KR-20260066170-A - Base Editor Comprising a Novel Cas12a Protein or a Variant Thereof, and Use Thereof for Genome Editing

Abstract

This specification discloses a base editing system comprising a novel dead-Cas12a protein that has not been previously disclosed. The above base editing system comprises a base editor protein and a programmable guide RNA. This specification discloses a base editing method using the above base editing system. The inventors of this specification have demonstrated that the above base editing system can edit nuclear base pairs as intended at the intended location. By using the base editing system and the base editing method disclosed in this specification: 1) it is possible to change A-T base pairs contained in a target site of an intracellular target nucleic acid (double-stranded nucleic acid) to G-C base pairs; or 2) it is possible to change C-G base pairs contained in a target site of an intracellular target nucleic acid (double-stranded nucleic acid) to T-A base pairs. Therefore, the above base editing system can be used to treat diseases caused by mutations in some base pairs within the genome.

Inventors

• 이봉희
• 바야르사이칸 델거
• 바야르사이칸 고비저럴
• 구옥재
• 강현아
• 이재석

Assignees

• 주식회사 엔세이지

Dates

Publication Date

20260512

Application Date

20240919

Priority Date

20230920

Claims (1)

1. Adenine-based editor composition comprising the following: Adenine base editor protein, or nucleic acid encoding said adenine base editor protein, Here, the adenine base editor protein comprises a dead-Cas12a protein, one or more adenine deaminases, and one or more nuclear localization signals, and The above dead-Cas12a protein comprises the amino acid sequence of SEQ ID NO. 266 or SEQ ID NO. 271; and Guide RNA or nucleic acid encoding the guide RNA, Here, the guide RNA comprises a scaffold and a guide domain, and The above scaffold comprises the nucleic acid sequence of SEQ ID NO. 33; Here, the scaffold interacts with the dead-Cas12a protein to cause the guide RNA to form a complex with the base editor, and The above guide domain targets a predetermined target nucleic acid.

Description

Base Editor Comprising a Novel Cas12a Protein or a Variant Thereof, and Use Thereof for Genome Editing This specification discloses technology related to CRISPR/Cas systems and base editing systems that apply the same. Background Technology - CRISPR/Cas12a System CRISPR/Cas12a System Overview The CRISPR/Cas12a system, also known as the CRISPR/Cpf1 system, is a CRISPR/Cas system belonging to the Type V A subtype of Class 2. The CRISPR/Cas12a system was first reported by the research team led by Feng Zhang at the Broad Institute. Along with the CRISPR/Cas9 system, the CRISPR/Cas12a system is a CRISPR/Cas system that has been the subject of extensive research. The above CRISPR/Cas12a system is also known to have target-specific cleavage activity against double-stranded nucleic acids, similar to the CRISPR/Cas9 system, and thus has high utility for gene editing in eukaryotic cells. Compared to the CRISPR/Cas9 system, the CRISPR/Cas12a system has the following characteristics: 1) the Cas12a protein has only one type of nucleic acid cleavage domain (RuvC domain), 2) the guide RNA consists only of crRNA, and 3) it cleaves nucleic acids in the form of sticky ends that generate a 4-5 nucleotide overhang when cutting genes. The aforementioned CRISPR/Cas12a system has advantages such as a simpler guide RNA structure compared to the CRISPR/Cas9 system and the ability to perform more precise gene editing due to a lower off-target cleavage rate, and many researchers are actively studying ways to utilize it. The following describes these CRISPR/Cas12a systems and their components in more detail. CRISPR/Cas12a classification The CRISPR/Cas12a system belongs to the A subtype of the type V CRISPR/Cas system in Class 2, and is also called the CRISPR/Cpf1 system. CRISPR/Cas12a System Configuration and Functions The CRISPR/Cas12a system comprises a Cas12a protein and a guide RNA thereof. The Cas12a protein and the guide RNA act as a complex and exhibit target-specific nucleic acid cleavage activity and incidental cleavage activity. The Cas12a protein recognizes a PAM sequence and functions to cleave the nucleic acid of the target sequence targeted by the guide RNA. The guide RNA functions to target the nucleic acid of the target sequence. Structure of Cas12a protein The Cas12a protein can be broadly divided into the REC (RECognition) lobe and the NUC (NUClease) lobe, and the NUC lobe is further divided into the RuvC domain, NUC domain, WED domain, PI (PAM Interacting) domain, and BH (Bridge Helix) domain. The specific structure of the Cas12a protein has already been reported by several researchers and is described in detail in Paul et al. (CRISPR-Cas12a: Functional overview and applications. Biomedical Journal, Vol. 43, Issue 1, pp. 8-17, 2020). Among these, the WED II, WED III, REC1, and PI domains play a crucial role in enabling the Cas12a protein to recognize PAM sequences. Meanwhile, the domains that play a crucial role in the Cas12a protein cleaving the nucleic acid of the target sequence are the RuvC domain and the NUC domain. Structure of guide RNA Unlike the guide RNA of the CRISPR/Cas12a system, the guide RNA of the CRISPR/Cas9 system consists of a single RNA molecule, which is called crRNA. The guide RNA includes a direct repeat (DR) and a guide domain. Specifically, the DR and the guide domain are connected sequentially in the direction from the 3' end to the 5' end of the guide RNA. The DR is a portion involved in the guide RNA interacting with the Cas12a protein to form a complex, and the guide domain is a portion that binds to the nucleic acid of the target sequence, enabling the CRISPR/Cas12a system to exhibit target-specific cleavage activity and incidental cleavage activity. Target-specific nucleic acid cleavage activity of the CRISPR/Cas12a system and PAM (Protospacer Adjacent Motif) The above CRISPR/Cas12a system possesses target-specific nucleic acid cleavage activity, and two conditions are required to exhibit such target-specific nucleic acid cleavage activity. First, there must be a nucleotide sequence of a certain length within the nucleic acid that can be recognized by the Cas12a protein. Second, there must be a sequence around the nucleotide sequence of the said certain length that can bind complementarily to the guide domain included in the guide RNA. When these two conditions are satisfied, such that 1) the Cas12a protein recognizes the nucleotide sequence of the said certain length, and 2) the guide domain binds complementarily to the sequence portion surrounding the nucleotide sequence of the said certain length, nucleic acid cleavage activity is exhibited. At this time, the nucleotide sequence of the said certain length recognized by the Cas12a protein is called a Protospacer Adjacent Motif (PAM) sequence. The above PAM sequence is a unique sequence determined by the above Cas12a protein. If the PAM sequence of the above Cas12a protein is known, it can be used to design a CRISPR/Cas12a system that