KR-20260067381-A - INHIBITION OF UNINTENDED MUTATIONS IN GENE EDITING

Abstract

A fusion protein and related molecules useful for performing base editing with reduced or no off-target mutations are provided. The fusion protein may comprise a first fragment containing a nucleobase deaminase or its catalytic domain, a second fragment containing a nucleobase deaminase inhibitory domain, and a protease cleavage site between the first fragment and the second fragment. Additionally, an improved prime editing system is provided, comprising a prime editing guide RNA with improved stability.

Inventors

• 천 지아
• 양 베이
• 양 리
• 황 싱쉬
• 왕 리제

Assignees

• 상하이테크 유니버시티

Dates

Publication Date

20260512

Application Date

20200203

Priority Date

20190202

Claims (13)

1. As a dual guide RNA system, A target single guide RNA comprising a first spacer having a sequence complementary to a target nucleic acid sequence adjacent to a first protospacer adjacent motif (PAM) site, A helper single guide RNA comprising a second spacer having a sequence complementary to a second nucleic acid sequence adjacent to a second PAM site, wherein the second spacer is a helper single guide RNA having a length of 8 to 15 bases. Clustered regularly interspersed short palindromic repeat (CRISPR)-associated (Cas) proteins, and Includes nucleobases, A double guide RNA system in which the second PAM region is 10 to 150 bases away from the first PAM region.
2. In claim 1, the second PAM site is a double guide RNA system located 34 to 91 bases away from the first PAM site.
3. In paragraph 2, the second PAM site is a dual guide RNA system upstream of the first PAM site.
4. In claim 1, the second spacer is a double guide RNA system having a length of 9 to 12 bases.
5. As a method for performing intracellular gene editing on a target nucleic acid sequence, Includes the step of introducing the following into the cell: A target single guide RNA comprising a first spacer having a sequence complementary to a target nucleic acid sequence adjacent to a first protospacer adjacent motif (PAM) site, A helper single guide RNA comprising a second spacer having a sequence complementary to a second nucleic acid sequence adjacent to a second PAM site, wherein the second spacer is a helper single guide RNA having a length of 8 to 15 bases. Clustered regularly interspersed short palindromic repeat (CRISPR)-associated (Cas) proteins, and nucleobases, A method in which the second PAM site is located 10 to 150 bases away from the first PAM site on the target nucleic acid sequence.
6. In paragraph 5, the method in which the second PAM site is 34 to 91 bases away from the first PAM site.
7. In paragraph 6, the method in which the second PAM section is upstream of the first PAM section.
8. In paragraph 5, the method wherein the second spacer is 9 to 12 base lengths.
9. In paragraph 5, a method in which Cas protein and nucleobase deaminase are introduced into a cell as one or more encoding polynucleotides.
10. In paragraph 5, the target single guide RNA and the helper single guide RNA are each introduced into the cell as RNA molecules or as coding DNA.
11. In claim 5, the nucleobase deaminase is fused to the inhibitory domain of the nucleobase deaminase or the second nucleobase deaminase through a protease cleavage site, and the method further comprises the step of introducing a protease capable of cleaving the cleavage site into a cell.
12. In paragraph 5, the cell is in a living organism.
13. In paragraph 12, the cell is in a method within a human subject.

Description

Inhibition of Unintended Mutations in Gene Editing Genome editing is a type of genetic engineering that uses engineered nucleases (molecular scissors) to insert, delete, or replace DNA in the genome of a living organism. The use of genome editing tools to genetically engineer the genomes of cells and living organisms has extensive applications in life science research, the development of biotechnology and agricultural technologies, and, most importantly, pharmaceutical and clinical innovation. For example, genome editing can be used to correct driver mutations that underlie genetic diseases and lead to the complete cure of these diseases in living organisms. Genome editing can also be used to manipulate crop genomes, increase crop yields, and confer resistance to environmental pollution or pathogen infections. Furthermore, microbial genome transformation through precise genome editing is crucial for the development of renewable bioenergy. The CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system has been the most powerful genome editing tool due to its concept of unprecedented editing efficiency, ease of use, and potential applicability in living organisms. Induced by guide RNA (gRNA), Cas nucleases can generate DNA double-strand breaks (DSBs) at targeted genomic regions within various cells (both cell lines and cells derived from living organisms). These DSBs are then repaired through endogenous DNA repair systems that can be utilized to perform desired genome editing. Generally, two major DNA repair pathways can be activated through DSBs, non-homologous end binding (NHEJ), and homologous-directed repair (HDR). NHEJ can introduce random insertions/deletions (indels) into genomic DNA regions surrounding DSBs, which can lead to open reading frame (ORF) shifts and ultimately gene inactivation. In contrast, when HDR is triggered, genomic DNA sequences at the target site can be substituted with sequences from exogenous donor DNA templates via homologous recombination mechanisms, which can cause the correction of gene mutations. However, the actual efficiency of HDR-mediated gene editing is low (normally < 5%) because the occurrence of homologous recombination is cell type-specific and cell cycle-dependent, and NHEJ is triggered more frequently than HDR. Therefore, the relatively low efficiency of HDR limits the translation of CRISRP/Cas genome editing tools in the field of precision gene therapy (disease-driven gene editing). A base editor (BE) that integrates the CRISPR/Cas system with the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) AID (activation-induced cytidine deaminase) family has recently been developed, which significantly enhances the efficiency of CRISPR/Cas-mediated gene editing. Through fusion with Cas9 nikase (nCas9) or catalytic dead Cpf1 (dCpf1 also known as dCas12a), the cytosine (C) deamination activity of APOBEC/AID family members can be intentionally induced to target bases within the genome and catalyze the substitution of C to thymine (T) at these bases. However, since members of the APOBEC/AID family can induce C-to-T base substitution mutations within single-stranded DNA (ssDNA) regions, the specificity of current base editing systems is compromised, thereby limiting the applicability of using BEs for therapeutic purposes, for example, to restore T-to-C mutations occurring in human diseases. For this reason, the creation of novel BEs that do not induce C-to-T mutations in other ssDNA regions but can specifically edit cytosines within target regions is desirable. Such novel BEs would enable more specific base editing in a variety of living organisms. Importantly, the high specificity of these BEs will facilitate potential clinical interpretation, particularly in gene therapies involving the restoration of disease-related T-to-C mutations. In some embodiments, the present disclosure provides a base editor useful for genome editing that causes a reduction or absence of off-target mutations common in current base editors. In some embodiments, a nucleobase deaminase inhibitor is cleavedly fused to a nucleobase deaminase involved in genome editing. In the presence of the nucleobase deaminase inhibitor, the nucleobase deaminase cannot react with (or may react less with) nucleotide molecules. At a target editing site, the nucleobase deaminase inhibitor can cleave to release a fully active nucleobase deaminase that can subsequently perform editing. Accordingly, in one embodiment, a fusion protein is provided comprising a first fragment comprising a nucleobase deaminase or a catalytic domain thereof, a second fragment comprising a nucleobase deaminase inhibitor, and a protease cleavage site between the first fragment and the second fragment. In some embodiments, the nucleobase deaminase is adenosine deaminase. In some embodiments, adenosine deaminase is tRNA-specific adenosine deaminase (TadA), adenosine deaminase tRNA