, the primary tumor cells are in a non-adherent state, and the organoids are not in contact with matrigel. As used herein, a "liquid medium" may be cell culture medium, PBS, or other cytosolic isotonic solution that maintains cells in a stable viable state for a period of time. In the present application, the "centrifugal incubation" is performed at normal temperature unless otherwise specified. The normal temperature in this application refers to the room temperature which is not usually controlled, and does not include the temperature which is extremely cold or extremely hot. It will be understood by those skilled in the art that room temperature as described herein is a wide range including 5 to 35 c, typically around 25 c, e.g., 10 c, 15 c, 20 c, 21 c, 22 c, 23 c, 24 c, 26 c, 27 c, 28 c, 29 c, 30 c, etc.

In this context, the term "adherent culture" is the culture of primary tumor cells in a medium comprising feeder cells in adherent culture. Starting from the addition of the primary tumor cells into the culture medium containing feeder layer cells cultured in an adherent manner, the primary tumor cells are gradually combined with the bottom or the wall of the culture container through adhesion factors, extracellular matrix and the like, namely, the cells adhere to the wall. Herein, all "adherent cultures" are static cultures, i.e. without centrifugation or shaking of the culture vessel holding the primary tumor cells, placed in an environment suitable for growth of the primary tumor cells. The term "matrigel culture" refers to co-culture of organoids in organoid medium after mixing the organoids with matrigel, which is a static culture.

As used herein, the term "cellular gene library" refers to a mixture of cells comprising different genotypes. In some embodiments, a gene library of cells can be made by batch knockout or knock-in of a population of cells. For example, a set of mixed lentiviral solutions comprising a Cas9 protein or protein coding sequence and sequences encoding sgrnas for different target genes, respectively, is introduced into primary tumor cells and expressed, thereby editing different genes in different cells to form a primary tumor cell library comprising different mutated genes. As used herein, the term "organoid gene library" refers to a mixture of organoids comprising cells of different genotypes.

As used herein, the term "Cas 9", "Cas 9 protein" or "Cas 9" enzyme refers to one of the endonucleases used in CRISPR (Clustered regularly interspaced short palindromic repeats) gene editing systems to make double strand breaks at specific positions of a target nucleic acid sequence. The CRISPR gene editing system further comprises a short RNA, referred to as gRNA. The gRNA comprises a sequence that is complementary to a target nucleic acid sequence and when it complementarily hybridizes to the target nucleic acid sequence can recruit Cas9 to a specific location of the target nucleic acid sequence. The term "spCas 9" refers to Cas9 protein derived from streptococcus pyogenes, such as Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang f.multiple Genome Engineering CRISPR/Cas systems.science.2013.15; 339(6121) 819-23 publication.

As used herein, the term "selectable marker gene" is a gene of known function and sequence by which a cell, virus or organism containing the "selectable marker gene" can be distinguished from a cell, virus or organism that does not contain the "selectable marker gene". Common "selectable marker genes" include luciferase genes, resistance genes, and the like. The substance capable of regulating physiological functions, such as RNA or protein transcribed or expressed by the "selection marker gene", is referred to as a "selection marker".

As used herein, the "high throughput screening" is the process of enriching or reducing the proportion of certain genotype cells in a cell mixture containing different genotypes by applying an external pressure to the cell mixture, and determining and analyzing the copy number of the different genotypes in the cell mixture after the external pressure is applied by sequencing or second generation sequencing ("NGS") methods, etc., to identify the functions of coding genes, non-coding RNAs and regulatory elements, thereby achieving the purpose of screening and analyzing a large number of genotypes simultaneously. The high throughput screening comprises a high throughput functional screening as described in, for example, CN 106637421A.

As used herein, the term "about" refers to the usual error range for individual values as would be readily known to one skilled in the art. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter itself. As used herein, the term "about" when preceding a numerical value means within 10% of the upper or lower numerical value. For example, "about 100" encompasses 90 and 110.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Method for gene editing organoids

At present, there are two organ-like editing methods, one is electrotransformation, the other is lentivirus infection, and through a series of pre-experiments, based on the higher efficiency of lentivirus transfection and the smaller damage to the organ-like, and the problem of limited initial organ quantity by the electrotransformation method is solved, so the method mainly adopts the lentivirus infection method. In addition, the inventor compares a method for simultaneously introducing the Cas9 protein and the sgRNA into organoids by using lentivirus in one step with a method for separately introducing the Cas9 protein and the sgRNA into organoids by a two-step method, and shows a remarkable advantage of the one-step method. The efficiency is higher, the flow is shorter, the damage to the organoid is smaller, the normal function of the organoid is kept to the maximum extent, and the subsequent experiment is not influenced. Accordingly, the present application provides a method of gene editing an organoid, comprising: mixing the lentivirus packaged with the Cas9 protein coding sequence and the gRNA coding sequence with organoids to form a mixture; co-culturing the lentivirus with the organoid mixture. In some embodiments, more than 1 gRNA is introduced and expressed simultaneously with the Cas9 protein coding sequence in organoids. In some embodiments, more than one gRNA is introduced into and expressed in organoids simultaneously with the Cas9 protein coding sequence and targets different gene sequences of interest, respectively. In some embodiments, the method results in one or more genetic modifications in the organoid genome. In some embodiments, the method results in organoid formation of a chimera.

In some embodiments, the organoids are cultured from stem cells isolated from tissue that have been differentiated by implantation into a three-dimensional framework or primary tumor cells isolated from PDX animal model tumors. Because the primary tumor cells separated by the PDX animal model are used for organoid culture, other factors are not needed to be added for assistance in the culture process, organoid growth is not influenced, and drug screening can be carried out on organoids, so that the effect of drugs on organoids is not influenced by the addition of auxiliary factors. Thus, in a preferred embodiment, the organoids are cultured from primary tumor cells isolated from PDX animal model tumors. In some embodiments, the PDX animal model is established on a rat model comprising severe immunodeficiency or a mouse model comprising severe immunodeficiency. In some embodiments the PDX animal model is established on an animal model selected from the group consisting of FPG rats, F334RG rats, Rag2 rats, SCID rats, Nude mice, Beige mice, Xid mice, SCID mice, NOD/SCID mice, BNX mice. In some embodiments, the organoids are cultured from primary tumor cells isolated from PDX mouse tumors. In some embodiments, the step of culturing primary tumor cells isolated from the PDX mouse tumor as the organoid comprises: the matrigel was mixed with primary tumor cells isolated from PDX mouse tumors and co-cultured. Wherein, in some embodiments, prior to mixing the lentivirus with the organoids, the organoids are treated with mild proteolytic enzyme, preferably Tryple, to separate from the matrigel and break into small organoids of uniform size. In some embodiments, when the primary tumor cells form a 3D structure, wherein the central lumen is surrounded by a monolayer of epithelium and cryptic regions, a mild solution of proteolytic enzymes is added, and the organoids are blasted and digested into uniformly sized organoids. In some particular embodiments, the mild proteolytic enzyme is TrypLE, which treats the organoids for about 3 to 15 minutes. In some embodiments, the treatment of the organoid with TrypLE is from 3 to 15 minutes. In some embodiments, the treatment of the organoid with TrypLE is between 4 and 14 minutes. In some embodiments, the time for TrypLE treatment of the organoid is 5 to 13 minutes. In some embodiments, the treatment of the organoid with TrypLE is between 6 and 12 minutes. In some embodiments, the treatment of the organoid with TrypLE is between 7 and 11 minutes. In some embodiments, the treatment of the organoid by TrypLE is for about 10 minutes, for example about 8 to 9 minutes.

In this context, TU refers to a shorthand for transduction units, some of which are capable of integrating into host chromosomes, such as lentiviruses (lentiviruses), so TU (transduction units) is used to indicate Lentivirus titers. Titer unit: TU/ml, refers to the number of biologically active viral particles contained per ml. TU is the "transduction unit" which represents the number of viral genomes that can infect and enter the target cell.

Typically, lentivirus TU can be determined by dilution counting or quantitative PCR.

In the present application, for example, a gradient dilution method can be used to determine the TU of lentiviruses. The virus titer is detected by using a gradient dilution method, and the steps are as follows: (1) inoculating 5000 cells of 2000-₂Culturing in a cell culture box; (2) after cell inoculation overnight, the cell culture medium containing concentrated virus stock solution with different volume is added according to the method of gradient dilution, and the mixture is placed at 37 ℃ and 5% CO₂Culturing in a cell culture box; (3) after the virus is infected for 24 hours, the cell culture medium is replaced and is placed in a 5% CO2 cell culture box for culture at 37 ℃; (4) after the virus is infected for 48h, cell stock solution is discarded, washed by PBS buffer solution, and then placed at 37 ℃ and 5% CO₂Carrying out static culture in a cell incubator; (5) after standing culture for 48-72 h, GFP fluorescence can be observed in cells successfully infected by the virus; (6) the fluorescence intensity of GFP is detected by a flow type or fluorescence spectrophotometer, the virus infection efficiency is evaluated by calculating the fluorescence proportion occupied by infected cells, and the virus titer is calculated according to the following formula I.

In some embodiments, the PDX mouse tumor isolated primary tumor cells are cultured in matrigel, wherein the PDX mouse tumor isolated primary tumor cells have a density of: 5E + 03/hole, preferably 1E + 04-5E + 04/hole, more preferably 1E + 05/hole.

The Cas9 protein is spCas 9. In some embodiments, the Cas9 protein coding sequence and the gRNA coding sequence are packaged into a lentivirus by the same lentivirus expression vector. In some embodiments, the Cas9 protein coding sequence and the gRNA coding sequence are located in different lentiviruses packaged by different lentiviral expression vectors. In some embodiments, the Cas9 protein coding sequence is packaged with a portion of the gRNA coding sequence into the same lentivirus by the same lentiviral expression vector and another portion of the gRNA coding sequence is packaged into another lentivirus by another lentiviral expression vector. In some embodiments, the lentiviral vector is selected from the group consisting of: pV2, pCMV, px458, and the like. In some embodiments, the lentiviral expression vector further comprises a selectable marker gene, preferably the selectable marker is a fluorescent protein. In some embodiments, the fluorescent protein is GFP. As can be seen from the examples, using the technical solution of the present application, the transfection efficiency of larger plasmids, such as pCRISPR-GFP plasmid exceeding 9KB in FIG. 8, can be improved. Thus, in some embodiments, the lentiviral vector is greater than 9KB, e.g., 9.5KB, 10KB, 11-30 KB, 12-25 KB, 13-20 KB, 14KB, and the like. Upon purification, lentiviruses can infect organoids in a better quantitative and more efficient manner.

In some embodiments, when the Cas9 enzyme and the gRNA are simultaneously introduced into an organoid and expressed by lentivirus transfection, the expression is driven by a promoter carried on the lentivirus. In some embodiments, the Cas9 protein coding sequence and the gRNA coding sequence are preceded by a foreign promoter that will initiate expression of the Cas9 protein and gRNA upon integration of the coding sequences into the host genome. In some particular embodiments, the exogenous promoter is selected from one of eukaryotic promoters. In some particular embodiments, the exogenous promoter is derived from a mammalian promoter. In some embodiments, the exogenous promoter is selected from the group consisting of: CMV, EF1a, beta actin gene promoter, SV40, PGK1, Ubc, CAG, TRE, UAS, U6. In some embodiments, the expression is performed by lentivirus site-directed integration of the Cas9 protein coding sequence and the gRNA coding sequence into the host cell genome, with the use of promoters naturally present in the host cell. . In some embodiments, the Cas9 protein and the gRNA are located in the same lentiviral vector. In some embodiments, the Cas9 protein is an spCas9 protein.

In some embodiments, a staining assisting agent is also added to the mixture when the lentivirus is mixed with the organoid, preferably the staining assisting agent is a TransDux Max transfection agent or a transfection agent identical to the active ingredient thereof.

In some embodiments, co-culturing the lentivirus with the organoid comprises: a centrifugal incubation step and a matrigel culture step. Wherein the centrifugation incubation step is to centrifuge the mixture, and matrigel is not added in the centrifugation incubation step; the matrigel culturing step is to mix the organoids in the mixture after centrifugation with matrigel and culture in a culture medium. In some embodiments, the centrifugation conditions are 100 to 800g, 0.5 to 3 hours; preferably 500-800 g, 1-2 h; most preferably 600g, 1 h.

In some embodiments, the co-culturing further comprises a standing incubation step between the centrifugation incubation step and the matrigel incubation step, the standing incubation step being a standing incubation of the mixture for 1-6 h; preferably 3-6 h; most preferably 4 h. In some embodiments, the centrifugation incubation, resting incubation and matrigel are performed at a temperature, humidity and carbon dioxide concentration that facilitates growth of the organoid. In some embodiments, the temperature that facilitates growth of the organoid is 37 ℃.

Method for constructing gene editing organoids

The present application also provides a method of gene editing an organoid, comprising: firstly, adding lentivirus containing a Cas9 protein coding sequence and an sgRNA coding sequence into a liquid matrix containing primary tumor cells; then co-culturing the lentivirus with the primary tumor cells; then screening the edited primary tumor cells; finally, culturing the screened primary tumor cells into organoids by using matrigel; wherein the lentivirus is added to a liquid medium comprising primary tumor cells, which are in a non-adherent state. In some embodiments, after the primary tumor cells are expanded and cultured using feeder cells, the feeder cells are removed to control the proportion of feeder cells to the total number of feeder cells and primary tumor cells to be 5% or less, preferably 4% or less, further preferably 3% or less, further preferably 2% or less, further preferably 1% or less. In some embodiments, the Cas9 protein is a spCas9 protein.

In some embodiments, the primary tumor cell is a primary tumor cell isolated from a PDX animal tumor. In some embodiments, the PDX animal belongs to a SCID rat, Nude mouse, Beige mouse, Xid mouse, SCID mouse, NOD/SCID mouse, or BNX mouse. In some embodiments, the primary tumor cell is a primary tumor cell isolated from a PDX mouse tumor. In some embodiments, the primary tumor cells are primary intestinal cancer cells isolated from a PDX mouse tumor formed from human intestinal cancer tissue isolated and inoculated into a critically ill immunodeficient mouse.

In some embodiments, more than 1 gRNA coding sequence is co-introduced into the primary tumor cell with the Cas9 protein coding sequence, and each sequence-encoded gRNA targets a different genetic site, resulting in one or more genetic modifications in the genome of the primary tumor cell. In some embodiments, the gRNA coding sequence introduced into the primary tumor cell simultaneously with the Cas9 protein coding sequence targets only the same genetic site.

In some embodiments, the Cas9 protein coding sequence and the gRNA coding sequence are packaged into a lentivirus by the same lentivirus expression vector. In some embodiments, the Cas9 protein coding sequence and the gRNA coding sequence are located in different lentiviruses packaged by different lentiviral expression vectors. In some embodiments, the Cas9 protein coding sequence is packaged with a portion of the gRNA coding sequence into the same lentivirus by the same lentiviral expression vector and another portion of the gRNA coding sequence is packaged into another lentivirus by another lentiviral expression vector. In some embodiments, the lentiviral vector is selected from the group consisting of: pV2, pCMV, px458, etc. In some embodiments, the lentiviral expression vector further comprises a selectable marker gene, preferably the selectable marker is a fluorescent protein. In some embodiments, the fluorescent protein is GFP.

In some embodiments, the Cas9 protein coding sequence and the gRNA coding sequence are expressed by promoters carried on lentiviruses. In some embodiments, the Cas9 protein coding sequence and the gRNA coding sequence are preceded by a foreign promoter that will initiate expression of Cas9 protein and gRNA upon integration of the coding sequences into the host genome. In some particular embodiments, the exogenous promoter is selected from one of eukaryotic promoters. In some embodiments, the exogenous promoter is selected from the group consisting of: CMV, EF1a, beta actin gene promoter, SV40, PGK1, Ubc, CAG, TRE, UAS, U6. In some embodiments, the expression is performed by lentivirus site-directed integration of the Cas9 protein coding sequence and the gRNA coding sequence into the host cell genome, with the use of promoters naturally present in the host cell.

In some embodiments, in the step of adding the lentivirus to a liquid matrix comprising primary tumor cells, a staining assisting agent is also added to the liquid matrix. In some embodiments, the staining assisting agent is polybrene. In some preferred embodiments, the reagent is a TransDux Max transfection reagent or a transfection reagent that is the same as the active ingredient thereof.

In some embodiments, the co-culturing the lentivirus with the primary tumor cells comprises a centrifugation incubation step and an adherent culture step, wherein the centrifugation incubation step is to centrifuge the liquid matrix containing the primary tumor cells added with the lentivirus, and the primary tumor cells are in a non-adherent state during the centrifugation incubation; and the adherent culture step is to add the centrifuged liquid matrix containing the lentivirus and the primary tumor cells into a culture medium containing feeder layer cells for adherent culture and perform static culture. In some embodiments, the feeder layer cells are selected from the group consisting of fibroblasts, uterine epithelial cells, rat liver cells, preferably fibroblasts, and further preferably mouse fibroblasts. In some embodiments, the centrifugation conditions are 200-1000 g, 1-2 h; preferably 600-1000 g, 1-2 h; most preferably 600g, 1 h.

In some embodiments, the co-culturing further comprises: and a standing incubation step is further included between the centrifugal incubation step and the adherent culture step, the standing incubation step is to perform standing incubation on the centrifuged liquid matrix containing the lentivirus and the primary tumor cells for 1-6 hours, preferably 2-4 hours, and further preferably 4 hours, and in the standing incubation step, the primary tumor cells are in a non-adherent state.

In some embodiments, the primary tumor cell that is edited when screened achieves a confluency of ≧ about 70%, such as about, e.g., 75%, 80%, 90%, 95, or 100% confluency. In some embodiments, because the lentiviral expression vector further comprises a screening marker gene, and the screening marker is a fluorescent protein, the screening is performed by flow cytometry based on fluorescence intensity.

In some embodiments, the step of culturing the screened primary tumor cells into organoids using matrigel comprises: mixing the primary tumor cells with matrigel, and co-culturing in organoid culture medium. In some embodiments, the primary tumor cells are co-cultured with an equivalent volume of matrigel.

Method of transfecting organoids

The present application also provides a method of transfecting an organoid comprising mixing a lentivirus with the organoid to form a mixture; and co-culturing the lentivirus with the organoid mixture. In some embodiments, a staining assisting agent is also added to the mixture when the lentivirus is mixed with the organoid. In some embodiments, the staining assisting agent is selected from polybrene. In some preferred embodiments, the staining assisting agent is a TransDux Max transfection agent or a transfection agent identical to the active ingredient thereof.

In some embodiments, the organoids are cultured from primary tumor cells isolated from a human tumor xenograft model (PDX) animal tumor. In some embodiments, the organoids are small organoids formed by protease digestion of a larger organoid. In some embodiments, each of said small organoids is treated with said mild proteolytic enzyme for a period of time ranging from about 3 to about 15 minutes in some embodiments. In some embodiments, the time for the mild protein digesting enzyme treatment is 10 minutes.

In some embodiments, co-culturing the mixture of lentivirus and organoid comprises a centrifugation incubation step and a matrigel culture step, wherein the centrifugation incubation step is centrifugation of the mixture without the addition of matrigel during the centrifugation incubation step; the matrigel culturing step is to mix the organoids in the mixture after centrifugation with matrigel and culture in a culture medium.

In some embodiments, the centrifugation is performed at 100 to 800g for 0.5 to 3 hours; preferably 500-800 g, 1-2 h; most preferably 600g, 1 h. In some embodiments, the co-culturing further comprises: a standing incubation step between the centrifugal incubation step and the matrigel culture step, wherein the standing incubation step is to perform standing incubation on the mixture for 1-6 hours; preferably 3-6 h; most preferably 4 h.

Gene-edited organoids and uses thereof

The present application also provides organoids edited using any of the foregoing methods; and uses of said organoids. In some embodiments, the organoid is a tumor organoid. In some embodiments, the tumor organoids are cultured from primary tumor cells isolated from PDX mouse tumors and then edited by the gene editing organoid method as described previously. In some embodiments, the tumor organoids are constructed starting from primary tumor cells by the methods of constructing gene-editing organoids as described above. In some embodiments, the organoid is an organoid 7 days after gene editing by any of the methods described above. In some embodiments, the organoid is an organoid after 10 days of gene editing by any of the methods described above. In some embodiments, the organoid is an organoid subjected to gene editing for 10 to 20 days by any of the methods described above, e.g., 11, 12, 13, 14, 15, 16, 17, 18, or 19 days after gene editing.

In some embodiments, the organoids can be used, for example, to find or study a driver of a tumor's resistance or sensitivity to a certain anti-cancer drug or to perform an anti-tumor drug-related assay. For example, in some embodiments, the tumor organ is an intestinal cancer tumor organoid, and thus it can be used to study the sensitivity and resistance mechanisms of intestinal cancer and to preliminarily test the effectiveness of drugs. Since organoids are closer to real tumors, compared with 2D cells, the time and cost of research on cell lines can be saved by directly using organoids for tumor function and treatment, which is helpful to break through the bottleneck of tumor treatment more quickly.Organoid library, and construction method and application thereof

The present application also provides methods of constructing organoid gene libraries using any of the methods described above. In some embodiments, the method comprises: organoids are edited or constructed using gRNA coding sequences targeting different gene sequences by the aforementioned "method for gene editing organoids" or "method for constructing gene editing organoids", respectively, to form a plurality of organoids having different mutations, respectively, and then the organoids having different mutations are mixed together to form an organoid gene library. In some embodiments, the method of constructing an organoid library comprises: using the aforementioned "method of gene editing organoids", a library containing organoids containing different mutations is obtained by simultaneously adding to the organoids a gRNA coding sequence having appropriate amounts of multiple target different gene sequences.

Accordingly, the present application also provides organoid gene libraries constructed using the above methods. In some embodiments, the organoid gene library is a tumor organoid library. In some embodiments, the organoid library is a colorectal cancer organoid library.

Thus, the application also provides uses of the organoid libraries described above, including uses for disease mechanism and therapeutic studies. The present invention also provides a method of high throughput screening using an organoid gene library as hereinbefore described, said high throughput screening being selected from the group consisting of: functional gene screening, drug target screening, drug sensitive gene screening and drug resistant gene screening.

According to the technical scheme, on one hand, the problem of obtaining a large number of organoid sources is solved, on the other hand, the organoid editing efficiency is improved, the screening process is simplified, and meanwhile, the problem of low organoid infection efficiency caused by large plasmids is solved. By using the technical scheme of the application, the accurately edited organoids can be obtained in a large quantity more easily, effective drug-resistant or sensitive driving genes are searched, and further support is provided for organoid-based gene function research, drug screening, research and development.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

It should be understood that the foregoing description, as well as the examples that follow, are intended to illustrate, but not to limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Examples

Example 1 organoid culture, passage and editing efficiency test method

1.1 organoid culture

Centrifuging the primary intestinal cancer cells for 5min by using 200 g; carefully aspirate the medium with a 1ml pipette tip without touching the cell pellet; the cells were washed by adding a 5ml pipette into a pre-cooled DMEM/F12(Gibco, cat # 12634-010) medium (containing 15mM HEPES Gibco cat # 15630-080, 1X Glutamax Gibco cat # 35050-061), and centrifuged at 200g for 5 min; carefully aspirate the medium with a 1ml pipette tip without touching the cell pellet; on ice, a medium (Accuroid, cat # 32031) was added to the primary cells to form a cell suspension 1, and the primary cell density was adjusted to suspend every 25ul of the cellsAdding matrigel (Corning, Cat: 356231) with the same volume as the cell suspension into the turbid solution 1 with the amount of 1E +05 primary cells, and uniformly mixing to form a cell suspension 2; preheating 24-well culture plate at 37 deg.C in advance for at least 30min, and adding 50 μ l of the cell suspension 2 at the center of each well without generating bubbles; standing at 37 deg.C for 10min, adding appropriate organoid culture medium into each well along the wall of the well, and placing the 24-well culture plate at 37 deg.C and 5% CO₂Culturing in an incubator (see FIG. 1); and (5) changing the liquid 2-3 days after the plate is paved, observing the growth state of the organoid, and carrying out passage 2-3 days after the liquid is changed.

1.2 organoid passages

The medium was aspirated off, 1ml of GCDR (STEMCELL, cat # 07174) was added to each well with a 1ml pipette tip, and digestion was carried out for 1 min; blowing and beating the organoid with a 1ml gun head, and transferring into a centrifuge tube after about 20 times per hole; shaking at room temperature at 20rpm for 10 min; 450g, centrifuging for 5 min; the supernatant was slowly aspirated with a 1ml pipette tip without touching the organoid pellet; adding 5-10ml of precooled DMEM/F12 culture medium, and cleaning organoids; centrifuging at 450g for 5 min; the supernatant was slowly aspirated off with a 1ml pipette tip; according to the following steps of 1: passage 2. Organoid medium (acculoid, cat # 32031) was added to the organoids on ice to form organoid and organoid medium mixture 1, followed by the addition of an equal volume of ice-melted matrigel (Corning, cat # 356231) to the mixture 1 to make mixture 2; adding mixture 2 into 24-well plate preheated at 37 deg.C for 30min, adding 50 μ l per well center, and standing at 37 deg.C for 10 min; each well was filled with 750. mu.l organoid medium along the plate wall, 37 ℃ 5% CO₂Culturing (see fig. 2); changing the liquid every 2 to 3 days, observing the growth state of organoids, and carrying out passage for 7 to 10 days after plating.

1.3 editing efficiency detection method

After the organoids are infected by electrotransfer or lentivirus for 10 days, the culture medium is sucked off, TryPLE is added according to 1 ml/hole, the organoids are blown and beaten, and the organoids are transferred into a centrifugal tube and digested for 5 min.

Extracting genome DNA; the procedure was followed with the TIANGEN kit blood/cell/tissue genomic DNA extraction kit (DP 304).

Amplifying a genome; q5 Hot Start Hi-Fi 2 Xbuffer (NEB, cat # M0494L) and 1. mu.g template were added and the protocol was followed.

Performing one-generation sequencing on the amplification product.

Example 2 comparison of electrotransformation and lentivirus infection in organoid editing

2.1 electrotransformation

Organoids with stable proliferation rate were selected, passaged once for about 7 days, and the medium in 24-well culture plates was aspirated off with a 1ml pipette tip.

Adding 0.5ml TryPLE (Life Technology, cat # 12604013) into each hole of a 1ml gun head, blowing and beating organoids for 20 times, adding into a centrifuge tube, digesting at 37 deg.C for 3-10min, and digesting the organoids to uniform size. Centrifuge at 450g for 5 min. The supernatant was slowly aspirated off with a 1ml pipette tip, and 5-10ml of opti-MEM (Gibco, cat # 31985-. 450g, and centrifuging for 5 min. The supernatant was slowly aspirated with a 1ml pipette tip, resuspended with 100. mu.l of opti-MEM with a 200. mu.l pipette tip, and then transferred to a 1mm electric rotor (BTX, cat. No.: 610) containing 10. mu.g of GFP mRNA (green fluorescent protein mRNA) per electric transfer condition, and the electric transfer was carried out with a BTX electric transfer apparatus using 2E +05 cells per electric transfer condition.

Adding 1ml organoid culture medium into the electrotransferred organoids, respectively, and centrifuging at room temperature for 5min at 450g for 40 min. The supernatant was slowly aspirated off with a 1ml pipette tip, 75. mu.l organoid medium was added per electrotransfer condition, mixed to form organoid and medium mixture 3, 75ul matrigel was added, mixed, added to a 24-well culture plate preheated at 37 ℃ for 30min, 50. mu.l was added to the center of each well, and then placed at 37 ℃ for 10 min. 750. mu.l organoid medium was added to each well along the walls of the well at 37 ℃ with 5% CO₂Culturing; fluorescence was observed for 48-96 h.

2.2 Lentiviral infection

Organoids with stable proliferation rates were selected, passaged once for about 7 days, and the medium was aspirated off with a 1ml pipette tip.

Adding 0.5ml of TryPLE into each hole of a 1ml gun head, blowing and beating the organoids for 20 times, adding into a centrifuge tube, digesting at 37 ℃ for 3-10min, and digesting the organoids into uniform size; centrifuging at 450g for 5 min; the supernatant was slowly aspirated off with a 1ml pipette tip, 5ml of DMEM/F12 medium was added, and the device was washedAn officer; the supernatant was slowly aspirated with a 1ml pipette tip; add 200. mu.l of concentrated virus (the virus packaged with the DNA coding sequences for RB1 sgRNA and GFP; titer 2E +08TU/ml) and 20. mu.l of organoid medium, and add the transfection aid TransDux (System Biosciences, cat # LV 860A-1); centrifuging for 1 hour at 600 g; standing and incubating for 4h at 37 ℃; adding 1ml organoid culture medium, mixing, centrifuging for 5min at 450 g; slowly sucking the supernatant with a 1ml pipette tip, re-suspending with 75. mu.l organoid culture medium, mixing to obtain a mixture 4 of organoid and culture medium, adding matrigel with the same volume as the mixture 4, mixing, adding into a 24-well culture plate preheated at 37 ℃ for 30min, adding 50. mu.l into the center of each well, and standing at 37 ℃ for 10 min. Each well was filled with 750. mu.l organoid medium at 37 ℃ with 5% CO along the walls of the well₂Culture, liquid change every 2 to 3 days, passage every 7 days, and continuous observation of fluorescence for 3 to 20 days (see fig. 3, fig. 4), and the editing efficiency was examined at 10 days and 20 days, respectively.

2.3 results and analysis

Lentivirus infection conditions and result analysis, (1) when the organoid is electrically transferred, organoid electric transfer efficiency and organoid growth state under different voltages are tested, and only 100V, 1ms and 2 pulses of 100V, 200V and 300V are used, the organoid growth state is better and has a small amount of fluorescence, so that under the condition of 100V, the pulse frequency is increased, the pulse time is reduced, and the organoid electric transfer efficiency is tested, and the result shows that the organoid only has the electric transfer efficiency under a few conditions, such as 100V, 0.5-1ms and 2-3 pulses, by the electric transfer method, see Table 1. (2) The lentivirus infects organoids, the fluorescence ratio can reach more than 50% when observed, and the detection editing efficiency can also reach about 50%. Compared with the lentivirus infection method, the latter method has obvious advantages, the fluorescence ratio after lentivirus infection is obviously higher than that of the electrotransformation observed from fluorescence (see figures 4 and 7), the editing efficiency is 58 percent, the advantages of the lentivirus infection are further verified, and therefore, the lentivirus infection mode is preferred for the organoid.

TABLE 1 results of organoid electrotransformation

Example 3 comparison of the one-step infection method with the two-step infection method

3.1 one-step infection method

Selecting organoids with stable proliferation speed, carrying out passage once in about 7 days, and sucking out the culture medium by using a 1ml gun head; adding 0.5ml of TryPLE into each hole of a 1ml gun head, blowing and beating the organoids for 20 times, adding into a centrifuge tube, digesting at 37 ℃ for 3-10min, and digesting the organoids into uniform size; centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml pipette tip, adding 5ml DMEM/F12 culture medium, and cleaning organoids; the supernatant was slowly aspirated with a 1ml pipette tip, 200. mu.l of concentrated virus (this virus was packaged with pCRISPR-GFP expression vector (FIG. 8) with the RB1 sgRNA coding sequence inserted; synthesized by Kinseri; titer 2E +08TU/ml) and 20. mu.l of organoid medium were added, and the transfection assisting reagent TransDux (System Biosciences, cat # LV860A-1) was added; centrifuging for 1 hour at 600 g; standing and incubating for 4h at 37 ℃; adding 1ml organoid culture medium, mixing, centrifuging for 5min at 450 g; slowly sucking the supernatant by using a 1ml gun head, respectively re-suspending by using 75 mu l organoid culture medium, uniformly mixing to obtain a mixture 5 of organoid and culture medium, adding matrigel which is melted on ice and has the same volume with the mixture 5, uniformly mixing, adding into a 24-hole culture plate preheated at 37 ℃ for 30min, adding 50 mu l of matrigel into the center of each hole, and standing at 37 ℃ for 10 min; each well was filled with 750. mu.l organoid medium at 37 ℃ with 5% CO along the walls of the well₂Culturing, changing the culture solution every 2 to 3 days, carrying out passage once every 7 days, observing fluorescence for 3 to 20 days, and detecting the editing efficiency at 10 days and 20 days respectively.

3.2 two-step infection method

Selecting organoids with stable proliferation speed, carrying out passage once in about 7 days, and removing the culture medium by suction; adding 0.5ml of TryPLE into each hole with 1ml of gun head, blowing and beating the organoid for 20 times, adding into a centrifuge tube, digesting at 37 ℃ for 3-10min, and digesting the organoid into uniform size; centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml pipette tip, adding 5ml DMEM/F12 culture medium, and cleaning organoids; the supernatant was slowly aspirated off with a 1ml pipette tip, 200. mu.l of concentrated virus (which contained expression vectors with inserted Cas9 and mCherry coding sequences; titer 2E +08TU/ml) and 20. mu.l of organoid medium were added, respectively, and the transfection-assisting reagent Tr was added according to the product instructionsansDux (System Biosciences, Cat.: LV 860A-1); centrifuging for 1 hour at 600 g; standing and incubating for 4h at 37 ℃; adding 1ml organoid culture medium, mixing, centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml gun head, respectively re-suspending by using 75 mu l organoid culture medium, uniformly mixing to form a mixture 6 of organoid and organoid culture medium, adding matrigel which is melted on ice and has the same volume with the mixture 6, uniformly mixing, adding into a 24-hole culture plate preheated at 37 ℃ for 30min, adding 50 mu l of matrigel into the center of each hole, and standing at 37 ℃ for 10 min; each well was filled with 750. mu.l organoid medium along the wall of 24-well culture plates at 37 ℃ with 5% CO₂Culturing, changing the culture solution once every 2 to 3 days, carrying out passage once every 7 days, and observing fluorescence;

removing the culture medium of the organoid infected for 10 days and sucking off the organoid; adding 0.5ml of TryPLE into each hole of a 1ml gun head, blowing and beating the organoids for 20 times, adding into a centrifuge tube, digesting for 3-10min at 37 ℃, and digesting the organoids into uniform size; centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml pipette tip, adding 5ml DMEM/F12 culture medium, and cleaning organoids; the supernatant was slowly aspirated off with a 1ml pipette tip, 200. mu.l of concentrated cas9 lentivirus (packaged with an expression vector with the RB1 sgRNA and GFP coding sequences inserted; Kinsley synthesis; titer 2E +08TU/ml) and 20. mu.l of organoid medium were added, respectively, and the transfection assisting reagent TransDux (System Biosciences, cat # LV860A-1) was added according to the product instructions; centrifuging for 1 hour at 600 g; standing and incubating for 4h at 37 ℃; adding 1ml organoid culture medium, mixing, centrifuging for 5min at 450 g; slowly sucking the supernatant by using a 1ml gun head, re-suspending by using 75 mu l organoid culture medium, uniformly mixing to form a mixture 7 of organoid and culture medium, adding matrigel which is melted on ice and has the same volume with the mixture, uniformly mixing, adding the mixture into a 24-hole culture plate preheated at 37 ℃ for 30min, adding 50 mu l of matrigel into the center of each hole, and standing at 37 ℃ for 10 min; each well was filled with 750. mu.l organoid medium along the wall of 24-well culture plates at 37 ℃ with 5% CO₂Culturing, changing the culture solution once every 2 to 3 days, carrying out passage once every 7 days, observing fluorescence, and detecting editing efficiency by infecting for 10 days and 20 days.

3.3 results and analysis

Organoids were edited using the same sgRNA, but using different infection steps, the results are shown in table 2, the one-step method was to infect organoids with a concentrated lentivirus into which both a Cas9 protein-encoding DNA sequence and a sgRNA-encoding DNA sequence were inserted, the two-step method was to infect organoids with a lentivirus packaged with a sgRNA expression vector, 10 days later, and then with a lentivirus packaged with a Cas9 expression vector, the two infection methods did not differ significantly from the fluorescence picture, as shown in fig. 4 and 6, respectively, but the one-step infection had advantages in the following respects, (1) the completion time: in the one-step method, only 10 days are needed from beginning to end, the fluorescence and the editing efficiency are stable, but in the two-step method, 12 days are needed from the first lentivirus infection to the second lentivirus infection, and because the two infections cause certain damage to organoids, the time for the editing efficiency to be stable is longer than 20 days; (2) editing efficiency: after 10 days of infection by the one-step method, the editing efficiency can reach 58%, is stable and has no difference with 20-day detection of infection, while the two-step method has no editing efficiency when 10 days of infection is completed, and the editing efficiency is only 6% when 20 days of infection is completed; compared with the two methods, the one-step method has the advantages of infecting organoids in both time and editing efficiency.

Table 2 two infection methods edit efficiency results

Infection method	Time of detection	Efficiency of editing
			One-step method	10 days	58％
Two-step process	20 days	6％

Example 4 comparison of organoids edited using 2D and 3D infection methods

4.1 infection of organoids by 2D

Selecting organoids with stable proliferation speed, carrying out passage once for about 7 days, sucking out organoid culture medium in 1 hole in a 24-hole culture plate by using a 1ml gun head, and adding 1ml of fresh organoid culture medium; mu.l of lentivirus (pCRISPR-GFP; Kinsry synthesis; shown in FIG. 8) packaged with vectors expressing both sgRNA and Cas9 was added at 37 ℃ with 5% CO₂Culturing for 24 h; the medium was aspirated off with a 1ml pipette tip, 1ml of fresh medium was added, 37 ℃ with 5% CO₂After 72h incubation, fluorescence was observed.

4.2 infection of organoids by 3D

Selecting organoids with stable proliferation speed, carrying out passage once in about 7 days, and sucking out the culture medium; adding 0.5ml TryPLE into each hole with 1ml gun head, blowing and beating organoids for 20 times, adding into a centrifuge tube, digesting at 37 deg.C for 3-10min, and digesting organoids into uniform size; centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml pipette tip, adding 5ml DMEM/F12 culture medium, and cleaning organoids; slowly absorbing the supernatant by using a 1ml pipette tip, respectively adding 200 mul of lentivirus packaged with vectors for simultaneously expressing sgRNA and Cas9 and 20 mul of organoid culture medium, adding a dyeing assistant reagent TransDux (System Biosciences, product number: LV860A-1), 600g, and centrifuging at normal temperature for 1h according to the product instruction; standing and incubating for 4h at 37 ℃; adding 1ml organoid culture medium, mixing, centrifuging for 5min at 450 g; slowly sucking the supernatant by using a 1ml gun head, re-suspending by using 75 mu l organoid culture medium, uniformly mixing to obtain a mixture 8 of organoid and culture medium, adding matrigel which is melted on ice and has the same volume with the mixture 8, uniformly mixing, adding into a 24-hole plate preheated at 37 ℃ for 30min, adding 50 mu l of matrigel into the center of each hole, and standing at 37 ℃ for 10 min; each well was filled with 750. mu.l organoid medium at 37 ℃ with 5% CO along the walls of the well₂The culture was changed every 2 to 3 days and passaged for 7 days, and fluorescence was observed, and the editing efficiency was examined on the 10 th day thereafter.

4.3 results and analysis

(1) The 2D infection mode is convenient to infect the organoid, the steps of organoid digestion, incubation and the like are not needed, the virus is directly added, but the infection efficiency is 0%, which indicates that the mode is not suitable for organoid infection; (2) organoids and lentiviruses are infected in a common centrifugal incubation manner (3D infection), the process is more complicated than that of 2D infection, but from the result, the efficiency of fluorescence and editing is good, the editing efficiency is shown in table 3, and the fluorescence picture is shown in fig. 4; from two infection modes, the organoid is infected in a 3D mode, and under the condition of not influencing the growth of the organoid, better infection efficiency can be obtained, and higher editing efficiency can be obtained at the same time

TABLE 3 editing efficiency of organoid 3D infection method

sgRNA targeting gene	Efficiency of editing
		RB1	58％
DAPK1	46％

Example 5 Lentiviral one-step infection method optimization for editing organoids

5.1 different lentivirus volume infections

Selecting organoids with stable proliferation speed, carrying out passage once for about 7 days, sucking out the culture medium by using a 1ml gun head, adding 0.5ml of TryPLE into each hole, blowing and beating the organoids for 20 times, adding the organoids into a centrifugal tube, digesting for 10min at 37 ℃, and digesting the organoids into uniform size; centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml pipette tip, adding 5-10ml DMEM/F12 culture medium, and cleaning organoids; the supernatant was slowly aspirated off with a 1ml pipette tip(ii) a In a total of 5 groups of organoids, 50. mu.l, 100. mu.l, 150. mu.l, 200. mu.l, 250. mu.l of concentrated virus (which is packaged with pCRISPR-GFP vector with the coding sequence for RB1 sgRNA inserted; Kinserin synthesis; titer 2E +08TU/ml) and 20. mu.l of organoid medium were added, respectively, and the staining assistant TransDux (System Biosciences, Cat.: LV860A-1) was added; centrifuging for 1 hour at 600 g; adding 1ml organoid culture medium respectively, mixing, centrifuging for 5min at 450 g; slowly sucking the supernatant by using a 1ml gun head, suspending each group of organoids by using 75 mu l of organoid culture medium respectively, uniformly mixing to form a mixture 10 of organoids and culture medium, adding matrigel which is melted on ice and has the same volume with the mixture 10, uniformly mixing, adding the mixture into a 24-hole culture plate preheated at 37 ℃ for 30min, adding 50 mu l of matrigel into the center of each hole, and standing at 37 ℃ for 10 min; each well was filled with 750. mu.l organoid medium at 37 ℃ with 5% CO along the walls of the well₂Incubation, fluorescence was observed for 3 to 10 days.

Results and analysis: results of different volumes of lentivirus infecting organoids are shown in Table 4

TABLE 4 efficiency of lentivirus infection of organoids at different volumes

Lentivirus virus volume	Results
		50μl	The fluorescence proportion is about 5%
100μl	The proportion of fluorescence is about 10%
		150μl	The fluorescence proportion is about 20%
200μl	The fluorescence proportion is about 40%
		250μl	The proportion of fluorescence is about 50%

It can be seen from the table that the infection efficiency of lentivirus less than 200 mul is not good, and the infection efficiency is gradually improved along with the increase of the lentivirus volume, but when 250 mul of lentivirus is added to infect the organoid, the organoid is gradually dead after being infected for one week, the initial number of organoid infection is also increased along with the increase of the virus amount to more than 200 mul, otherwise, the organoid is gradually dead along with the increase of the culture time, so that 200 mul of lentivirus is selectively added to infect the organoid.

5.2 incubation: increased 37 ℃ incubation of infected organoids

Selecting organoids with stable proliferation speed, carrying out passage once for about 7 days, sucking out the culture medium by using a 1ml gun head, adding 0.5ml of TryPLE into each hole, blowing and beating the organoids for 20 times, adding the organoids into a centrifugal tube, digesting for 3-10min at 37 ℃, and digesting the organoids into uniform size; centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml pipette tip, adding 5-10ml DMEM/F12 culture medium, and cleaning organoids; the supernatant was slowly aspirated with a 1ml pipette tip, and 200. mu.l of concentrated virus (packed with pCRISPR-GFP vector with RB1 sgRNA inserted; Kinsley synthesis; titer 2E +08TU/ml) and 20. mu.l of organoid medium were added to each group, respectively, and the transfection aid reagent TransDux (System Biosciences, cat # LV860A-1) was added. Four organoids in total, -600g, 1h centrifugation; standing and incubating the four groups of organoids at 37 ℃ for 1h, 2h, 4h and 6h respectively; adding 1ml organoid culture medium respectively, mixing, centrifuging for 5min at 450 g; slowly sucking the supernatant with a 1ml gun head, suspending each group of organoids with 75 μ l organoid culture medium, mixing, adding 75ul matrigel, and mixing; adding into 24-well culture plate preheated at 37 deg.C for 30min, adding 50 μ l per well, and standing at 37 deg.C for 10 min; each well was filled with 750. mu.l organoid medium at 37 ℃ with 5% CO along the walls of the well₂Incubation, fluorescence was observed for 3 to 10 days.

Results and analysis: the results of the incubation step with the addition of 37 ℃ are shown in Table 5

TABLE 5 Effect of different incubation times on organoid infestation

Compared with the result without the standing incubation (as shown in 5.1), after the standing incubation step is added, as the incubation time is increased, 200 μ l of lentivirus infects the organoid, the fluorescence ratio reaches more than 50%, from the result, the short-time standing incubation does not obviously affect the improvement of the organoid infection efficiency, and the long-time standing incubation affects the growth state of the organoid, so the standing incubation at 37 ℃ for 4 hours is the optimal time. When the standing incubation step is not added, only the infection efficiency is increased by centrifuging the lentivirus and the organoid, the infection efficiency is increased along with the increase of the quantity of the lentivirus by the method, when the quantity of the lentivirus reaches 250 microliter, the infection efficiency is only about 50 percent, and the organoid slowly dies after the lentivirus is infected for one week, which shows that although the infection efficiency is improved, the late growth of the organoid is influenced. After the incubation step at 37 ℃ is added, the infection efficiency of 200 mul of lentivirus can reach more than 50%, the growth of organoids can not be influenced, and the subsequent experiment of infecting organoids can not be influenced under the condition of solving the infection efficiency, so that the increase of the step is helpful for improving the infection efficiency of organoids.

5.3 organoid transfection volumes: digestion time optimization

Organoids with stable proliferation rates were selected and passaged once about 7 days. Four organoids in total, the medium in each well was aspirated with a 1ml pipette tip; 0.5ml of TryPLE was added to each well with a 1ml tip, organoids were blown 20 times, added to a centrifuge tube, and digested at 37 ℃. The digestion time of 4 groups of organs is 3min, 5min, 10min and 15min respectively. Centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml pipette tip, adding 5ml DMEM/F12 culture medium, and cleaning organoids; the supernatant was slowly aspirated off with a 1ml pipette tip and 200. mu.l of each was addedl of concentrated virus (which was packaged with the pCRISPR-GFP vector with the RB1 sgRNA inserted; Kinsery synthesis; titre 2E +08TU/ml) and 20. mu.l of organoid medium, and the transfection assisting reagent TransDux (System Biosciences, cat # LV860A-1) was added. Centrifuging for 1 hour at 600 g; standing and incubating for 4h at 37 ℃; adding 1ml organoid culture medium, mixing, centrifuging for 5min at 450 g; the supernatant was slowly aspirated off with a 1ml pipette tip, and each group was suspended with 75. mu.l organoid medium per group, mixed well, added with 75. mu.l matrigel, mixed well and added to a 24-well plate preheated at 37 ℃ for 30 min. The addition position was in the center of each well and the amount added was 50. mu.l. Standing at 37 deg.C for 10min, adding 750 μ l organoid culture medium into each well along the wall of the well, and culturing at 37 deg.C with 5% CO₂Culturing, changing the medium every 2 to 3 days, and carrying out passage once every 7 days, and observing fluorescence.

Results and analysis: lentiviral infection was performed with organoids of different digestion times and fluorescence was observed 7 days later with results shown in Table 6.

TABLE 6 results of organoid infestation at different digestion times

From the results of different digestion times, the organoids should be digested to be uniform in size, so that the phenomenon of nonuniform infection can not occur during infection, but the phenomenon of massive death after infection can occur after the organoids are digested for too long time, so that the growth of the organoids is influenced, the digestion time mentioned in the current literature is in a range of 3-15min, which is determined according to the initial amount of the organoids, the amount of the added digestive juice and the initial organoid size, so that the digestion time cannot be used as a sole measurement factor, only the organoids are observed to be uniform in size, and the infection efficiency is also higher.

5.4 optimization of centrifugation conditions

Organoids with stable proliferation rates were selected and passaged once about 7 days. Discarding the culture medium, adding 0.5ml TryPLE into each well, transferring the organoid digestive juice into a centrifuge tube, digesting at 37 ℃ for 3-10min, and centrifuging at normal temperature of 450g for 5 min; the supernatant was slowly aspirated off with a 1ml pipette tip, add5ml of DMEM/F12 culture medium is added, and organoids are cleaned; the supernatant was slowly aspirated off with a 1ml pipette tip, 200. mu.l of concentrated virus and 20. mu.l of organoid medium were added, respectively, and the transfection aid reagent TransDux (System Biosciences, cat: LV860A-1) was added. Dividing the organoids into four groups, wherein the centrifugal speed and the time of each group are respectively 200g and 1.5 h; 450g, 1 h; 600g, 1 h; 800g and 1 h. After centrifugation, standing and incubating for 4h at 37 ℃; adding 1ml organoid culture medium, mixing, centrifuging at 450g for 5 min; slowly sucking out supernatant with 1ml pipette tip, re-suspending with 75 μ l culture medium respectively, adding 75ul matrigel, mixing, adding into 24-well plate, 50 μ l per well, standing at 37 deg.C for 10min, adding 750 μ l culture medium along the wall of each well, 37 deg.C, 5% CO₂Culture, changing the medium every 2 to 3 days, subculturing every 7 days, and observing fluorescence.

Results and analysis: different centrifugation conditions have different influences on the infection efficiency of the organoid, and the result shows that the organoid and the virus cannot be completely centrifuged to the bottom of the tube after centrifugation at 200g for 1.5h, and the fluorescence ratio is less than 10%; the fluorescence ratio is about 20% at 450g and 1 h; at 800g and 1h, in the later culture process of the organoid, the growth speed obviously becomes slow, and the fluorescence ratio is about 30 percent; after 600g is centrifuged for 1h, the growth speed of the organoid is not influenced, and the fluorescence ratio is more than 50%, so that higher editing efficiency can be achieved.

5.5 comparison of edit efficiency for different time Point detection

Organoids with stable proliferation rates were selected and passaged once about 7 days. The culture medium is divided into two groups, and the culture medium in each hole is sucked by a 1ml gun head; adding 0.5ml of TryPLE into each hole with 1ml of gun head, blowing and beating the organoid for 20 times, adding into a centrifuge tube, digesting at 37 ℃ for 3-10min, and digesting the organoid into uniform size; centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml pipette tip, adding 5ml DMEM/F12 culture medium, and cleaning organoids; the supernatant was slowly aspirated off with a 1ml pipette tip, 200. mu.l of concentrated virus (packed with pCRISPR-GFP vector with RB1 sgRNA inserted; Kinsley synthesis; titer 2E +08TU/ml) and 20. mu.l of organoid medium were added, respectively, and the transfection aid reagent TransDux (System Biosciences, cat # LV860A-1) was added; centrifuging for 1 hour at 600 g; standing and incubating for 4h at 37 ℃; adding 1ml organoid for cultureMixing, centrifuging for 5min at 450 g; the supernatant was slowly aspirated off with a 1ml pipette tip, each organoid group was suspended with 75. mu.l organoid medium, mixed well, added with 75. mu.l matrigel, mixed well and added to a 24-well plate preheated at 37 ℃ for 30min, and 50. mu.l was added to the center of each well. Standing at 37 deg.C for 10 min; each well was filled with 750. mu.l organoid medium at 37 ℃ with 5% CO along the walls of the well₂Culture, fluid change every 2 to 3 days, passage once every 7 days, and fluorescence was observed for 3 to 20 days. The two groups of organoids were tested for editing efficiency at 10 days and 20 days, respectively.

Results and analysis: after the organoid was infected, fluorescence was stabilized around day 7 after infection, so the editing efficiency of the first group was examined on day 10, followed by 10 days of culture, and the editing efficiency of the second group was examined, with the results shown in table 7.

TABLE 7 detection of organoid editing efficiency at different times

Time	Efficiency of editing
		10 days after infection	45％
20 days after infection	46％

From the detection result, the editing efficiency of the organoid is basically consistent between 10 days and 20 days after the organoid is infected by the lentivirus, which indicates that the organoid is stable after 10 days of infection by the lentivirus, and experiments such as subsequent screening can be carried out without continuously spending more time to wait for the stable editing efficiency.

5.6 stability testing of Lentiviral infection organoid procedures

5.6.1 Lentiviral infection of organoids of different samples

Two groups of organoids with stable proliferation rates were selected, sample 1 (the organoids used were as in examples 1-4) and sample 2, respectively, with different mutations, and infected with lentivirus. The specific process is as follows:

subculturing the organoid once in about 7 days, sucking out the culture medium in each hole by using a 1ml gun head, adding 0.5ml TryPLE into each hole, blowing and beating the organoid for 20 times, adding into a centrifuge tube, digesting at 37 ℃ for 3-10min, and digesting the organoid into uniform size; centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml pipette tip, adding 5ml DMEM/F12 culture medium, and cleaning organoids; the supernatant was slowly aspirated off with a 1ml pipette tip, and 200. mu.l of sgRNA-packaged concentrated virus and 20. mu.l of organoid medium were added to each group of organoids, respectively, and the transfection aid reagent TransDux (System Biosciences, Cat.: LV860A-1) was added thereto; centrifuging for 1 hour at 600 g; standing and incubating for 4h at 37 ℃; adding 1ml organoid culture medium, mixing, centrifuging for 5min at 450 g; slowly sucking out supernatant with 1ml gun head, re-suspending with 75 μ l organoid culture medium respectively, mixing, adding 75ul matrigel, mixing, adding into 24-well culture plate preheated at 37 deg.C for 30min, adding 50 μ l per well, standing at 37 deg.C for 10 min; each well was filled with 750. mu.l organoid medium at 37 ℃ with 5% CO along the walls of the well₂Culture, fluid change every 2 to 3 days, passage every 7 days, observed for 3 to 20 days of fluorescence, at day 10 to detect editing efficiency.

Results and analysis: the organoid editing efficiency for two different samples using the method of the present application is shown in table 8, and the fluorescence images of the organoids are shown in fig. 4 and 5. From the results, it can be seen that the lentiviral gene editing method of the present application has similar editing efficiency on organoids of different mutants. Therefore, the organoid infection process of the present application is used for gene editing, the editing efficiency is stable, and too large editing efficiency difference can not occur due to the change of organoid mutant types.

TABLE 8 different sample editing efficiency

Organoid sample	Efficiency of editing
		Sample 1	58％
Sample 2	49％

5.6.2 infection of organoids with different sgRNAs

Organoids with stable proliferation rates were selected and passaged once about 7 days. . Absorbing the culture medium of each hole with 1ml of gun head, adding 0.5ml of TryPLE into each hole, blowing and beating the organoid for 20 times, adding into a centrifuge tube, digesting at 37 ℃ for 3-10min, and digesting the organoid into uniform size; centrifuging at 450g for 5 min; slowly sucking the supernatant by using a 1ml pipette tip, adding 5ml DMEM/F12 culture medium, and cleaning organoids; the supernatant was slowly aspirated with a 1ml pipette tip, and 200. mu.l of vector packaging expressing both sgRNA and Cas9 (titer 2E +08TU/ml), and 20. mu.l of organoid medium were added to each group of organoids, respectively. The concentrated virus added to the two groups of organoids contained the DNA coding sequences of sgRNA RB1 and sgRNA DAPK1, respectively, along with the addition of the transfection assisting agent TransDux (System Biosciences, Cat.: LV 860A-1); centrifuging for 1 hour at 600 g; standing and incubating for 4h at 37 ℃; adding 1ml organoid culture medium, mixing, centrifuging for 5min at 450 g; slowly sucking out supernatant with 1ml gun head, re-suspending with 75 μ l organoid culture medium respectively, mixing, adding 75ul matrigel, mixing, adding into 24-well culture plate preheated at 37 deg.C for 30min, adding 50 μ l at the center of each well, and standing at 37 deg.C for 10 min; each well was filled with 750. mu.l organoid medium at 37 ℃ with 5% CO along the walls of the well₂Culture, liquid change every 2 to 3 days, passage every 7 days, fluorescence observed for 3 to 20 days, and detection of editing efficiency at 10 days, respectively.

Results and analysis: the editing efficiency after different sgrnas infect the same kind of organ is shown in table 9.

Table 9 detection of editing efficiency of different sgrnas

sgRNA-targeted gene	Efficiency of editing
		RB1	58％
DAPK1	46％

Therefore, when different sgrnas are used for infecting organoids, the organoid infection process is basically stable, and the editing efficiency and the infection efficiency are not greatly changed along with the change of the sgrnas.

Claims

1. A method of gene editing an organoid, comprising:

co-culturing the lentivirus with an organoid in the mixture.

2. The method of claim 1, wherein said organoids are cultured from primary tumor cells isolated from a human tumor xenograft model (PDX) animal tumor,

3. The method of claim 2, wherein prior to mixing the lentivirus with the organoids, the organoids are treated with mild proteolytic enzyme, preferably Tryple, to separate from the matrigel and break into small organoids of uniform size.

4. The method according to any one of claims 1 to 3, wherein a staining assisting agent is also added to the mixture when the lentivirus is mixed with the organoid, preferably the staining assisting agent is TransDux Max transfection agent or a transfection agent identical to the active principle thereof.

5. A method of transfecting an organoid with a lentivirus, comprising:

mixing lentivirus with organoids to form a mixture;

co-culturing the lentivirus with the organoid mixture.

6. An organoid edited using the method of any one of claims 1 to 5.

7. An organoid edited after 10 days using the method of any one of claims 1 to 5.

8. An organoid gene library consisting of organoids according to claim 6 or 7.

9. Use of the organoid of claim 6 or 7 for disease mechanism and therapeutic studies.

10. A method of high throughput screening using the organoid gene library of claim 8, said high throughput screening being selected from the group consisting of: any one of functional gene screening, drug target screening, drug sensitive gene screening and drug resistant gene screening.