KR102264829B1 - Reporter System for Assessing Cleavage Activity of CRISPR/Cas9 - Google Patents

Abstract

The present invention relates to a novel reporter system capable of evaluating the cleavage efficiency of gene scissors (CRISPR / Cas9), and more specifically, a first promoter - a gene scissors target sequence - Lac repressor - Poly (poly) A - Second promoter (promoter) - Lac operator (operator) - Reporter (reporter) gene - Poly (poly) A is operably linked in sequence Evaluation of cleavage efficiency of the gene scissors, characterized in that configured A reporter construct and a method for evaluating the cleavage efficiency of gene scissors using the same.
According to the present invention, since the reporter systems for evaluating the efficiency of conventional gene editing depend on a frameshift in the protein coding region, it is difficult to measure the activity of the gene scissors when a 3n base insertion or deletion occurs. Thus, the present invention has the advantage of being able to evaluate the gene scissoring efficiency regardless of the frame shift. Therefore, using the reporter system of the present invention, sgRNA with high efficiency can be simply selected, and gene editing can be efficiently performed in cells and animal models using this.

Description

Reporter system for evaluating the cleavage efficiency of gene scissors {Reporter System for Assessing Cleavage Activity of CRISPR/Cas9}

The present invention relates to a novel reporter system capable of evaluating the cleavage efficiency of gene scissors (CRISPR / Cas9), and more specifically, a first promoter - a gene scissors target sequence - Lac repressor - Poly (poly) A - Second promoter (promoter) - Lac operator (operator) - Reporter (reporter) gene - Poly (poly) A is operably linked in sequence Evaluation of cleavage efficiency of the gene scissors, characterized in that configured It relates to a reporter construct and a method for evaluating cleavage efficiency of gene scissors using the same.

Genetic scissors (CRISPR/Cas9) are derived from the acquired immune system of prokaryotic cells and are widely used for gene editing. The CRISPR/Cas9 system consists of a Cas9 endonuclease and a single guide RNA (sgRNA). sgRNA is a form in which CRISPR RNA (crRNA) having 20 sequences capable of recognizing a target sequence and trans-activating CRISPR RNA (tracrRNA) forming a structure are combined. Cas9 binds to a nucleotide sequence in a specific region of the genome by sgRNA and cleaves the -3 base pair (bp) in front of the 5'-NGG-3' protospacer adjacent motif (PAM) in the target sequence. The cut portion of the target DNA is repaired through non-homologous end joining (NHEJ) or homology directed repair (HDR) processes. In the process of DNA repair of non-homologous end joining (NHEJ), random insertion or deletion of nucleotide sequences occurs between cleavage sites, resulting in frameshifts and premature mutations in the protein coding portion of the gene. Eventually, gene knockout is induced. Homologous recombination (HDR) DNA repair requires a DNA template to repair DNA, and if plasmids or ssODN (single-strand DNA oligonucleotides) are provided exogenously, specific nucleotide sequences can be accurately substituted in the HDR repair process. . Due to these characteristics, the CRISPR/Cas9 system is being used as a new gene editing method.

In order to improve the gene editing efficiency using the CRISPR/Cas9 system, it is important to select a target gene-specific and highly efficient sgRNA with a 20 bp guide sequence--- ¹ . Many studies have been reported to select sgRNA and measure its activity. For example, in silico method--- ^2-5 , PCR ⁶ , Indel Detection by Amplicon Aanalysis (IDAA) ⁷ , surveyor assay ⁸ , T7E1 ⁸ , various methods such as next generation sequencing (NGS) There is this. Although the in silico sgRNA design technology has improved a lot, experimental verification is required due to the complexity of the intracellular environment, sanger sequencing, next generation sequencing (NGS), surveyor nuclease assay that cuts heteroduplex mismatch sequences, and T7E1 Assays can be affected by PCR specificity or polymorphism. Various surrogate reporter systems have been developed to measure the degree of genetic variation of target genes by CRISPR/Cas9. The method of measuring the activity of sgRNA by measuring the expression level of EGFP or antibiotic resistance genes was mainly used to determine the incidence of frameshift genetic mutations in the protein coding region by gene scissors- ^9-13 . However, since these systems depend on a frameshift in the protein coding region, it is difficult to measure the activity of sgRNA when 3n base insertion or deletion occurs. Another surrogate reporter system, when the EGFP gene expressed by the CMV promoter is inserted into the gene of the genome cut by gene scissors, measures the expression level of EGFP using poly A of the inserted gene by FACS to measure the efficiency of guide RNA. Confirmed ⁹ .

Therefore, the present inventors have made intensive research efforts to overcome the problems of the prior art. As a result, when using a reporter expression control system using two promoters and a lac repressor, the efficiency of gene scissors can be measured without depending on the frameshift of the protein coding region. It was confirmed that it is possible, and the present invention was completed.

KR 1013804100000 B KR 1016575040000 B US2019032092 (A1)

Therefore, the main object of the present invention is to provide a novel reporter system that can evaluate the efficiency of gene editing without depending on the frameshift of the protein coding region.

Another object of the present invention is to provide a method for evaluating the efficiency of gene scissors using the reporter system.

As used herein, "gene scissors (CRISPR/Cas9)" refers to a target sequence cleavage system composed of a Cas9 endonuclease and a single guide RNA (sgRNA). "Reporter gene" means any sequence that produces a protein product that is readily measured in routine assays. "Reporter construct" refers to a nucleic acid construct in which a plurality of nucleotide sequence elements designed to function as a reporter gene under specific conditions are linked.

As used herein, "operably linked" permits the juxtaposition of two or more components (sequence elements), each component having a normal function, wherein the component exhibits a function exerted in one of the components at least one of the components. means that they are arranged in a state that allows the possibility of adjustment

According to one aspect of the present invention, the present invention is a reporter construct capable of evaluating the cleavage efficiency of gene scissors (CRISPR/Cas9), a first promoter - a gene scissors target sequence - Lac repressor ) - poly (poly) A - second promoter (promoter) - Lac operator (operator) - reporter (reporter) gene - poly (poly) A cleavage efficiency of the gene scissors, characterized in that the operably linked in sequence A reporter construct for evaluation is provided.

In the present invention, two promoters are used to position the gene scissors target sequence and Lac repressor after the first promoter, and Lac operator and reporter genes are located after the second promoter. By doing this, the cleavage of the gene scissors regulates the expression of a protein ( LacI ) that regulates reporter gene expression so that the activity of the gene scissors can be measured.

In the present invention, the first promoter and the second promoter may be any promoter that can be constitutively expressed in a cell. Preferably, the first promoter is the EF1 alpha promoter and the second promoter is the CMV promoter. It provides a reporter structure for evaluating the cleavage efficiency of the gene scissors, characterized in that.

In the present invention, the gene scissors target sequence has a sequence that can be recognized by a gene scissors (CRISPR/Cas9) system, specifically, a sequence that can be specifically recognized by sgRNA forming a complex with Cas9 A reporter construct for evaluating the cleavage efficiency of gene scissors is provided.

In the present invention, the reporter gene may be any reporter gene expressing a fluorescent or luminescent protein, but preferably a gene expressing luciferase or EGFP (Enhanced Green Fluorescence Protein). It provides a reporter construct for evaluating the cleavage efficiency of

In the present invention, the reporter construct preferably provides a reporter construct for evaluating cleavage efficiency of gene scissors, characterized in that it has the cleavage map of FIG. 1 . In the cleavage map of FIG. 1, Luciferase is only an example of a reporter gene.

In the present invention, when the reporter gene is EGFP, there is provided a reporter structure for evaluating cleavage efficiency of gene scissors, characterized in that the FKBP sequence is inserted after EGFP in order to reduce the background expression of EGFP. By inserting the FKBP sequence as described above, EGFP-FKBP is expressed to promote EGFP degradation. A cleavage map of the reporter construct is shown in Fig. 5a.

In the present invention, there is provided a reporter structure for evaluating cleavage efficiency of gene scissors, characterized in that a non-specific M13 nucleotide sequence of 20 base pairs is inserted before LacI in order to eliminate changes in the activity of the gene scissors according to the sequence arrangement of the target. A cleavage map of the reporter construct is shown in Fig. 4d.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising a reporter construct for evaluating the cleavage efficiency of the gene scissors according to the present invention.

In the present invention, the recombinant vector is preferably deposited under Accession No. KCTC14041BP (EF1a-LacI-oo-CMV-LUC) or Accession No. KCTC14042BP (EF1a-LacI-oo-CMV-GFP). provides a vector. The recombinant vectors were released on 2019.11.21. It was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC: Korean Collection for Type Cultures) on the same date.

According to another aspect of the present invention, the present invention provides a host cell comprising the recombinant vector according to the present invention.

In the present invention, the cell may be any cell (prokaryotic cell or eukaryotic cell) capable of expressing the recombinant vector, but is preferably a eukaryotic cell including yeast, animal cell, human cell, etc. host cells are provided.

According to another aspect of the present invention, the present invention comprises the steps of introducing a vector capable of both transcription of sgRNA and protein expression of Cas9 into the host cell according to the present invention; incubating the cells under conditions in which sgRNA transcription and Cas9 protein are expressed; and measuring the level of reporter gene expression in the cell, wherein the increased level of reporter gene expression correlates with increased scissor-induced cleavage of the target sequence. It provides an evaluation method.

In the present invention, preferably by measuring the expression level of the reporter gene, there is provided a method for evaluating the cleavage efficiency of a gene scissors, characterized in that high-efficiency sgRNA is selected.

In the present invention, preferably a helix-turn-helix motif (Helix-turn-Helix motif) is removed (non-functional) a reporter construct that produces LacI is a gene scissors characterized in that it is used as a control. A method for evaluating the cutting efficiency of

In the present invention, preferably, in order to overcome the problem of increasing the background of the reporter according to the length of the target sequence, the efficiency of sgRNA is measured and evaluated as a relative value to the control that is not cut with gene scissors. To provide a method for evaluating the cleavage efficiency of gene scissors.

Hereinafter, the present invention will be described in more detail.

Genetic scissors (CRISPR/Cas9) recognize and cut a specific part of the genome according to the specificity of sgRNA (single guide RNA). The efficiency of gene editing by gene editing varies depending on sgRNA, and to check the efficiency of gene editing, various methods including PCR, T7E1 assay, Surveyor nuclease assay, next generation sequencing (NGS), and surrogate reporter system are used. have. In the present invention, a reporter technology for measuring the efficiency of gene scissors that can measure the efficiency of a large number of sgRNAs simply and accurately has been developed. The reporter system suppresses luciferase or EGFP reporter expression by binding to the lac repressor (LacI) protein by the EF1α promoter and binding to the lac operator (LacO) located behind the CMV promoter. When the gene scissors cut the sgRNA target sequence that exists between the EF1α promoter and the lacI gene, the expression of the lac repressor is suppressed and the reporter is expressed. The efficiency of DNA cleavage by the gene scissors can be measured by measuring the activity of the reporter expressed by the gene scissors cutting. The reporter system of the present invention can measure the activity of the gene scissors simply and accurately compared to the existing method of measuring the gene scissors efficiency, and using this, the gene scissors can be utilized for gene editing.

The present invention used a reporter expression control system using a lac repressor without depending on the frameshift of the protein coding region. ^14,15 LacI causes the expression of the lac repressor protein by the EF1 α promoter, and this protein binds to the lac operator (LacO) in front of luciferase or EGFP, thereby suppressing the expression of luciferase or EGFP reporter. However, in the present invention, when the target sequence of CRISPR-Cas9 inserted between the EF1 α promoter and LacI is cut by CRISPR-Cas9, LacI by the EF1 α promoter is not expressed, but luciferase or EGFP is expressed. In this way, by measuring the luciferase activity or the expression level of the EGFP reporter, the efficiency of cleavage of the target sequence by CRISPR-Cas9 can be measured.

The reporter invented in the present invention measured the efficiency by designing sgRNA with different efficiencies for the target gene, and similar results were confirmed when the efficiency of the gene scissors measured by the reporter was compared with the results of other experimental methods. Therefore, it is considered that a highly efficient sgRNA can be simply selected using the reporter of the present invention, and gene editing can be efficiently performed in cells and animal models using this.

In the present invention, in order to measure the efficiency of gene editing , a technique for regulating gene expression using LacI was used. In the reporter plasmid measuring the efficiency of gene editing, the LacI gene is expressed by the EF1 alpha promoter, which produces the lac repressor protein. The lac repressor inhibits luciferase expression by binding to the Lac operator located behind the CMV promoter. When the sgRNA target sequence located between the EF1 alpha promoter and Lac I gene is cut by gene scissors, lac repressor protein expression is suppressed and luciferase expression is activated. The cleavage efficiency of the gene scissors can be evaluated by measuring the LacI -luciferase reporter activity (Fig. 1A).

To check whether the expression of luciferase is regulated by the expression of lac repressor, non-functional lacI was prepared. Helix-turn-Helix (HTH) of the lac repressor required for binding to the Lac operator, and a control reporter expressing non-functional LacI (NF- LacI ) were produced by removing the DNA binding domain (FIG. 1B). When the lac repressor did not function, it was confirmed that the activity of luciferase increased by about 100 times, and it was found that the expression of luciferase was regulated according to the degree of LacI expression (FIG. 1C).

There are many reports on the design of sgRNA of gene scissors, among which, based on three reported studies, a ^{guide RNA having a wide range of efficiencies for 3,5,16} , PTEN gene was designed and developed in the present invention. The efficiency of gene editing was measured using a LacI-luciferase reporter (Figure 1D). Among the 12 sgRNAs, when 6 sgRNAs with different efficiencies were selected and the concentration of the gene scissors (0 to 1800 ng) was changed to check whether the expression of the reporter occurs by the gene scissors, the reporter expression increases according to the concentration of the gene scissors. could confirm that (Figure 1E)

In the present invention, 12 sgRNAs were designed in the PTEN gene to check whether the efficiency measurement value of the gene scissors reporter is similar to the result measured by other experimental methods. (Figure 2A). Among them, surveyor analysis was performed on 6 sgRNAs. (Fig. 2B). The correlation between the results of the reporter and surveyor analysis was high at R=0.9702 ( FIG. 2C ), and when the correlation was analyzed by analyzing the genetic variation for 12 sgRNAs by the next-generation sequencing method, the correlation was high at R=0.7529. In order to check whether similar results are obtained for other genes, 12 sgRNAs were designed from the TP53 gene and the correlation between the results of measuring the efficiency of gene editing using the scissors reporter and next-generation sequencing method was high, R=0.8626.

There is a report that if the chromosome is condensed by histone, the access of the sgRNA and Cas nuclease complex to the target DNA strand is inhibited, thereby affecting the cleavage efficiency of the target. ²⁰ The degree of condensing of chromosomes by histones can be measured by the sensitivity of DNA degrading enzymes. The degree of chromosomal condensation is measured by DNase hypersensitivity. If the peak value is 0, it is heterochromatin, if it is 1, it is euchromatin. Among the sequences classified according to the DNase hypersensitivity peak value used in the Cpf1 paper, 20 cas9 target sequences with NGG PAM were randomly selected- ¹⁸ . In the present invention, a total of 20 target sequences randomly selected by classifying 10 each into heterochromatin (condensed) and euchromatin (unwrapped) according to the sensitivity of DNA degrading enzymes are cloned into a reporter at once, and then the efficiency of sgRNA for each target is measured as a reporter As a result of measuring and comparing the on-phase and actual genomes, the correlation for the 20 targets was R=0.6143 (FIG. 3A), but when the correlation was analyzed by dividing it into heterochromatin and euchromatin groups, the differences were R=0.3622 and R=0.8539, respectively. has occurred (Fig. 3B and Fig. 3C). Through this, it was confirmed that factors that affect the cleavage efficiency as well as the characteristics of the target sequence exist when the CRISPR-Cas system operates in the genome. In this reporter system, the efficiency of sgRNA according to the sequence is accurately measured, but It was found that the influence on other genetic factors was not representative.

To confirm the performance of this reporter system , using PTEN Doench_4 sgRNA with the highest efficiency selected on the reporter targeting the PTEN gene, two factors expected to affect the expression of the lac repressor in the reporter; The reporter activity was measured by inducing a change in the nucleotide sequence according to the number of targets and the position of the target (FIG. 4A, FIG. 4D). First, as a result of confirming the reporter performance according to the number of targets, the expression of luciferase increased as the number of targets increased when only the reporter and Cas9 were present without sgRNA (Fig. 4B). It is presumed that as the number of targets increases, the expression level of LacI changes as the distance between the EF1α promoter and LacI increases, thereby affecting the expression of luciferase. However, when the efficiency of Doench_4 sgRNA was corrected with a value relative to the control that was not cut with gene scissors, a difference in reporter activity according to the number of targets could be excluded (Fig. 4C). Therefore, there was no difference in reporter efficiency according to the number of targets. In addition, it was confirmed whether the activity of the reporter was changed when the target had the same number of targets but the arrangement of the targets was different (FIG. 4D). In order to prevent the target sequence that can affect transcription or translation from being positioned at the end, a common sequence of 20 base pairs (M13 forward) was inserted just before LacI. First, as a result of checking the performance of the reporter according to the positional arrangement of the target, there was no change when only the reporter and Cas9 were present without sgRNA (Fig. 4E), and the cleavage efficiency of the gene scissors could be measured regardless of the arrangement (Fig. 4F). Based on these results, it was found that this reporter system accurately measures the efficiency regardless of the target length and alignment order.

In the present invention, it was attempted to develop a reporter system capable of measuring the efficiency of sgRNA not only through a method of measuring luciferase but also through the expression of EGFP (FIG. 5A). In the LacI -EGFP reporter system, the background expression of EGFP was too strong in the reporter alone without gene editing, and the FKBP-derived destabilizing domain was added after EGFP to rapidly degrade EGFP, so that the EGFP background could be removed ²¹ . In the reporter expressing non-functional LacI (NF- LacI ), it was confirmed that the expression of EGFP was increased by about 30-fold (FIG. 5B). By measuring the efficiency of six sgRNAs in the reporter, it was found that the expression of EGFP was regulated according to the efficiency of gene scissors (FIG. 5C). When comparing the sgRNA efficiency measured using the luciferase reporter and the LacI -EGFP reporter, the correlation was R=0.9218 ( FIG. 5D ). Through this, it was confirmed that the efficiency of sgRNA can be measured exactly like the LacI -luciferase reporter even using the LacI-EGFP reporter.

Nutlin is an MDM2 inhibitor, and cells that normally express TP53 are all killed by Nutlin, and cells that do not express TP53 continue to survive without responding to Nutlin. In order to confirm whether the sgRNA selected in the reporter system developed in the present invention is applicable to the study of gene editing in cells, the efficiency of sgRNA for the TP53 gene was applied to actual human liver organoids. Among the total of 12 sgRNAs measured by the reporter, 6 sgRNAs with different efficiencies were selected and delivered to human liver organoids, followed by treatment with 40 μM Nutlin (Fig. 6A). As a result of measuring the survival rate of human liver organoids after Nutlin treatment through ATP assay, it was found that there was a difference in the survival rate according to the efficiency of the sgRNAs measured on the reporter (Fig. 6B, Fig. 6C). These results mean that gRNAs with excellent efficiency can be selected using the reporter developed in the present invention, and this will be utilized for gene editing in cells.

According to the present invention, since the reporter systems for evaluating the efficiency of conventional gene editing depend on a frameshift in the protein coding region, it is difficult to measure the activity of the gene scissors when a 3n base insertion or deletion occurs. Thus, the present invention has the advantage of being able to evaluate the gene scissoring efficiency regardless of the frame shift. Therefore, using the reporter system of the present invention, it is possible to simply select highly efficient sgRNA and use it to efficiently perform gene editing in cells and animal models.

The reporter system manufactured according to the present invention can perform the efficiency of gene editing simply and accurately by replacing techniques such as surveyor assay, next generation sequencing, and surrogate reporter system used to check the efficiency of gene editing. In addition, it is expected that multiple guide RNAs can be tested at once and selected in the order of highest efficiency, which can be used to regulate gene expression in cells and animals.

Figure 1. LacI verify the performance of the works, and LacI -luciferase Reporter Reporter -luciferase
(A) Schematic diagram of the LacI-luciferase reporter. When the target sequences are inserted between the EF1α promoter and the LacI sequence, the LacI gene is expressed by the EF1α promoter when CRISPR is not present, thereby suppressing the expression of the luciferase present thereafter. When the target sequence is cleaved by CRISPR, LacI expression is suppressed and luciferase is expressed. pA: polyadenylation sequence, oo: double lac operator, CMV: cytomegalovirus
(B) Non-functional LacI fabrication. To confirm the expression of the reporter luciferase by LacI , a non-functional LacI (NF LacI ) in which the Helix-turn-Helix (HTH) domain, which is a DNA binding domain, was removed was prepared.
(C) Non-functional LacI -luciferase reporter alone was delivered to the 293T cell line, and then the luciferase expression of the reporter was measured 48 hours later. The experiment was repeated three times. ( ***p<0.001.)
(D) Measuring the efficiency of 12 sgRNAs for the human PTEN gene using a reporter. After cloning 12 target sequences of human PTEN sgRNA selected 4 each from the Doench, GeGKO, and Low groups into the reporter, the reporter luciferase value was measured for each sgRNA in the 293T cell line. Relative luciferase activity compared to Renilla was calculated, replaced with a fold value for the relative luciferase value of the reporter alone, and displayed on the y-axis. The experiment was repeated three times.
(E) Changes in luciferase values between sgRNAs according to the concentration of CRISPR. As a result of (D), six sgRNAs were selected, and as the CRISPR concentration increased, the difference in the luciferase value on the reporter between the sgRNAs increased. The concentration of sgRNA per 2.5 x 10 ⁵ cells is shown on the X axis.
Figure 2. Efficiency of sgRNAs was measured using LacI-luciferase reporter
(A) Twelve sgRNAs selected from the Human PTEN locus. (The red arrow indicates the position and direction of the sgRNAs belonging to the Doench group, the blue arrow is the GeCKO group, and the black arrow is the Low group). Representative next-generation sequencing data was expressed as indels of the target site by sgRNA.
(B) Representative Surveyor assay results for 6 sgRNAs in the PTEN gene. Six sgRNAs selected as a result of Figure 1-(D) were delivered to each 293T cell line, and then, 48 hours later, genetic variation was analyzed using surveyor assay.
(C) Correlation of sgRNA efficiency measured by Surveyor assay and LacI-luciferase reporter. In Figure 1-(D), the efficiency of six sgRNAs for the human PTEN gene measured by the LacI- luciferase reporter was measured by a surveyor assay method, and the correlation between the two experimental methods was compared.
(D) Correlation between LacI- luciferase reporter and sgRNA efficiency by next-generation sequencing. By analyzing the reporter activity of 12 sgRNAs for the human PTEN gene and the indels occurrence rate analyzed by NGS, the correlation between the two methods was expressed as R value.
(E) Twelve sgRNAs selected from the Human TP53 locus. The red arrow indicates the position and direction of the sgRNAs belonging to the Doench group, the blue arrow to the GeCKO group, and the black arrow to the Low group. In the representative next-generation sequencing data, the indel of the target site by sgRNA was expressed.
(F) Correlation between LacI- luciferase reporter and sgRNA efficiency by next-generation sequencing method. By analyzing the reporter activity of 12 sgRNAs for the human TP53 gene and the indels generation rate analyzed by NGS, the correlation between the two methods was expressed as R value.
Figure 3. Measuring the efficiency of sgRNA targeting regions with different DNase hypersensitivity using a LacI-luciferase reporter
(A) Analysis of the correlation between LacI- luciferase reporter measurements and NGS-analyzed indels ratio for sgRNAs targeting a total of 20 different regions, each of which is present in euchromatin and heterochromatin. Red dots are euchromatin, blue dots are heterochromatin.
(B) Analysis of the correlation between LacI- luciferase reporter measurements and indels ratio analyzed by NGS for 10 sgRNAs targeting euchromatin.
(C) Correlation of indels ratio analyzed by NGS and LacI-luciferase reporter measurements for 10 sgRNAs targeting heterochromatin
Figure 4. Measurement efficiency of LacI- luciferase reporter according to target length and alignment position
(A) Schematic diagram of the number of target sequences between the EF1α promoter and LacI. Among the sgRNAs for the human PTEN gene, when the sgRNA target sequence showing the highest efficiency on the reporter was added alone (1) and when inserted with other target sequences (6, 12) were designed.
(B) As a result of measuring the relative luciferase activity of the reporter alone in the 293T cell line, the luciferase expression increased as the number of targets increased. ***p<0.001 All experiments were repeated three times.
(C) In the 293T cell line, the luciferase activity when the reporter cleaved the reporter in the presence of sgRNA was calculated as a fold value for reporters with different target lengths and the reporter was displayed on the y-axis. The target length is indicated on the X axis. Although the target length was different, the efficiency of sgRNA on the reporter was similar. All experiments were repeated three times. (ns:not significant)
(D) Schematic diagram of the alignment position of the target between the EF1α promoter and LacI. Among the sgRNAs for the human PTEN gene, the sgRNA showing the highest efficiency on the reporter was designed when the first, middle, and last sgRNAs were arranged. In order not to affect the expression of LacI according to the sequence change, a non-specific 20base pair of M13 sequence was added before LacI.
(E) As a result of measuring the relative luciferase activity of the reporter alone in the 293T cell line, the alignment position of the target does not affect the expression of the reporter luciferase. (ns:not significant, n=3)
(F) In the 293T cell line, the luciferase activity when the reporter cleaved the reporter in the presence of sgRNA was calculated as a fold value for the reporter alone in the reporter with a different target alignment position, and it is shown on the y axis. The position of the target is indicated on the X axis. Even if the target alignment position was different, the efficiency of sgRNA on the reporter was similar. All experiments were repeated three times (ns: not significant, n=3).
Figure 5. Verify performance by constructing a LacI-EGFP reporter
(A) Schematic diagram of the LacI-EGFP reporter. The principle of operation is similar to that of the LacI- luciferase reporter, and the FKBP sequence is inserted after EGFP so that it can be degraded to eliminate the EGFP background expression of the reporter.
(B) To confirm that EGFP expression is regulated by LacI , NF LacI- EGFP reporter was constructed and EGFP expression was confirmed by measuring MEAN values through FACS analysis (***p<0.001, n=3).
(C) Efficiency of six sgRNAs selected through a LacI- luciferase reporter for the human PTEN gene was measured through EGFP expression.
(D) Correlation analysis of sgRNA efficiency measurements in LacI- Luciferase reporter and LacI-EGFP reporter
Figure 6. In vivo verification of the efficiency of sgRNA for the human TP53 gene measured on the reporter
(A) Each of the six human TP53 sgRNAs selected from the reporter was delivered to the human liver organoid, and then treated with nutlin, an mdm2 inhibitor, to compare the viability of each sgRNA compared to the normal expression of TP53 (control).
(B) The liver organoid of (A) was measured for cell viability through ATP assay.
(C) Correlation analysis between the efficiency measured on the reporter of 6 sgRNAs and cell viability measured through ATP assay.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

Example 1: Construction of a reporter plasmid capable of evaluating the efficiency of gene editing

LacI -luciferase Reporter plasmid (EF1α -mutiple target sites- lacI -polyA- CMV- lac O -luciferase-polyA) is in Dr. Van Andel Research Institute It was constructed using CMV-lacO- Luciferase and EF1α- LacI plasmids provided by Peter W. Laird ¹⁵ . EF1α-multiple target sites (EcoRI, BlpI) - LacI -poly A was EF1α-multiple target sites, LacI, and then the poly A PCR amplification using the In-Fusion cloning HD inserted into the NotI cleavage site of the CMV- lacO -Luciferase became LacI -luciferase prepared above The reporter plasmid (EF1α -mutiple target sites- lacI -polyA-CMV- lac O -luciferase-polyA) was deposited with the Korean Collection for Type Cultures with accession number KCTC14041BP (EF1a-LacI-oo-CMV-). LUC).

LacI - EGFP reporter plasmid (EF1α -mutiple target sites- LacI -polyA- CMV- lacO -EGFP-FKBP-polyA) is LacI - removing by cutting a luciferase reporter plasmid with luciferase HindШ / XbaI, and inserted into the PCR-amplified EGFP was produced. After PCR amplification of non-functional LacI (NF- LacI ) in which the HTH (helix-turn-helix) portion, which is the DNA binding domain of LacI , was removed, luciferase or LacI -EGFP reporter was cut and inserted with BlpI/NheI. To suppress the background expression of EGFP, the self-degrading FKBP12 derived destabilizing domain (FKBP) sequence was PCR amplified and then inserted into the BsrgI and PacI sites to prepare a reporter expressing the EGFP-FKBP fusion protein. The production cost LacI - EGFP reporter plasmid (EF1α -mutiple target sites- LacI -polyA- CMV- lacO -EGFP-FKBP-polyA) is deposited on the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center (Korean Collection for Type Cultures) Number KCTC14042BP (EF1a -LacI-oo-CMV-GFP).

In Table 1, the primers used to construct the reporter of the present invention are specified.

name direction order EF1a-multiple target sites Forward SEQ ID NO: 1 EF1a-multiple target sites Reverse SEQ ID NO: 2 LacIGY Forward SEQ ID NO: 3 LacIGY Reverse SEQ ID NO: 4 poly A Forward SEQ ID NO: 5 poly A Reverse SEQ ID NO: 6 LacIGY GFP Forward SEQ ID NO: 7 LacIGY GFP Reverse SEQ ID NO: 8 NF LacIGY Forward SEQ ID NO: 9 NF LacIGY Reverse SEQ ID NO: 10

Example 2: sgRNA design

In silico (CRISPRko; https://portals.broadinstitute.org ), sgRNAs expected to have high efficiency by targeting the PTEN or TP53 gene to select sgRNAs with various efficiencies to confirm the function as a reporter measuring the efficiency of sgRNA /gpp/public/analysis-tools/sgrna-design , named Doench) ³ and the sgRNA sequence used for genome-scale CRISPR library screening (GeCKO) - ¹⁶ The GC ratio of the target sequence of 20 base pairs was less than 25%, so 4 were directly designed based on the sgRNA, which is expected to have the lowest efficiency (named Low) - ⁵ .

In order to insert the target sequence for the designed sgRNA into the reporter, primer annealing or PCR with one or several target sequences linked in series with the 23 base pair designed between the EF1α promoter and LacI of the LacI -luciferase reporter or LacI-EGFP reporter was synthesized through, and then inserted into a reporter plasmid digested with EcoRI/Blp1. ^{A guide RNA was inserted into the vector (px459, addgene) 17} in which sgRNA transcription and Cas9 protein expression can occur together using restriction enzyme (bbs1).

Experimental Example 1: The activity of gene scissors LacI - Measured with luciferase reporter

After delivering luciferase reporter plasmid (200ng), renilla (100 ng), and px459 (sgRNA+Cas9) plasmid (1800ng) to 2.5 x 10 ^{5 HEK293T cell line using Lipofectamine 3000, firefly luciferase through luminocensce (610nm) 48 hours later} and renilla luciferase activity were measured, and the relative activity of firefly luciferase to renilla was calculated to measure the efficiency of each sgRNA. , The cleavage efficiency between the sgRNA candidates was compared by calculating the fold value compared to the luciferase value when only the px459 control (Cas9 without sgRNA) plasmid and the lacI-luciferase reporter were present. In order to find the optimal concentration of sgRNA and Cas9 in the reporter system, the px459 (sgRNA+Cas9) plasmid (0 ng to 1800 ng) was tested and it was confirmed that 1800 ng was appropriate.

Experimental Example 2: Surveryor analysis

A reporter having a target sequence for 6 g RNAs (Doench_3, Doench_4, GeCKO_3, GeCKO_4, Low_1, Low_4) targeting the ^{PTEN gene and px459 (sgRNA+Cas9) plasmids were applied to 2.5 x 10 5} HEK293T cells using Lipofectamine3000. Then, 2000ng each was delivered, and 48 hours later, all cells were rolled up and genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega). Each 100ng of the extracted genomicDNA was subjected to PCR using primers designed to include each target sequence, and then treated with surveyor nuclease according to the usage of the Surveyor® Mutation Detection Kit (idt), followed by electrophoresis, and then the strand cut by nuclease. The relative amounts of these were quantified using imageJ.

Experimental Example 3: NGS analysis (targeted deep sequencing)

The sgRNA expression plasmid (px459) targeting PTEN was transferred to HEK293T cells using Lipofectamine3000, and after 48 hours, genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega). Then, using 100 ng of genomic DNA, a total of 160 bp was amplified by PCR by 80 bp before and after the site where the target sequence was delivered by Cas9. The amplified DNA product was purified using the QIAquick® Gel Extraction Kit (Qiagen), and the Illumina adapter was attached to both strands of the DNA through PCR on 20 ng of the product. This was purified once more using QIAquick® Gel Extraction Kit (Qiagen), and then next-generation sequencing was performed using Hiseq. For analysis data, the primary data is separated under suitable conditions (comparison range=50, minimum frequency=1, WT marker=5) using Cas-Analyzer, and then the secondary condition (read counts cut off=1/1000 of total) Read counts), the data separated once more were compared with the normal sequence to analyze the ratio of actual Cas9 cleavage.

Experimental Example 4: Euchromatin and Heterochromatin (DNase sensitive site) selection

The degree of condensing of chromosomes by histones can be measured by the sensitivity of DNA-degrading enzymes, and DNase-sequencing of the sensitivity to DNA-degrading enzymes in the HEK293T cell line presented in the Encyclopedia of DNA Elements (ENCODE) for specific sequence sites was performed. Sensitivity to DNA degrading enzyme was classified as 0 or 1 according to the match between the specific sequence and the sequence containing the peak using the data indicated by the peak after analysis through ^{18- 18} . In this data, a total of 20 target sequences randomly selected by classifying 10 each into heterochromatin (condensed) and euchromatin (unwrapped) according to the sensitivity to DNA degrading enzyme among the parts containing the target sequences for Cas9 with NGG PAM. was inserted into a LacI -luciferase reporter plasmid, and the efficiency of sgRNA for each target was measured on the reporter through the activity of luciferase, and measured on the actual genome through targeted deep sequencing (NGS) and compared.

Experimental Example 5: FACS analysis

In order to suppress the background expression of EGFP in the EGFP reporter, the back of the EGFP sequence was cut with Bsrg1 and Pac1, and then the FKBP sequence that made itself degraded was inserted through PCR. Six target sequences targeting PTEN selected by the luciferase reporter were generated. At one time, it was inserted between the EF1α promoter and LacI through PCR in the same way as the LacI-luciferase reporter. Then, the HEK293T cell line was transferred together with the sgRNA plasmid (px459) targeting each target sequence using lipofectamine3000, and 48 hours later, FACS analysis was performed to determine the difference in the increased EGFP expression in the presence of CRISPR compared to the EGFP expression in the reporter alone. It was analyzed by measuring the change in the MEAN value.

Experimental Example 6: Organoid culture and Nutlin-3 selection

Human liver organoid culture medium {ADF-12 (+1% penicillin/streptomycin, 1% GlutaMAX, 10Mm HEPES, 10% Rspondin1, 25ng/ml hHGF, 100ng/ml hFGF10, 1X B27 supplement, 1mM N- Cetylcysteine, 10nM gastrin, 10mM nicotinamide, 1X N2 supplement, 50ng/ml hEGF, 10μM Forkskolin, 5μM A83-01) were cultured in 24 wells (80% confluency of 50μl BME metrix) and TP53 TP53 selected through LacI -luciferase reporter Plasmids (px459) expressing six target sgRNAs (Doench_2, Doench_3, GeCKO_3, GeCKO_4, Low_1, Low_3) were respectively transferred using Lipofectamine3000, ^{then treated with 40 μM Nutlin 19} and 24 hours later, and organoid viability for 6 days. was observed. For the survival rate of human liver organoids, CellTiter-Glo luminescent assay (Promega) was used to measure the amount of ATP. Briefly, the organoid culture plate was equilibrated at room temperature, the cell culture medium was removed, and CellTiter-Glo reagent was mixed with PBS (1:1) and added, followed by ^{measurement using SpectraMax ⓡ} Paradigm ^ⓡ (Molecular Devices).

Although the present invention has been described with reference to the above embodiments, it will be understood that these are merely exemplary, and that various modifications and equivalent other embodiments are possible therefrom by those skilled in the art to which the present invention pertains. . Therefore, the true technical protection scope of the present invention will have to be determined by the technical matters of the appended claims.

[references]

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Korea Institute of Bioscience and Biotechnology KCTC14041BP 20191121 Korea Institute of Bioscience and Biotechnology KCTC14042BP 20191121

<110> KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES <120> Reporter System for Assessing Cleavage Activity of CRISPR/Cas9 <130> PN190436 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> EF1a-multiple target sites Forward primer <400> 1 gtacttggag cggccgcggc tccggtgccc gtcagtg 37 <210> 2 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> EF1a-multiple target sites Reverse primer <400> 2 ttgcccatgg tggcggctta gcggcgcgcc gaattctcac gacacctgaa atggaa 56 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LacIGY Forward primer <400> 3 cgccaccatg ggcaaatatg taac 24 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LacIGY Reverse primer <400> 4 tcacaccttc ctcttcttct ta 22 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> poly A Forward primer <400> 5 aagaggaagg tgtgagcaga catgataaga tacat 35 <210> 6 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> poly A Reverse primer <400> 6 gtatatctgg cggccgctac cacatttgta gaggttt 37 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> LacIGY GFP Forward primer <400> 7 agtcaagctt ccaccatggt gagcaagggc gagga 35 <210> 8 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> LacIGY GFP Reverse primer <400> 8 agcttctaga ttaattaatt acttgtacag ctcgtcca 38 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> NF LacIGY Forward primer <400> 9 gctaagccgc caccggagtt gccacctcca gtct 34 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> NF LacIGY Reverse primer <400> 10 gctagctgca tgagtgaatc agccaa 26

Claims (15)

As a reporter construct capable of evaluating the cleavage efficiency of the gene scissors composed of Cas9 nuclease (endonuclease) and sgRNA (single guide RNA), a first promoter - multiple target sequences - Lac repress Repressor - poly (poly) A - second promoter (promoter) - Lac operator (operator) - reporter (reporter) gene - poly (poly) A reporter construct, characterized in that it is operably linked in sequence A composition for evaluating the cutting efficiency of gene scissors comprising a. The composition of claim 1, wherein the first promoter is an EF1 alpha promoter and the second promoter is a CMV promoter. The composition for evaluating the cutting efficiency of the gene scissors according to claim 1, wherein the gene scissors target sequence has a sequence that can be specifically recognized by sgRNA forming a complex with Cas9. The composition of claim 1, wherein the reporter gene is a gene expressing luciferase or EGFP (Enhanced Green Fluorescence Protein). The composition for evaluating cleavage efficiency of gene scissors according to claim 1, wherein the reporter structure has the cleavage map of FIG. According to claim 1, wherein when the reporter gene is EGFP (Enhanced Green Fluorescence Protein), in order to reduce the background expression of EGFP, the FKBP sequence is inserted after EGFP. The composition for evaluating the cutting efficiency of gene scissors. According to claim 1, wherein in order to eliminate the change in the activity of the gene scissors according to the sequence arrangement of the target Lac repressor (repressor, LacI ) in front of the non-specific M13 nucleotide sequence of 20 base pairs is inserted characterized in that the cutting efficiency evaluation of the gene scissors for composition. A recombinant vector comprising a reporter construct included in the composition for evaluating the cleavage efficiency of gene scissors according to any one of claims 1 to 7. The recombinant vector according to claim 8, wherein the recombinant vector is deposited under Accession No. KCTC14041BP (EF1a-LacI-oo-CMV-LUC) or Accession No. KCTC14042BP (EF1a-LacI-oo-CMV-GFP). 10. A host cell comprising the recombinant vector according to claim 8 or 9. 11. The host cell of claim 10, wherein the cell is a eukaryotic cell. 12. A method comprising: introducing a vector capable of sgRNA transcription and Cas9 protein expression into the host cell according to claim 10 or 11;
incubating the cells under conditions in which sgRNA transcription and Cas9 protein are expressed; and
measuring the level of reporter gene expression in the cell;
wherein the increased level of reporter gene expression is correlated with increased scissor-induced cleavage of the target sequence. The method according to claim 12, wherein the high-efficiency sgRNA is selected by measuring the expression level of the reporter gene. 13. The cleavage of the gene scissors according to claim 12, wherein a reporter construct that produces non-functional LacI from which a helix-turn-Helix motif has been removed is used as a control. Efficiency evaluation method. The method according to claim 12, wherein the efficiency of sgRNA is measured and evaluated as a relative value to a control that is not cut with gene scissors in order to overcome the problem that the background of the reporter itself increases according to the length of the target sequence. A method for evaluating the cutting efficiency of gene scissors.

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