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Proteome obtained by splitting Cas9 protein and application of proteome

A proteomics and protein technology, applied in the field of proteomics, can solve the problems of reduced splicing efficiency and no longer applicable, and achieve the effects of improving gene editing efficiency and safety, wide selection range, and low off-target rate

Pending Publication Date: 2021-04-27
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, after coupling the deaminase module and the efficiency enhancement module to obtain a larger single base editing system, the length of the protein has changed, and the splicing efficiency of some split sites has been greatly reduced due to the excessively long Cas9N or Cas9C. no longer applicable

Method used

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  • Proteome obtained by splitting Cas9 protein and application of proteome
  • Proteome obtained by splitting Cas9 protein and application of proteome
  • Proteome obtained by splitting Cas9 protein and application of proteome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087]Example 1 Split of Cas9 protein

[0088]The CAS9 protein was split into two different amino acid sequences CAS9N (N end) and CAS9C (E-terminal). Among them, the CAS9 protein is SPCas9 (H840A) protein, and its amino acid sequence is shown in SEQ ID NO.1, and the encoding nucleotide sequence is shown in SEQ IDNO.2.

[0089]The split positions are: between the 994th to 995 bits, between the 1,05 to 1006 bits, between the 1024th to 1025 bits, and between the 1032 to 1033 bits, the unino acid sequences obtained by each of the detachment fragments obtained and The nucleotide sequence encoding it is shown in Table 1.

[0090]Table 1:

[0091]

Embodiment 2

[0092]Example 2 Construction of AAV carrier

[0093]First, experimental method

[0094]1. Split the peptide RMA INTEIN gene (whose nucleotide sequence such as SEQ ID NO.7 is shown, which is shown in SEQ ID No. 6, as shown in SEQ ID NO. The RMAC, the sequence is the nucleotide sequence fragment of the first to 306th position of the nucleotide sequence shown in SEQ IDNO 7, respectively, and the nucleotide sequence of the No. 307 to 462 (corresponding amino acid fragment, respectively, respectively, respectively, respectively.) SEQ ID NO. 6 The amino acid fragment of 1 to 102 of the amino acid sequence shown and the amino acid fragment of No. 103 to 154) of the amino acid sequences.

[0095]2. According to the Cas9 split method of Table 1, Prime Editor (PE) is constructed, and the nucleotide sequence is shown in SEQ IDNO.10, wherein the sequence of the first to 27th base is a nucleoside encoding a nuclear positioning signal peptide. The sequence of acid sequences (encoded amino terminal sequenc...

Embodiment 3

[0108]Example 3 Splitted PE system can effectively perform editing of a karace point point

[0109]First, experimental method

[0110]The split-PE system was constructed in the method of Example 2 containing the expression vector group containing reverse transcriptase: PAAV-EF1α-Pen-INTEINN and PAAV-EF1α-INTEINC-PEC, wherein the expression carrier group is SPCas9 protein, respectively, according to respectively. Split from the following four sites: Between the 994th to 995 bits, between the 1,05-006 bits, between the 1024th to 1025 bits or between the 1st 332 to 1033, the peptide is RMA INTEIN.

[0111]HEK293T cells containing M1EMGFP steady expressed, M1EMGFP for EMGFP (Nucleotide Sequence, SEQ ID NO.11) Sequence Codon CAG mutant to TAG, due to the EMGFP mutation, causes the translation to terminate in advance, only Tag is edited to CAG to restore the normal expression of GFP, so the ability of gene editing can be detected by statistics of GFP positive cells and accurate and convenientness ...

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Abstract

The invention discloses a proteome obtained by splitting Cas9 protein and application of the proteome, and provides a Cas9 splitting method. A protein peptide segment obtained by splitting Cas9 can be reassembled into Cas9 protein with functional activity in a target cell or organ by various splicing methods such as intein. The splitting site provided by the invention is superior to a reported site, the activity of Cas9 after in-vivo recombination is higher, and the off-target rate is lower; due to the fact that the split protein is smaller, the limitation of the carrying capacity of carriers such as AAV is smaller, the selection range of the carriers is wider, the application range is wider, and the gene editing efficiency and safety can be effectively improved; and the single Cas9N or Cas9C does not have a complete function, the effect of regulating the Cas9 protein function can be achieved by further regulating the adding time sequence of the Cas9N and the Cas9C or regulating the proportion of the Cas9N and the Cas9C, and important significance is achieved for further application of CRISPR.

Description

Technical field[0001]The present invention relates to the field of biotechnology, and more particularly to a protein group and its application thereof in a Cas9 protein resolution.Background technique[0002]The CRISPR / CAS (Clustered Regular "system is an antiviral acquired immune mechanism derived from bacteria and antique bacteria. The CRISPR / CAS system exerts an immunogen in the form of an RNA protein complex, wherein the effector protein includes CAS9, CAS12A, CAS12B, CASX, and the like. The scientist was transformed into a single-stranded guidance RNA (Guide RNA, GRNA). The Cas protein first identifies the sequence of PA m (Protospacer Adjacement Motif) on the genome, and the GRNA specifically identifies and complements each other with the target site, thereby activating the Cas protein to perform the function of the endonuclease to cut DNA double strand, realize a specific site Gene editing. Self-development has become a wide range of applications in various organisms since ...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/11C12N15/864A61K48/00
CPCC12N9/22C12N15/86A61K48/005C07K2319/00C12N2750/14143
Inventor 黄军就支胜尧
Owner SUN YAT SEN UNIV
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