KR20210030805A - Composition for enhancing gene editing efficiency by homologous recombination comprising autophagy inducing agent - Google Patents

Abstract

The present invention relates to a composition comprising an autophagy inducer for enhancing efficiency of gene editing through homologous recombination and a method for enhancing efficiency of gene editing. The technique according to the present invention can remarkably enhance the efficiency of homologous recombination gene editing, for example, CRISPR gene editing, to up to about 10 fold by inducing autophagy in human cells and animal cells. In addition, taking advantage of an intrinsic autophagy mechanism of cells, the technique has a wide range of applications, especially in vivo, and thus can overcome the limits that conventional techniques of enhancing homologous recombination efficiency have. Furthermore, the technique of the present invention allows accurate and effective gene editing and thus is expected to find advantageous applications in various fields including bioscience research, gene therapy, and new drug development.

Description

Composition for enhancing gene editing efficiency by homologous recombination comprising autophagy inducing agent}

The present invention relates to a composition for increasing gene editing efficiency by homologous recombination comprising an autophage inducing agent and a method for increasing gene editing efficiency.

In many cases, genetic diseases in humans are caused by mutations in specific genes, and most mutations induce normal functioning of genes. In this case, when treating genetic diseases using gene editing technology, it is rarely possible to improve the condition by means of gene knockout, and it is necessary to accurately correct the mutation and change it to the wild type. Therefore, the increase in gene editing technology by homologous recombination is essential in the treatment of genetic diseases.

As such gene editing technology, genetic scissors technology has been continuously developed and used. The genetic scissors cause a specific double-strand cut at a desired location on the genome, and the cut is performed by the cell's own mechanism. ) Or non-homologous end joining (NHEJ).

For example, it is known that DNA double helices cut by CRISPR-CAS, a third-generation genetic scissors, are repaired by non-homologous ends in most cases. However, repairs by nonhomologous end-links cause unpredictable mutations, and thus the genome cannot be corrected in the exact desired form. On the other hand, the homologous recombination mechanism uses the donor DNA sequence to correct the gene, so it has the advantage of correcting the genome exactly in the desired shape, but it works only in a specific cell cycle (G2-S) and occurs only in cells with active division. Is known. Therefore, in gene editing using CRISPR-Cas, the efficiency of rust-in using homologous recombination is very low, and it appears even lower in differentiated cells in which cell division is inhibited.

Until recently, various methods have been attempted to increase the efficiency of gene melting using such homologous recombination. For example, by fixing the cells to the G2-S group using a chemical substance that fixes the cell cycle, gene correction is performed, or by using a chemical substance or protein that inhibits non-homologous end connection, the double helix is cut and then proceeds to a homologous recombination mechanism. It has been reported that a method of inducing or transferring a donor DNA by binding to a cas protein has been reported (Mol Cells. 2018 Nov 30; 41(11):943-952.). However, these methods may have some effect in cell lines, but are difficult to use in vivo. Specifically, chemicals used for cell cycle fixation can cause large changes in cells, leading to large deletions in genes, and non-homologous end-linking inhibitors also have the potential to induce unwanted mutations by inhibiting normal intracellular repair mechanisms. However, the method of linking the donor DNA has a disadvantage that it is possible to experiment in a cell line, but it is difficult to transfer it to the tissue.

Therefore, it is necessary to develop a method that can increase the efficiency of gene editing by homologous recombination efficiently and safely not only in vitro but also in vivo.

The present inventors have endeavored to develop a technology that can effectively increase the efficiency of homologous recombination safely in vivo when gene editing using genetic scissors. As a result, when inducing autophagy, an intrinsic mechanism of cells, human cells and animal cells It was confirmed that the homologous recombination efficiency increased significantly, and the present invention was completed based on this.

Accordingly, an object of the present invention is to provide a composition for enhancing gene editing efficiency by homologous recombination, including an autophagy inducing agent.

In addition, another object of the present invention is to provide a method for increasing gene editing efficiency by homologous recombination, including the step of inducing autophagy.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a composition for enhancing gene correction efficiency by homologous recombination, including an autophagy inducing agent.

In one embodiment of the present invention, the autophagy inducing agent may be a nutrient-deficient medium or mTOR (mammalian target of rapamycin) inhibitor.

In another embodiment of the present invention, the nutrient-deficient medium may be a Hanks' balanced salt solution (HBSS).

In another embodiment of the present invention, the mTOR inhibitor may be selected from the group consisting of Rapamycin, Torin 1, Torin 2, and PP242.

In another embodiment of the present invention, the gene correction may be performed through genetic scissors selected from the group consisting of Zinc Finger Nuclease, TALENs, and CRISPR.

In addition, the present invention provides a method for increasing gene editing efficiency by homologous recombination, including the step of inducing autophagy.

In one embodiment of the present invention, the autophagy induction may be achieved by culturing cells in a nutrient-deficient medium.

In another embodiment of the present invention, the nutrient-deficient medium may be a Hanks' balanced salt solution (HBSS).

In another embodiment of the present invention, the autophagy induction may be performed by treating cells with a mammalian target of rapamycin (mTOR) inhibitor.

In another embodiment of the present invention, the mTOR inhibitor may be selected from the group consisting of Rapamycin, Torin 1, Torin 2, and PP242.

In another embodiment of the present invention, the gene correction may be performed through genetic scissors selected from the group consisting of Zinc Finger Nuclease, TALENs, and CRISPR.

The technology according to the present invention can significantly increase the efficiency of homologous recombination gene editing by, for example, CRISPR gene scissors, by inducing autophagy in human cells and animal cells, up to about 10 times, and also utilizes the intrinsic autophagy mechanism of cells. By doing so, the range that can be applied is wide, and especially in vivo , the limitations of the existing homologous recombination efficiency enhancement technology can be overcome. Furthermore, the technology of the present invention is expected to be useful in various fields such as life science research, gene therapy, and new drug development by enabling accurate and efficient gene correction.

1 schematically shows a schematic diagram for inserting a sequence encoding an EGFP fluorescent protein having a homologous sequence attached to the N-terminus of LMNA, an intrinsic gene of human cells, using CRISPR gene scissors.
2 is a brief illustration of an experimental method for confirming the change in gene editing efficiency due to homologous recombination after autophagy induction in an embodiment of the present invention.
3 is a human cell line (HEK293T, U2OS, HeLa) and mouse cell line (MEF) cultured in HBSS medium and induced gene correction by homologous recombination, and then confirmed whether autophagy was induced through Western blot and flow cytometric analysis. This is the result of measuring the homologous recombination efficiency (HDR efficiency).
Figure 4 is a result of measuring the homologous recombination efficiency (HDR efficiency) through flow cytometry after treating a human cell line (HeLa) and a mouse cell line (MEF) with rapamycin and Torin-1, respectively, and inducing gene correction by homologous recombination. to be.

As a result of research efforts to overcome the limitations of the prior art and to develop a technology that can effectively increase the efficiency of gene editing through homologous recombination, the present inventors experimentally found that the efficiency of homologous recombination can be significantly increased through induction of intracellular autophagy. By confirming with, the present invention was completed.

Accordingly, the present invention provides a composition for enhancing gene editing efficiency by homologous recombination, including an autophagy inducer.

In addition, the present invention provides a method for increasing gene editing efficiency by homologous recombination, including the step of inducing autophagy.

The present inventors have confirmed that the efficiency of gene editing through homologous recombination is remarkably increased when autophagy is induced in cells through specific examples.

More specifically, in one embodiment of the present invention, in order to experimentally measure the efficiency of homologous recombination induced by CRISPR gene scissors, CRISPR/CAS-9 gene scissors were manufactured and used as an endogenous gene present in human cells. A donor DNA was prepared by inserting the gene sequence encoding the EGFP fluorescent protein into the LMNA gene, and CRISPR/CAS9 gene scissors and donor DNA were also prepared for the LMNA gene (MmLmna) of mouse cells in the same manner (Example 1 Reference).

In another embodiment of the present invention, it was verified whether or not the efficiency of homologous recombination gene editing by CRISPR gene scissors increases when autophagy is induced in human cells and mouse cells. To this end, human cells and mouse cells were each cultured in HBSS medium to create an environment deficient in nutrients, or mTOR inhibitors Rapamycin and Torin 1 were each treated to induce autophagy. In addition, CRISPR/CAS 9, sgRNA, and donor DNA prepared in Example 1 were transfected into cells to induce homologous recombination gene correction by CRISPR gene scissors, and the homologous recombination efficiency was measured. As a result, it was confirmed that when autophagy was induced in both human cells and mouse cells, the homologous recombination efficiency was increased by about 10 times compared to the control (see Examples 2-1 and 2-2).

From the above results, it was found that the efficiency of gene editing through homologous recombination can be effectively increased by inducing an intrinsic mechanism of cells called autophagy.

The term "autophagy" used in the present invention is also referred to as autophagy. It is a mechanism that supplements nutrients in situations where growth is unfavorable by decomposing organelles in their own cells by being induced under stress conditions such as lack of nutrient energy. It is a very important process for maintaining the homeostasis of cells, and therefore, studies have reported that the activity of autophagy is abnormally increased or decreased in various diseases including cancer. If autophagy due to nutrient energy deficiency in cells is induced for a long time, it may proceed to apoptosis, but in the present invention, autophagy was induced only for a short time and the autophagy inducing element was removed, so that the CRISPR- It is possible to effectively increase the efficiency of homologous recombination by CAS gene scissors.

In the present invention, the method of inducing autophagy may be to induce a condition of lack of nutrient energy in cells, and specifically, cells with a nutrient-deficient or deficient medium, preferably HBSS (Hanks' balanced salt solution) Although it can be made by culturing in the same medium, the type of medium is not limited thereto, and a person skilled in the art can induce autophagy by inducing nutrient energy deficiency conditions in cells by selecting an appropriate medium and culture conditions.

In the present invention, as another method of inducing autophagy, autophagy can be induced by treating cells with an inhibitor of mTOR, which is the highest signaling kinase of autophage. As the mTOR inhibitor, various substances used in the relevant technical field may be used, preferably those selected from the group consisting of Rapamycin, Torin 1, Torin 2, and PP242, but are not limited thereto.

As used herein, the term "gene correction through homologous recombination" refers to a series of processes in which a DNA double helix cut by a genetic scissors is repaired through a homologous recombination mechanism, which is an intracellular DNA repair mechanism.

The genetic scissors may include all those used for gene editing and editing techniques in the relevant technical field, preferably zinc finger nuclease (Zinc Finger Nuclease), TALENs (TALENs), CRISPR-CAS (CRISPR-CAS) Alternatively, it may be CRISPR-Cpf1, more preferably CRISPR-CAS, but is not limited thereto.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example 1. Exemplary genes To LMNA EGFP To insert Crisper Genetic scissors and donor DNA production

The present inventors attempted to insert a fluorescent protein into an endogenous gene present in human cells in order to effectively measure the efficiency of homologous gene recombination induced by CRISPR gene scissors. 1 schematically shows a schematic diagram of inserting a fluorescent protein using CRISPR gene scissors.

More specifically, as shown in FIG. 1, the N-terminus of the human LMNA gene encoding LaminA, a nuclear structural protein expressed in a large amount in human cells, was cut with CRISPR gene scissors, and the homology sequence was Insertion of the EGFP sequence by homologous recombination can be induced by transferring the EGFP sequence attached to the. When homologous recombination occurs, a protein bound to EGFP and LaminA is expressed, which can be analyzed through flow cytometry to measure the efficiency of homologous recombination. In addition, CRISPR gene scissors and donor DNA were prepared for the mouse LMNA gene (MmLmna) in order to carry out the same experiment in the mouse cell line.

Example 2. Human cell And in animal cells Autophagy Phenomenon-induced Sangdong Reunion Increase efficiency Confirm

In order to verify whether the homologous recombination efficiency is changed by the autophagy phenomenon, the present inventors performed an experiment by the method shown in FIG. 2. As a method to induce autophagy in cells, an inhibitor of receptor protein that induces a nutrient-energy-deficient environment or transmits intracellular autophagy signaling can be used. Therefore, as shown in Figure 2, in order to create a nutrient-deficient condition, cells were cultured in HBSS medium or treated with mTOR inhibitor Rapamycin or Torin-1 to induce autophagy, and prepared in Example 1 above. CRISPR/Cas9, sgRNA, and donor DNA were transfected into cells to induce gene correction through homologous recombination. Subsequently, the homologous recombination efficiency was measured by analyzing the degree of fluorescence generation by the EGFP protein through flow cytometry (FACS) on the transfected cell line.

2-1. Due to a nutrient-deficient environment Autophagy Judo

As described above, the cell line was first cultured in HBSS medium to induce autophagy due to a nutrient-deficient environment, and then the intracellular homologous recombination efficiency was measured according to the above method.

As a result, as shown in FIG. 3, human cell lines (HEK293T, U2OS, HeLa) and mouse cell lines (MEF) were each cultured in HBSS medium. It was confirmed that phage was induced. In addition, as a result of measuring the homologous recombination efficiency in the cells through flow cytometry, it was confirmed that the homologous recombination efficiency was significantly increased by about 3 to 8 times compared to the control (Ctrl) in all of the four cell lines.

2-2. Rapamycin or Torin -1 by processing Autophagy Judo

In addition to the results of Example 2-1, flow cytometry was performed after treatment with Rapamycin and Torin 1, which are used as compounds for inducing autophagy in human HeLa cell lines and mouse MEF cell lines, respectively. Through the homologous recombination efficiency was measured.

As a result, it was confirmed that, as shown in FIG. 4, when Rapamycin and Torin 1 were treated in HeLa and MEF cell lines, respectively, the homologous recombination efficiency was significantly increased by about 2.5 to 9 times compared to the control group.

From the results of Example 2, it was found that the efficiency of gene editing by homologous recombination was remarkably increased through the induction of autophagy in human and animal cells.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.

Claims (11)

Autophagy (autophagy) comprising an inducer, homologous recombination (Homologous Recombination) by a composition for increasing the efficiency of gene editing.
The method of claim 1,
The autophagy inducer is a nutrient-deficient medium or mTOR (mammalian target of rapamycin) inhibitor, characterized in that the composition.
The method of claim 2,
The nutrient-deficient medium is HBSS (Hanks' balanced salt solution), characterized in that, the composition.
The method of claim 2,
The mTOR inhibitor is characterized in that it is selected from the group consisting of Rapamycin, Torin 1, Torin 2, and PP242.
The method of claim 1,
The gene correction is characterized in that occurs through a genetic scissors selected from the group consisting of zinc finger nuclease (Zinc Finger Nuclease), TALENs (TALENs) and CRISPR (CRISPR), the composition.
A method for increasing gene editing efficiency by homologous recombination, comprising the step of inducing autophagy.
The method of claim 6,
The autophagy induction is characterized in that by culturing the cells in a nutrient-deficient medium.
The method of claim 7,
The nutrient-deficient medium is HBSS (Hanks' balanced salt solution), characterized in that, augmentation method.
The method of claim 6,
The autophagy induction is characterized in that the cell is treated with an mTOR (mammalian target of rapamycin) inhibitor.
The method of claim 9,
The mTOR inhibitor is characterized in that selected from the group consisting of rapamycin, torin 1 (Torin 1), torin 2 (Torin 2) and PP242, augmentation method.
The method of claim 6,
The gene correction is characterized in that through the gene scissors selected from the group consisting of zinc finger nuclease, TALENs and CRISPR, augmentation method.

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