KR-20260067993-A - Method for controlling CRISPR-Cas system using a DNA regulator that responds to miRNA

KR20260067993AKR 20260067993 AKR20260067993 AKR 20260067993AKR-20260067993-A

Abstract

The present invention relates to a method for regulating a CRISPR-Cas system using an inhibitory DNA aptamer that responds to miRNA. Specifically, the present invention relates to a DNA aptamer for regulating CRISPR-Cas system activity, wherein the aptamer is composed of two DNA strands, strand 1 and strand 2, and strand 1 is composed of a seed region containing a sequence identical to the target strand base sequence of a target DNA that is a target for gene editing by CRISPR-Cas, a PAM (Protospacer Adjacent Motif) region, and a stem region containing a sequence identical to miRNA; and strand 2 is composed of a PAM region, a stem region composed of a sequence complementary to the stem region of strand 1, and a toehold region containing a sequence complementary to the miRNA sequence. The present invention relates to a method for cell-specifically regulating the activity of the CRISPR-Cas system using the DNA aptamer for regulating the activity of the CRISPR-Cas system.

Inventors

정철희
윤다영

Assignees

고려대학교 산학협력단

Dates

Publication Date: 20260513
Application Date: 20251029
Priority Date: 20241105

Claims (15)

As a DNA aptamer for regulating CRISPR-Cas system activity, The above aptamer consists of two DNA strands, strand 1 and strand 2, and The above strand 1 is composed of a seed region containing a sequence identical to the target strand base sequence of the target DNA that is the target of gene editing by CRISPR-Cas, a PAM (Protospacer Adjacent Motif) region, and a stem region containing a sequence identical to miRNA, and A DNA aptamer for regulating CRISPR-Cas system activity, characterized in that strand 2 comprises a PAM region, a stem region composed of a sequence complementary to the stem region of strand 1, and a toehold region comprising a sequence complementary to the miRNA sequence.
In paragraph 1, The above DNA aptamer for regulating CRISPR-Cas system activity is characterized by inhibiting the cleavage of target DNA by binding to a Ribonucleoprotein (RNP) complex and inhibiting the activity of the CRISPR-Cas system under conditions in the absence of miRNA.
In paragraph 1, The DNA aptamer for regulating CRISPR-Cas system activity is characterized in that, under conditions where miRNA is present, the tohold region of strand 2 binds complementarily to the miRNA, causing strand displacement, so that the stem regions of strand 1 and strand 2, which were complementarily bound, are separated into a single strand, and the DNA aptamer is separated from the RNP complex, thereby restoring the activity of the CRISPR-Cas system that had been inhibited, and causing the RNP complex to recognize and cleave the target DNA.
In paragraph 1, The above DNA aptamer for regulating CRISPR-Cas system activity is characterized by being able to specifically regulate the activity of the CRISPR-Cas system in cells expressing miRNA.
In paragraph 4, A DNA aptamer for regulating CRISPR-Cas system activity, characterized in that the cell expressing the above miRNA is a cancer cell or an aging cell.
In paragraph 1, A DNA aptamer for regulating CRISPR-Cas system activity, characterized in that the target DNA is DNA encoding telomerase reverse transcriptase (TERT).
In paragraph 1, A DNA aptamer for regulating CRISPR-Cas system activity, characterized in that the miRNA is miR21, which is overexpressed in cancer cells.
In paragraph 1, The above-mentioned DNA aptamer for regulating CRISPR-Cas system activity is, A DNA aptamer for regulating CRISPR-Cas system activity, characterized by comprising: any one strand 1 selected from the group consisting of the nucleotide sequences of SEQ ID NOs 1 to 5; and a strand 2 consisting of the nucleotide sequence of SEQ ID NO. 6.
In paragraph 1, A DNA aptamer for regulating CRISPR-Cas system activity, characterized in that a Locked Nucleic Acid (LNA) is inserted or substituted in the tohold region of strand 2.
In Paragraph 9, A DNA aptamer for regulating CRISPR-Cas system activity having an inserted or substituted LNA (Locked Nucleic Acid) is characterized by enhancing the reactivation of the CRISPR-Cas system by increasing the binding affinity between miRNA and the tohold of strand 2.
A method for specifically regulating the activity of a CRISPR system using a DNA aptamer for regulating the activity of a CRISPR-Cas system according to any one of claims 1 to 10.
In Paragraph 11, The above method is characterized by specifically regulating the activity of the CRISPR-Cas system in cells expressing miRNA.
In Paragraph 12, A method characterized in that the cell expressing the above miRNA is a cancer cell or an aging cell.
In Paragraph 11, A method characterized in that the above-described DNA aptamer for regulating CRISPR-Cas system activity binds to an RNP (Ribonucleoprotein) complex and inhibits the activity of the CRISPR-Cas system under conditions in the absence of miRNA.
In Paragraph 11, A method characterized in that, under conditions where miRNA is present, the DNA aptamer for regulating the activity of the CRISPR-Cas system detaches from the RNP complex and reactivates the activity of the CRISPR-Cas system that had been inhibited.

Description

Method for controlling the CRISPR-CAS system using a DNA regulator that responds to miRNA The present invention relates to a novel method for regulating a CRISPR-Cas system using a DNA regulator that binds to a Ribonucleoprotein (RNP) complex formed by Cas9 nuclease and gRNA and responds to miRNA. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9) is a gene editing technology derived from the adaptive immunity of microorganisms that cleaves target dsDNA (double-strand deoxyribonucleotide) present within biological genomes. This system consists of gRNA and Cas9 nuclease; the gRNA comprises a 20nt (nucleotide) guide sequence that binds complementarily to the target sequence and a scaffold sequence for Cas9 binding. The Cas9 nuclease possesses an REC lobe that recognizes the gRNA scaffold sequence and a NUC lobe with nuclease function. The NUC lobe contains HNH and RuvC domains, respectively, capable of cleaving the target strand and non-target strand. The target recognition and cleavage process by CRISPR-Cas9 is as follows. After Cas9 and gRNA form an RNP complex, they recognize the 5'-NGG-3' PAM (Protospacer Adjacent Motif) sequence located on the non-target strand of the target sequence. By recognizing the target strand complementary to the guide sequence located upstream of the PAM, they induce a DNA Double Strand Break (DSB). Since DNA Double Strand Breaks are highly dangerous to cells, they are repaired through intracellular repair mechanisms; during this process, gene editing occurs, such as gene removal through the insertion of random nucleotide sequences or gene insertion using homologous template strands. The CRISPR system is attracting attention as a therapeutic gene editing technology due to its advantages, such as simple manipulation, high efficiency, and the ability to perform various gene manipulations including substitution, deletion, and insertion through diverse variants. However, there are still difficulties in regulating cell-specific activity. Meanwhile, miRNAs have the advantage of being expressed endogenously within cells, resulting in low toxicity and eliminating the need for exogenous introduction. Furthermore, miRNAs are small, non-coding RNA molecules that play a crucial role in the post-transcriptional regulation of genes, and their potential as biomarkers for various diseases, particularly cancer and cardiovascular disease, is widely recognized. Therefore, developing methods to detect miRNAs sensitively and specifically is critical in the field of molecular diagnostics. Accordingly, the inventors conducted research to develop a CRISPR system differentiated from other regulators by using non-toxic, endogenously expressed miRNAs as a solution to the problems of existing CRISPR systems. To this end, the inventors completed the present invention by developing a method to specifically control the activity of the CRISPR-Cas9 RNP form using independent DNA regulators that respond to miRNAs. Figure 1 is a schematic diagram showing the CRISPR-Cas regulatory action using the miRNA-responsive DNA regulator of the present invention, indicating that the activity of CRISPR-Cas is activated only when a specific miRNA is present. Figure 2 illustrates the process of regulating CRISPR-Cas activity using the DNA regulator of the present invention, wherein (A) the characteristics of the target dsDNA are shown, the target DNA is composed of a target strand (TS) and a non-target strand (NTS), the target strand contains a target sequence complementary to sgRNA, and the non-target strand contains an NGG PAM sequence, (B) the structure of the DNA aptamer, which is the DNA regulator of the present invention, is shown schematically, consisting of two separate strands, strand 1 contains a PAM sequence and a seed sequence, and strand 2 contains a tohold sequence and a stem sequence complementary to a specific miRNA, (C) the mechanism of CRISPR-Cas9 regulation by the DNA regulator of the present invention, and the reactivation of the second step is induced only in the presence of the target miRNA. Figure 3 confirms the inhibitory effect of CRISPR-Cas9 activity according to the seed length of the DNA aptamer, which is the DNA regulator of the present invention, (A) shows the region that plays the most important role in inhibiting RNP cleavage activity in the DNA aptamer region of the present invention, (B) confirms the CRISPR-Cas9 activity inhibitory ability of the DNA regulator of the present invention as a result of in vitro cleavage analysis, where the molar ratio of RNP to DNA regulator was 1:1, NC represents the negative control, and PC represents the positive control, (C) shows the ratio of cleaved target DNA quantified as a graph, and ND represents not detected. Figure 4 shows the analysis of CRISPR-Cas9 reactivation efficiency according to the tohold length of the DNA aptamer, which is the DNA regulator of the present invention, where (A) the tohold is the