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CN-120399091-B - CBE editing system and application

CN120399091BCN 120399091 BCN120399091 BCN 120399091BCN-120399091-B

Abstract

The invention belongs to the technical field of gene editing, and discloses CBE editing protein and application. The CBE editing protein fusion de-ubiquitinated protein disclosed by the invention. Compared with other single base editors such as ABE, the CBE single base editors have unstable proteins and can be rapidly degraded in cells. By fusing the deubiquitinated protein, the stability of the CBE single-base editor protein is improved, and therefore the editing efficiency of the CBE is improved.

Inventors

  • XIAO ZHENDONG
  • ZHANG QI
  • Diao Liting
  • ZENG JIN
  • Ren Yahan

Assignees

  • 中山大学附属第三医院

Dates

Publication Date
20260512
Application Date
20250425

Claims (7)

  1. 1. The CBE editing protein is characterized in that the CBE editing protein is fused with protein expressed by a deubiquitination gene, and the nucleotide sequence of the coding protein is shown as SEQ ID NO. 2.
  2. 2. An expression vector expressing the CBE editing protein of claim 1.
  3. 3. The expression vector of claim 2, comprising a nucleotide sequence set forth in SEQ ID NO. 1.
  4. 4. A nucleic acid encoding the CBE editing protein of claim 1.
  5. 5. A CBE editing system comprising the CBE editing protein of claim 1.
  6. 6. Use of the CBE editing protein of claim 1, the expression vector of claim 2 or 3, the nucleic acid of claim 4, the CBE editing system of claim 5 for gene editing for diagnosis and treatment of non-disease.
  7. 7. The use of claim 6, wherein the editing is of a C to T gene at a specific site.

Description

CBE editing system and application Technical Field The invention relates to the technical field of gene editing, in particular to a CBE editing system and application. Background The gene editing technology is a genetic engineering technology for realizing specific DNA fragment modification by carrying out site-directed editing on a target gene. In the prior art, gene editing was achieved at the earliest by specifically recognizing a specific base sequence using Zinc Finger Protein (ZFP). Following ZFNs, TALEN technology derived from AvrBs3 protein emerged. However, ZFN and TALENs editing techniques have the limitations of complex design/screening process, high off-target rate, difficult package delivery, etc. Then, the CRISPR technique is a technique for achieving gene editing by cleaving a nucleic acid at a specific position using an RNA-guided endonuclease. The CRISPR-Cas system, collectively known as a regularly clustered short palindromic repeat and CRISPR-associated protein (Cas), consists of Cas nuclease and two separate RNA components, one programmable crRNA (CRISPR RNA) and one immobilized tracrRNA (transactivating crRNA). Cas1-Cas2 proteins are capable of cleaving the invading phage DNA into small fragments, which are then integrated as spacers into a CRISPR array. Subsequently, the CRISPR array is transcribed to produce crrnas and complementary tracrRNA, which form a double stranded RNA structure, recruiting Cas proteins for cleavage. In the vicinity of crRNA targeting sequences on invasive DNA, the Protospacer Adjacent Motif (PAM) plays a crucial role in the adaptation and interference phases, and CRISPR-Cas complexes recognize these sequences during target DNA binding. The CRISPR-Cas9 technology is a first choice technology for genetic operations such as gene knockout, gene knock-in, large fragment deletion and the like of plants, animals and microorganisms due to the characteristics of simplicity and high efficiency. CRISPR-Cas systems fall mainly into two broad categories, one being polyprotein effector complexes comprising type I, III, IV and the other being single Cas protein complexes comprising type II, V, VI. In comparison to the former, the two-class systems (type II Cas9, type V Cas12a, type VI Cas13a and Cas13b systems) have greater potential in genome editing applications. Wherein, compared to other systems requiring multiple Cas proteins, the type II system has only one Cas protein of Cas 9. Cas9 proteins rely on crRNA and tracrRNA fusion and cleavage of formed sgrnas (single guide RNAs), playing a role in RNA-guided DNA recognition, making them useful in genome engineering. Cas9 comprises domains homologous to HNH and RuvC endonucleases, wherein the HNH domain cleaves a complementary DNA strand and the RuvC-like domain cleaves a non-complementary DNA strand. In 2012, jinek et al reported for the first time that the CRISPR-Cas9 system could be engineered into a programmable RNA-guided DNA endonuclease. Subsequently Cong et al achieved targeted gene editing in mammalian cells for the first time using the engineered CRISPR-Cas9 system. After that, the CRISPR-Cas9 technology is rapidly becoming the first choice technology for genetic operations such as plant, animal, microorganism gene knockout, gene knock-in, large fragment deletion and the like due to the characteristics of simplicity and high efficiency. As research is in progress, researchers have developed a host of new editing tools by mining other types of CRISPR systems, making the CRISPR-Cas system the dominant gene editing tool. However, CRISPR-Cas systems suffer from some non-negligible problems, such as inefficiency, and non-targeted cleavage, lower safety, etc. Komor et al report a single base editing technique that fuses spCas9 with a cytosine deaminase, uses the precise ability of spCas9 to target DNA to direct the cytosine deaminase to a target sequence, and then converts cytosine C within a mutation window to thymine T, also referred to as a Cytosine Base Editor (CBE) later, under the catalysis of the cytosine deaminase. The CBE system consists mainly of nCas or dCas9 and cytidine deaminase 1 (apodec 1), where spontaneous deamination of cytosine is the main source of C-G to T-a conversion. Komor et al report BE1 (rAPOBEC-XTENdCas) whose principle is to let dCAS 9N-terminal form a fusion protein with APOBEC1, and then the gRNA directs the fusion protein to the target site, APOBEC1 deaminates the C of the single-stranded DNA in the R loop to U, and subsequently completes the C-to-T conversion via DNA replication. However, further studies have found that the in vitro average apparent editing efficiency of the BE1 system is 44%, but only 0.8% to 7.7% in mammalian cells. To increase editing efficiency, uracil DNA glycosylase inhibitor (UGI) was fused to the C-terminus of BE1, and a second generation base editor BE2 (APOBEC-XTEN-dCAS 9-UGI) was constructed, increasing UGI increased editing efficiency by approximately 3-fold