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CN-121975769-A - Novel gene editing protein Cas12a-X, mutant Cas12a-X1 thereof and application thereof

CN121975769ACN 121975769 ACN121975769 ACN 121975769ACN-121975769-A

Abstract

The invention provides a novel gene editing protein Cas12a-X, a mutant Cas12a-X1 thereof and application thereof. The invention discovers a novel gene editing protein Cas12a-X from human intestinal microbial metagenome, and the homology of the novel gene editing protein Cas12a-X with common LbCas a is 48%. The novel gene editor shows remarkable in-vitro and in-vivo editing activity guided by crRNA, has the editing efficiency equivalent to LbCas a and has high application value. Based on the structural and functional characteristics of the wild Cas12a-X protein, the amino acid site of the key structural domain is mutated to obtain the Cas12a-X1 three-mutation variant. In vivo experiments prove that the gene editing efficiency of the mutant is improved by at least 1.2 times compared with that of the wild Cas12 a-X. The invention expands the application range of the CRISPR-Cas12a system and provides a new tool for the fields of precise medicine, disease treatment, variety improvement and the like.

Inventors

  • HU WENPING
  • WANG MING
  • SUN ZHAOLIN
  • CHENG XINRUI
  • LI NING
  • JIANG LIN
  • MA YUEHUI

Assignees

  • 中国农业科学院北京畜牧兽医研究所
  • 北京首农股份有限公司

Dates

Publication Date
20260505
Application Date
20251208

Claims (10)

  1. 1. The gene editing protein Cas12a-X is characterized in that the amino acid sequence of the gene editing protein Cas12a-X is shown as SEQ ID NO. 1.
  2. 2. The mutant Cas12a-X1 of the gene-editing protein of claim 1, having an amino acid sequence as shown in SEQ ID No. 2.
  3. 3. A novel gene editor comprising the gene-editing protein of claim 1 or the mutant of claim 2, and a functional protein or polypeptide; The functional protein or polypeptide is selected from the group consisting of an epitope tag, a reporter gene sequence, a nuclear localization signal sequence, a transmembrane peptide, a targeting moiety, a transcriptional activation domain, a transcriptional repression domain, a nuclease domain, a domain having activity selected from the group consisting of deaminase, reverse transcriptase, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, and nucleic acid binding activity.
  4. 4. A nucleic acid encoding the gene editor of claim 3, or a nucleotide sequence having more than 70% sequence homology with said nucleic acid.
  5. 5. A complex for gene editing, characterized in that the complex comprises a protein component and a nucleic acid component; The protein component is the gene editing protein of claim 1 or the mutant of claim 2 and the gene editor of claim 3; The nucleic acid component comprises a PAM sequence characterized by TTTN and a targeting sequence capable of hybridizing with a target sequence, and The nucleic acid component contains a nucleotide sequence of a guide RNA.
  6. 6. A biological material comprising the nucleic acid or protein of claim 4 or the complex of claim 5, wherein the biological material comprises an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium, or a cell line.
  7. 7. A delivery composition comprising a delivery vehicle and a component selected from the group consisting of the gene-editing protein of claim 1, the mutant of claim 2, the gene-editor of claim 3, or a variant thereof modified by binding to other various elements, the nucleic acid of claim 4, the complex of claim 5, and the biomaterial of claim 6; The variants include a base editor BE, a guide editor PE, a transcriptional regulatory editor, and an apparent regulatory editor.
  8. 8. A medicament, reagent or kit comprising the gene-editing protein of claim 1, the mutant of claim 2, the gene-editor of claim 3 or a variant of said gene-editor modified by binding to other types of elements, the nucleic acid of claim 4, the complex of claim 5, the biomaterial of claim 6, the delivery composition of claim 7; The variant comprises the elements base editor BE, guide editor PE, transcriptional regulatory editor and apparent regulatory editor.
  9. 9. The gene-editing protein of claim 1, the mutant of claim 2, the gene-editor of claim 3 or the variant of the gene-editor modified after binding to other classes of elements, the nucleic acid of claim 4, the complex of claim 5, the biological material of claim 6, the delivery composition of claim 7, the use of any of the following drugs, agents or kits of claim 8: 1) The method is used for gene editing, including DNA editing, precise editing, transcription regulation and control editing and epigenetic regulation and control; 2) Gene editing for microorganisms or animal and plant cells, embryos; 3) For preparing a gene editing microorganism or an animal or plant; the use is for non-disease diagnosis and treatment purposes; The variant comprises an element of a base editor BE, a guide editor PE, a transcriptional regulatory editor or an apparent regulatory editor.
  10. 10. A method for modifying a target gene, comprising modifying the target gene with one of the gene-editing protein according to claim 1, the mutant according to claim 2, the gene-editor according to claim 3 or a variant modified by binding the gene-editor to other various elements, the nucleic acid according to claim 4, the complex according to claim 5, the biological material according to claim 6, the delivery composition according to claim 7, the drug, the reagent or the kit according to claim 8; the methods are for non-disease diagnosis and treatment purposes; The variant comprises an element of a base editor BE, a guide editor PE, a transcriptional regulatory editor or an apparent regulatory editor.

Description

Novel gene editing protein Cas12a-X, mutant Cas12a-X1 thereof and application thereof Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a novel gene editing protein Cas12a-X, a mutant Cas12a-X1 thereof and application thereof. Background The gene editing technology is taken as an important tool of modern biology, and the development of the gene editing technology has profound effects on the fields of life sciences, medicine, agriculture and the like. CRISPR-Cas systems, an emerging gene editing technology, are receiving great attention for their efficiency, accuracy and ease of manipulation. The origin of the CRISPR-Cas system can be traced back to the 90 s of the 20 th century, when scientists discovered an adaptive immune system of bacteria and archaea that was able to degrade DNA of invasive viruses or phages by RNA-guided means, i.e. Zinc Finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were developed, but their design and production processes were complex, limiting use. As research is advanced, the CRISPR-Cas system is engineered as a highly efficient gene editing tool whose core elements include Cas protein and single stranded guide RNAs (sgrnas), the specificity of which is determined by the double RNA guide structure. CRISPR-Cas systems can be divided into one type of system and two types of systems depending on the number of Cas proteins. Among them, CRISPR-Cas9 is the most widely used system due to its simple and efficient editing capability. The CRISPR/Cas9 technology achieves precise gene knockout or knock-in by sgrnas guiding Cas9 nucleases to target DNA sequences. In addition, the CRISPR-Cas9 system also derives a plurality of technologies, such as a base editor BE, a guide editor PE, apparent genetic and transcriptional regulation and the like, which can realize precise gene modification in a plurality of forms and meet different gene editing requirements. The gene editing technology is very widely applied by precisely controlling genetic information. In the medical field, the method brings hope for radical treatment by repairing genetic mutation into genetic diseases such as sickle cell anemia, cystic fibrosis and the like, enables cancer immunotherapy, for example edits PD-1 or CAR genes of T cells to enhance tumor killing capacity, and in the agricultural field, improves stress resistance (such as bacterial blight resistant rice) and nutritional value (such as golden rice) by optimizing crop genes, and accelerates animal breeding (such as high lean-meat percentage pig species) to ensure grain safety. In the aspects of industry and environmental protection, the technology promotes microorganisms to efficiently synthesize medicines (such as artemisinin) and biofuels and assist in environmental remediation, such as engineering bacteria for designing degradable plastics or cultivating heavy metal enriched plants. In basic science research, gene editing analyzes the core life mechanisms of development, metabolism and the like, and builds a disease model to accelerate drug development. In addition, it has potential in ecological protection, such as enhancing coral heat resistance to cope with climate change, or controlling invasive species using gene driven techniques. Biological breeding represented by gene editing technology is a key technology in China, and industrial development is promoted through technological innovation. In general, the gene editing technology is becoming a core driving force for promoting medical innovation, agricultural transformation and sustainable development by virtue of accuracy, high efficiency and universality, and will release far-reaching transformation potential in the future under the framework of technology iteration and global ethics consensus. Although the most widely used CRISPR-Cas9 technology has significant advantages, the practical application faces a plurality of challenges including Off-target effect (Off-TARGET EFFECTS) that Cas9 can still cut Off non-target sites when sgRNA is partially complementary (such as 3-5 base mismatch) with non-target DNA, delivery efficiency and vector limitation that Cas9 protein is oversized, spCas9 (about 4.2 kb) is difficult to package into common viral vectors (such as AAV capacity of only 4.7 kb) and needs to be split into a dual vector system (such as separating Cas9 from sgRNA), resulting in reduced in vivo delivery efficiency (less than 20% of mouse liver transfection), and immunogenicity risk that about 50% of human population has anti-Cas 9 antibodies due to contact with Streptococcus (SpCas 9 source), immune cell activation (such as IL-6 elevation) and rapid removal of CAR 9 expression cells in editing cell therapy are observed in clinical trials. Long-term expression of Cas9 may trigger T cell mediated immune attacks, for example, in non-human primates, cas9 initiates elevation of liver injury markers ALT/AST a