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CN-121975867-A - Protein editing system and application thereof

CN121975867ACN 121975867 ACN121975867 ACN 121975867ACN-121975867-A

Abstract

The invention provides a protein editing system and application thereof, which belongs to the technical field of biology, and the invention utilizes mutant protein K92A to reserve the characteristic of the recruited PARN protein for degrading mRNA tail and combines the strong binding capacity of a lambda N-BoxB system to successfully construct the protein editing system. The protein editing system can be developed into a product for protein editing, has been successfully applied to knockdown expression of various proteins and pathogens, can avoid irreversible damage on a gene layer caused by gene editing, provides a new solution for rapid application of gene knockdown expression, and has a wide application prospect.

Inventors

  • QIU YANG
  • Xu Xiqiu
  • LIU LEI
  • MENG XIANGYU
  • Huang Qiunan

Assignees

  • 中国科学院武汉病毒研究所

Dates

Publication Date
20260505
Application Date
20260212

Claims (10)

  1. 1. A protein editing system is characterized by comprising effector plasmids and targeting plasmids; The effector plasmid is used for expressing fusion protein lambdan-K92A, wherein K92A on the fusion protein lambdan-K92A is a mutant of respiratory syncytial virus M2-1 protein, and can be specifically combined with PARN protein; The targeting plasmid is used for transcribing and generating chimeric RNA by editable constructing a vector plasmid with BoxB and a target gene, wherein the target gene represents a 3' UTR region sequence positioned at the downstream of a target protein coding region, the chimeric RNA can transcribe and generate guide RNA, the chimeric RNA is BoxB RNA transcribed and is an RNA sequence formed by chimeric guide RNA, and the guide RNA can specifically bind to target protein messenger RNA; The fusion protein λN-K92A and the chimeric RNA are capable of binding to form a stable λN-BoxB complex, causing the PARN protein specifically bound on the λN-BoxB complex to degrade the poly (A) tail of the messenger RNA of the target protein.
  2. 2. The protein editing system of claim 1, further comprising targeting a negative control plasmid.
  3. 3. The protein editing system according to claim 1, wherein the 3' UTR region sequence represented by the target gene on the targeting plasmid is a downstream 3' UTR region sequence selected from the entire 3' UTR regions to be closest to the stop codon.
  4. 4. The protein editing system according to claim 1, wherein the effector plasmid is constructed by modifying a PRK-HA- λN vector, wherein the modification comprises replacing a λN sequence with a coding sequence in which a mutant K92A of λN and M2-1 proteins are sequentially linked, wherein the effector plasmid can express HA protein and fusion protein λN-K92A simultaneously, and wherein the effector plasmid is abbreviated as PRK- λN-K92A.
  5. 5. The protein editing system according to claim 1, wherein the targeting plasmid is constructed by modifying a CRISPR V2 vector, the modification comprises replacing a Cas9 element with an EGFP reporter gene and replacing a gRNA sequence downstream of a U6 promoter with a sequence connected with a target gene in sequence BoxB, the targeting plasmid can simultaneously express green fluorescent protein GFP and transcribe chimeric RNA, and the targeting plasmid is BoxB-target gene for short.
  6. 6. The protein editing system according to claim 5, wherein the sequence of BoxB connected with the target gene sequence comprises a BoxB sequence and a target gene sequence which are connected in sequence, the constructed targeting plasmid is 2X BoxB-target gene for short, and the chimeric RNA transcribed from the constructed 2X BoxB-target gene is an RNA sequence formed by chimeric of a 2-segment BoxB RNA hairpin structure and guide RNA transcribed from the target gene.
  7. 7. The protein editing system according to claim 5, wherein the targeting plasmid constructed when the target protein is SLC7A11 protein is 2X BoxB-SLC7A11, on the 2X BoxB-SLC7A11, SLC7A11 represents a 3' UTR sequence positioned downstream of SLC7A11 protein coding region, and can transcribe to generate guide RNA which specifically binds to SLC7A11 protein messenger RNA, and on the 2X BoxB-SLC7A11, the 5' end and the 3' end of SLC7A11 sequence respectively contain EcoRI and NheI restriction sites.
  8. 8. A method for using the protein editing system is characterized by comprising the steps of 1) designing and screening target gene sequences, 2) constructing the target gene sequences on target plasmids and verifying whether the construction of the target plasmids is correct, 3) cotransfecting effector plasmids and the target plasmids into target cells, and 4) culturing for 24-48 hours after cotransfecting and collecting cell samples for verifying the expression effect of the target proteins.
  9. 9. A product for protein editing, characterized by comprising the protein editing system according to any one of claims 1 to 7 for specifically knocking down the expression of a target protein by constructing a targeting plasmid carrying the target gene in an editable manner.
  10. 10. Use of a protein editing system as claimed in any one of claims 1 to 7, or a method of using a protein editing system as claimed in claim 8, or a product for protein editing as claimed in claim 9, for knocking down expression of a protein of interest using a host cell.

Description

Protein editing system and application thereof Technical Field The invention relates to the field of biotechnology, in particular to a protein editing system and application thereof, which are particularly suitable for carrying out specific knockdown editing on specific genes or proteins in vitro. Background The current common techniques for achieving functional inactivation or inhibition of gene expression include two major categories, one being gene editing (e.g., CRISPR and TALENs), which can be implemented by nuclease cleavage to alter a programmable plasmid system, and the other being gene knockdown (e.g., RNAi), which can be implemented at the RNA level for reversible post-transcriptional gene silencing. However, these techniques have respective limitations. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology guided by guide RNAs (grnas) enables accurate gene knockdown, but has off-target effects caused by non-specific cleavage. The off-target effect of transcription activator-like effector (TALE) protein mediated TALENs (Transcription Activator-Like Effector Nucleases) technology is lower than CRISPR, but the DNA-level cleavage efficiency is lower than CRISPR, and the complex design and high cost limit its application. More importantly, gene editing which depends on a DNA double strand break mechanism has the effect that the gene editing is permanent and irreversible, and the strategy of 'one-step in place' is too severe to meet the application scene of needing temporary, reversible or fine-adjustment of dosage. In contrast, the reaction mechanism of the small interfering RNA (siRNA) or short hairpin RNA (shRNA) -mediated RNAi (RNA Interference) technology is relatively mild and reversible, but is highly dependent on the endogenous mechanism of cells, is easily affected by cell types and delivery methods, and generally faces the restriction and trouble of off-target effects (including cross reactions of siRNA/shRNA with non-perfect complementarity to non-targeted mRNA, interference to endogenous microRNA pathways), and has yet to be improved in terms of gene silencing efficiency, specificity, controllability, operational simplicity, and the like. The development of a new gene research product is urgently needed in the field, and ideal promotion characteristics include not only being capable of efficiently utilizing natural mechanisms (such as RNA processing channels) in cells, but also being capable of realizing efficient, controllable, reversible, editable and relatively mild gene expression inhibition so as to meet urgent demands on gene silencing diversity in basic research and clinical application. Therefore, the invention aims to develop a brand new gene silencing product, and solves the technical problems of off-target effect, potential risk of exogenous enzyme, incapability of editing or high programming complexity and the like existing in the existing gene silencing by utilizing a cell endogenous mechanism. During the course of Respiratory Syncytial Virus (RSV) virus studies, the M2 gene of RSV was found to be a gene with 2 Open Reading Frames (ORFs) that encode 2 distinct proteins, the M2-1 protein and the M2-2 protein, respectively, i.e., the M2-1 protein is encoded by the first open reading frame of the M2 gene of the RSV genome, with 194 amino acids (aa) in full length, and a highly structurally and functionally homologous M2-1 protein also being present in human metapneumovirus. The M2-1 protein helps virus RNA polymerase such as RSV to cross gene termination signals, ensures that downstream genes can still be transcribed effectively, and plays a core role in resisting termination. The host body invaded by the virus also comprises 2 host proteins PARN protein and SLC7A11 protein, wherein PARN (Poly (A) -specific Ribonuclease, polyadenylic acid specific ribonuclease) protein is ribonuclease which plays a role in regulation by degrading the Poly (A) tail of messenger RNA, wherein the Poly (A) tail is a polyadenylic acid sequence at the 3' end of mRNA, the length and integrity of the Poly (A) tail are closely related to mRNA stability and translation efficiency, PARN protein can be used as one of ' goats ' expressed by host cell genes by degrading the Poly (A) tail, SLC7A11 (Solute LINKER CARRIER FAMILY 7 member 11,SLC7A11) protein is a member 11 of solute carrier family 7, SLC7A11 protein is a transporter located on a cell membrane, intracellular glutamic acid is transported to outside cells, extracellular cystine is transported to inside cells, and the final purpose is playing a role in resisting oxidative stress and iron death by promoting the synthesis of glutathione (H), the most important antioxidant in cells. The lambdan-BoxB system is an RNA-protein interaction system developed based on a transcription anti-termination mechanism of phage lambda, the core consists of an N protein (lambdan protein) of lambda phage and a BoxB RNA stem-loop structure specif