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CN-121991924-A - LbCas12a protein mutant and preparation method and application thereof

CN121991924ACN 121991924 ACN121991924 ACN 121991924ACN-121991924-A

Abstract

The application belongs to the technical field of biology, and particularly relates to LbCas a protein mutant and a preparation method and application thereof. The application provides a group of engineered LbCas a protein mutants, which remarkably expands the recognition range of TNTN PAM sequences and improves trans-cutting efficiency by introducing arginine substitution mutation in a PAM recognition region (such as D156R) and an alpha-helical bridge region (residues 47-51). These mutants provide a high performance tool for the application of CRISPR-Cas12a technology in gene editing and high temperature compatible diagnostics.

Inventors

  • JIANG LINGXIAO
  • CAO YUNSHAN
  • LIN JIAQIONG
  • HUANG YONGXIN
  • HUANG ZHENYI

Assignees

  • 南方医科大学珠江医院

Dates

Publication Date
20260508
Application Date
20260128

Claims (10)

  1. 1. A set of LbCas a protein mutants, wherein said mutants further comprise one of the following two conditions on the basis of a LbCas a protein comprising a RVRR mutation: (1) Introducing at least one arginine substitution in the region corresponding to amino acid residues 47 to 51 of the wild-type LbCas a protein; (2) Also comprising a D156R mutation and at least one arginine substitution introduced in the region of the amino acid residues corresponding to positions 47 to 51 of the wild-type LbCas a protein; The RVRR mutated LbCas a protein has the G532R, K538V, Y R and K595R mutations compared to the wild-type LbCas a protein.
  2. 2. The LbCas a protein mutant according to claim 1 wherein the arginine substitution is selected from at least one of the following mutations Y47R, K48R, G R, V R or K51R.
  3. 3. The LbCas a protein mutant according to any one of claims 1 to 2, wherein the mutation site of the mutant comprises one set of the following combinations: (1) Y47R, G532R, K538V, Y R and K595R; (2) K48R, G532R, K538V, Y R and K595R; (3) g49R, G532R, K538V, Y R and K595R; (4) V50R, G532R, K538V, Y R and K595R; (5) K51R, G532R, K538V, Y R and K595R; (6) d156R, Y47R, G532R, K538V, Y542R and K595R; (7) d156R, K48R, G532R, K538V, Y542R and K595R; (8) d156R, G49R, G532R, K538V, Y542R and K595R; (9) d156R, V50R, G532R, K538V, Y542R and K595R; (10) d156R, K51R, G532R, K538V, Y542R and K595R.
  4. 4. A nucleic acid molecule encoding a LbCas a protein mutant according to any one of claims 1 to 3.
  5. 5. A vector comprising the nucleic acid molecule of claim 4.
  6. 6. A cell comprising the vector of claim 5 or expressing the LbCas a protein mutant of any one of claims 1 to 3.
  7. 7. A process for producing a LbCas a protein mutant as claimed in any one of claims 1 to 3, which comprises fermenting the cell as claimed in claim 6, and And separating LbCas a protein mutant from the fermentation product.
  8. 8. The method of claim 7, wherein culturing one or more of the following conditions are met: (1) Culturing until OD600 of the culture system is 0.5-0.7, adding IPTG to induce expression, wherein final concentration of IPTG in initial system of induced expression is 0.1-0.3 mmol/L, and (2) The culture temperature is 14-16 ℃, and the time for inducing expression is 14-20 h.
  9. 9. A test kit comprising a LbCas a protein mutant according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4, a vector according to claim 5 or a cell according to claim 6.
  10. 10. A method for detecting salmonella, comprising detecting salmonella in a sample using the LbCas a protein mutant of any one of claims 1 to 3 or the kit of claim 9.

Description

LbCas12a protein mutant and preparation method and application thereof Technical Field The application belongs to the technical field of biology, and particularly relates to LbCas a protein mutant and a preparation method and application thereof. Background The CRISPR-Cas12a system is a key technology in gene editing and molecular diagnosis, has the core advantages of trans-cleavage activity after targeted activation, and lays a foundation for high-sensitivity nucleic acid detection. The system has wide application prospects in the fields of basic research, clinical diagnosis, biotechnology and the like, and particularly has great potential in the aspects of pathogen rapid detection, gene function analysis, accurate medical treatment and the like. With the continuous development of molecular diagnostic techniques, the performance requirements for CRISPR-Cas systems are also continually increasing. Existing CRISPR-Cas12a technology faces many challenges in practical applications. Taking the commonly used example of the larenospira Cas12a, its activation is usually dependent on recognition of a stringent 5'-TTTV-3' PAM sequence, which severely limits its targeting range in the genome. Although researchers have engineered PAM recognition regions of Cas12a by protein engineering means to develop variants, these variants still have significant limitations in PAM compatibility, catalytic activity, and temperature adaptability. Most variants only recognize specific types of PAM sequences and stability under different temperature conditions is to be improved. The core technical problems faced by the current CRISPR-Cas12a technology include limited PAM recognition range, difficulty in efficiently recognizing and cutting non-classical PAM sequences, insufficient trans-cutting activity, low catalytic efficiency especially under non-classical PAM, insufficient thermal stability, and difficulty in maintaining stable activity in a wider temperature range. These problems severely limit the wide application of CRISPR-Cas12a technology in the fields of gene editing and molecular diagnostics, especially in detection applications where high sensitivity and multi-scenario adaptation are required. Disclosure of Invention Based on this, an embodiment of the application provides LbCas a protein mutant, and a preparation method and application thereof. In one aspect, the application provides a set of LbCas a protein mutants, which, on the basis of LbCas a protein comprising a RVRR mutation, further comprise one of the following two conditions: (1) Introducing at least one arginine substitution in the region corresponding to amino acid residues 47 to 51 of the wild-type LbCas a protein; (2) Also comprising a D156R mutation and at least one arginine substitution introduced in the region of the amino acid residues corresponding to positions 47 to 51 of the wild-type LbCas a protein; The RVRR mutated LbCas a protein has the G532R, K538V, Y R and K595R mutations compared to the wild-type LbCas a protein. In some of these embodiments, the arginine substitution is selected from at least one of the following mutations Y47R, K48R, G49R, V R or K51R. In some of these embodiments, the mutant has mutation sites Y47R, G532R, K538V, Y542R and K595R. In some of these embodiments, the mutant has mutation sites K48R, G532R, K538V, Y R and K595R. In some of these embodiments, the mutant has mutation sites G49R, G532R, K538V, Y R and K595R. In some of these embodiments, the mutant has mutation sites V50R, G532R, K538V, Y R and K595R. In some of these embodiments, the mutant has mutation sites K51R, G532R, K538V, Y R and K595R. In some of these embodiments, the mutant has mutation sites D156R, Y47R, G532R, K538V, Y R and K595R. In some of these embodiments, the mutant has mutation sites D156R, K48R, G532R, K538V, Y R and K595R. In some of these embodiments, the mutant has mutation sites D156R, G, R, G, 532, R, K, 538V, Y, 542R and K595R. In some of these embodiments, the mutant has mutation sites D156R, V50R, G532R, K538V, Y R and K595R. In some of these embodiments, the mutant has mutation sites D156R, K51R, G532R, K538V, Y R and K595R. In another aspect, the application provides a nucleic acid molecule encoding the LbCas a protein mutant. In another aspect, the application provides a vector comprising said nucleic acid molecule. In another aspect, the application provides a cell comprising said vector or said LbCas a protein mutant. In another aspect, the application provides a method for preparing the LbCas a protein mutant comprising fermenting the cell, and And separating LbCas a protein mutant from the fermentation product. In some embodiments, the method comprises culturing to OD600 of 0.5-0.7, adding IPTG for induction expression, wherein the final concentration of IPTG in the initial system of induction expression is 0.1-0.3 mmol/L, and In some embodiments, the method of preparation comprises culturing at a temperature of 14-16 ℃ for a time