KR-20260062200-A - Methods and gene editing components for ultra-precision single-base editing

Abstract

The present invention relates to a method for ultra-precision single base editing and a gene editing component. It proposes an innovative gene editing method based on the combination with a deamination enzyme by developing a guide RNA (gRNA) for high-accuracy base editing. The gRNA of the present invention includes a mismatch region and a shielding region for a single base substitution region. It was confirmed that by utilizing a gRNA containing a mismatch region between the gRNA and the target DNA sequence, the deamination enzyme is improved to accurately recognize adenine, which is the base conversion target of the single base substitution region, thereby inducing accurate target base substitution without substitution of other bases within the target sequence. Thus, the combination of the engineered gRNA and the base substitution enzyme can be usefully utilized as a next-generation single base editor capable of inducing ultra-precision single base substitution.

Inventors

• 이승환
• 바돈 이사벨 웬
• 김윤경

Assignees

• 중앙대학교 산학협력단

Dates

Publication Date

20260507

Application Date

20241025

Claims (20)

1. It includes a mismatched base region for a single base substitution region; and a shielding region, The mismatched base region for the above single base substitution region is characterized by the following: i) The single base substitution region further comprises adenine that is a base conversion target; and 0 to 6 bases from the adenine in the 3' or 5'direction; and ii) The above mismatched base region does not include a base corresponding to the base of the above single base substitution region, or includes a base that cannot form a complementary bond; A single base editing guide RNA (gRNA), wherein the shielding region comprises a base complementary to a single base substitution region other than a mismatched base region.
2. In paragraph 1, A guide RNA (gRNA) for single base editing, wherein the single base substitution region comprises an adenine that is a base conversion target; and zero to two bases from the adenine in the 3' or 5' direction.
3. In paragraph 1, A single base editing guide RNA (gRNA), wherein the above gRNA comprises a form that includes a mismatched base region and a shielding region for a single base substitution region in succession.
4. In paragraph 1, A single base editing guide RNA (gRNA), wherein the above gRNA includes a truncated form modified to form a protruding structure in a single base substitution region.
5. In paragraph 1, A guide RNA (gRNA) for single base editing, wherein the mismatched base region for the single base substitution region and the shielding region are connected to the 5' end of the gRNA.
6. In paragraph 1, A single-base editing guide RNA (gRNA), characterized in that the single-base substitution region has two or more adenines.
7. In paragraph 1, A single-base editing guide RNA (gRNA) in which the number of bases in the mismatch region is 5 bp to 10 bp.
8. In paragraph 1, The single-base editing guide RNA (gRNA) comprises any one base sequence selected from the group consisting of SEQ ID NOs 8 to 18.
9. In paragraph 1, A single-base editing guide RNA (gRNA) characterized by having 40% or more of the total bases included in the single-base substitution region.
10. In paragraph 1, A single-base editing guide RNA (gRNA), wherein the above single-base editing is the substitution of adenine with guanine.
11. A composition for single base editing comprising the gRNA of claim 1 and a deamination enzyme.
12. In Paragraph 11, A composition for single base editing in which the above-mentioned deamination enzyme deaminates adenine.
13. In Paragraph 11, A composition for single salt editing in which the above-mentioned deamination enzyme is TadA 8e or ADAR.
14. In Paragraph 13, A composition for single base editing, wherein the above TadA 8e comprises the amino acid sequence represented by SEQ ID NO. 20; and the above ADAR comprises the amino acid sequence represented by SEQ ID NO. 22.
15. In Paragraph 11, A composition for single base editing, wherein the single base editing described above is the substitution of adenine with guanine.
16. A single-base editing vector comprising a base sequence representing the gRNA of claim 1 and a base sequence encoding a deamination enzyme.
17. In Paragraph 16, A single-base editing vector, wherein the vector is any one selected from the group consisting of an adeno-associated virus vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, and a herpes simplex virus vector.
18. In Paragraph 16, A vector for single base editing, wherein the above single base editing is the substitution of adenine with guanine.
19. In Paragraph 16, The above vector is a single base editing vector comprising a base sequence represented by SEQ ID NO. 23 or SEQ ID NO. 25.
20. A method for editing a single nucleotide, comprising the step of infecting a host cell with the vector of claim 16.

Description

Methods and gene editing components for ultra-precision single-base editing The present invention relates to a method for ultra-precision single base editing and a gene editing component. Since the birth of gene editing technology in the 1970s, various gene editing tools have been developed. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, which is currently being actively researched as a genome editing tool, is a type of adaptive immune system in which microorganisms that survive infection by bacteriophages, etc., store a portion of the infected DNA sequence (approximately 20 bases) in the form of a spacer, recognize it upon reinfection, and induce double-strand breaks in the invading DNA. Among them, the CRISPR/Cas9 (CRISPR-associated 9) system is the most widely used because it requires only a single polypeptide to cause double-strand breaks at target base sites, and its functions are divided into a guide RNA that acts as a guide to the target base site and a Cas9 nuclease that causes double-strand breaks at the target site, thus having the advantages of a simple principle and low design cost compared to previous generation genome editing tools such as ZFN and TALEN. For the CRISPR system to induce double-strand breaks at a target site, in addition to the complementary match between the guide RNA and the target bases, an interaction between the Cas9 nuclease and the Protospacer Adjacent Motif (PAM) located next to the target site is required. The PAM is a short sequence located right next to the target site and serves as an important criterion for distinguishing between foreign and self DNA in the CRISPR system. The CRISPR/Cas9 system possesses a 5’-NGG PAM sequence, which acts as an obstacle by limiting the range of regions in the genome that can be selected as targets. Therefore, CRISPR/Cas systems with PAMs of different sequences have been studied to expand the range of selectable targets. Meanwhile, a gene editor using the Adenine base editor (ABE8e), the latest CRISPR-based single base editing technology, consists of nCas9 (TadA8e-nCas9(D10A)) with the functional domain of TadA8e deaminase linked to it, and guide RNA. The TadA8e deaminase linked to the CRISPR module was improved from tRNA-adenosine deaminase and has the function of replacing A bases with G bases on target DNA. While the most recent base editing technologies based on such CRISPR systems have been reported to have limitations in biological applications due to the broad editing window within the target sequence, little is known about gene editing technologies that increase efficiency by narrowing the editing window while overcoming these technical limitations. Figure 1 shows a schematic diagram of an innovative gene editing method based on deamination enzymes and guide RNA engineering. FIG. 2a shows a schematic diagram of a single base editor (TadA8e-nCas9(D10A)) that operates on fusion-engineered gRNA-based target sequence DNA containing a shielding region, and FIG. 2b shows the target site of the single base editor for the hDNMT1 gene. Figures 2c and 2d show a schematic diagram of the TadA8e-nCas9 (D10A) system, a single base editing system using a fusion-engineered gRNA containing a 3' terminal “shielding region (nucleotide sequence portion shown in pink),” and the efficiency (%) of inducing a single base substitution within the target sequence for each. Figure 2c is a schematic diagram showing an engineered gRNA sequence and structure including a shielding region at the 3' end. Figure 2d shows the results of base editing induction efficiency (%) within the target sequence when using TadA8e-nCas9 (D10A), a base editing system utilizing engineered gRNA with a shielding region at the 3' end. For each engineered gRNA, the base substitution region representing the efficiency of editing from an A base to a G base within the target sequence is shown (n=2). Figures 3a and 3b show a schematic diagram of the TadA8e-nCas9 (D10A) system, a base editing system using fusion-engineered gRNA containing a 5' terminal “shielding region (base sequence portion shown in pink),” and the efficiency (%) of inducing a single base substitution within the target base sequence for each. Figure 3a is a schematic diagram showing an engineered gRNA sequence and structure including a 5' terminal shielding region. Figure 3b shows the base editing induction efficiency (%) in the target sequence when using TadA8e-nCas9 (D10A), a base editing system utilizing engineered gRNA containing a 5' end shielding region. For each engineered gRNA, the base substitution region representing the efficiency of editing from an A base to a G base in the target sequence is shown (n=1). Figures 3c and 3d show a schematic diagram of the TadA8e-nCas9 (D10A) system, a base editing system using engineered gRNA containing a “shielding region (base sequence portion shown in pink)” of various structures at the 5' end, and the efficiency (%) of inducing a single