CRISPR RNA mutant and application thereof in gene editing and nucleic acid detection

Abstract

The invention relates to the technical field of biology, in particular to a CRISPR RNA mutant and application of the CRISPR RNA mutant in gene editing and nucleic acid detection. The invention provides a crRNA (Ribonucleic Acid) mutant capable of regulating and controlling the activity of LbCas12a. The gene expression regulation based on the crRNA mutant is not influenced by leakage inhibition and is more controllable, and meanwhile, the precise regulation of the gene expression level can be realized. The base editing based on the crRNA mutant can be compatible with various deaminases, an editing window is narrower, and more accurate editing can be realized. Based on gene editing driven by homologous recombination of the crRNA mutant, higher transformation and editing efficiency can be obtained in bacteria. On the basis of nucleic acid detection of the crRNA mutant, one-pot detection without modification, additional substances and target site replacement can be realized, and meanwhile, a semi-quantitative result which does not need to be calculated and processed and can be directly read is output.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to CRISPR...RNA mutants and applications thereof in gene editing and nucleic acid detection. Background Art

[0002] CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) is a powerful nucleic acid positioning and cutting tool, in which Cas12a can specifically identify target DNA under the guidance of CRISPRRNA (crRNA) and specifically cut the target DNA. After completing a specific cut, the accessory cutting activity of Cas12a is activated, and the single-stranded DNA present in the surrounding area is non-specifically cut. This performance has been widely used in gene editing and nucleic acid detection. The gene editing method based on Cas12a is easy to construct, has strong specificity, and excellent effect. It can achieve gene expression regulation, base editing, homologous recombination-driven gene editing, etc. in a variety of organisms, which is of great significance for disease treatment, plant yield increase, and engineering strain development. The nucleic acid detection method based on Cas12a can get rid of the dependence of traditional qPCR-based nucleic acid detection methods on laboratories and professional instruments, achieve accurate and instant detection, and provide a powerful means for infectious disease prevention and control and diagnosis.

[0003] However, wild-type Cas12a and its crRNA performance are difficult to adapt to various application scenarios. For example, in gene expression regulation, wild-type Cas12a and its crRNA have leakage suppression problems (i.e. once Cas12a and crRNA are introduced in vivo, whether they are constitutive expression or inducible expression, whether or not inducers are added, gene expression at the target site will be affected), and it is difficult to achieve the regulation of gene expression levels. In base editing, wild-type Cas12a and its crRNA will edit multiple bases at the target site, and specific bases cannot be accurately located. In homologous recombination-driven gene editing, the specific cutting activity of wild-type Cas12a and its crRNA is too strong, and it is easy to cause biological death when applied to prokaryotes, and conversion and editing efficiency are low. In nucleic acid detection, wild-type Cas12a and its crRNA are difficult to be compatible with nucleic acid amplification, and the two are combined in one step to cause the detection system to be low in sensitivity, and quantitative results cannot be output at the same time. Therefore, it is necessary to regulate the activity of Cas12a to solve the problems it faces in gene editing and nucleic acid detection.

[0004] The commonly used methods for regulating the activity of Cas12a currently include Cas12a and crRNA modification. Cas12a modification usually involves mutating some residues in the protein, and crRNA modification usually involves extending, truncating, and chemically modifying the nucleic acid chain. Although these methods have been successful in specific applications, they still have the problems of high cost, cumbersome procedures, and lack of universality. Therefore, it is necessary to develop a simple and effective method for regulating the performance of Cas12a to optimize its performance in the fields of expression regulation, base editing, homologous recombination-driven gene editing, and nucleic acid detection. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide CRISPR RNA mutants and their applications in gene editing and nucleic acid detection.

[0006] The present invention provides a mutant of a CRISPR RNA repeat sequence, wherein the CRISPR RNA repeat sequence is as shown in SEQ ID NO: 1, and the mutant is mutated at at least one site in the 1st, 5th, 11th, 12th, 13th, 14th, and 15th nucleic acid positions.

[0007] In the present invention, the mutation in the mutant includes at least one of the following:

[0008] The U at position 1 mutates to C or G;

[0009] The U at position 5 mutated to G;

[0010] The U at position 11 mutated to A, C, or G;

[0011] The A at position 12 mutates to C, G, or U;

[0012] The A at position 13 mutated to C, G, or U;

[0013] The G at position 14 mutated to C, G, or U;

[0014] The U at position 15 is mutated to C, G or A.

[0015] In some embodiments, the mutant has 2 mutation sites, including:

[0016] U01C and U05G, U01C and U11A, U01C and U11C, U01C and U11G, U01C and A12C, U01C and A12G, U01C and A12U, U01C and A13U, U01C and A13G, U01C and A13C, U01C and G14A, U01C and G14C, U01C and G14U, U01C and U15C, U01C and U15A, U01C and U15G, U01G and U05G, U01 G and U11A, U01G and U11C, U01G and U11G, U01G and A12C, U01G and A12G, U01G and A12U, U01 G and A13U, U01G and A13G, U01G and A13C, U01G and G14A, U01G and G14C, U01G and G14U, U01G and U15C, U01G and U15A, U01G and U15G, U05G and U11A, U05G and U11C, U05G and U11G, U05G and A12C, U05G and A12G, U05G and A12U, U05G and A13U, U05G and A13G, U05G and A13C, U05G and G14A, U05G and G14C, U05G and G14U, U05G and U15C, U05G and U15A, U05G and U15G, U11A and A12C, U11A and A12G, U11A and A12U, U11A and A13 U, U11A and A13G, U11A and A13C, U11A and G14A, U11A and G14C, U11A and G14U, U11A and U15C, U11A and U15A, U11A and U15G, U11C and A12C, U11C and A12G, U11C and A12U, U11C and A13U, U11C and A13G, U11C and A13C, U11C and G14A, U11C and G14C, U11C and G14U, U11C and U15C, U11C and U15A, U11C and U15G, U11G and A12C, U11G and A12G, U11G and A12U, U11G and A13U, U11G and A13G, U11G and A13C, U11G and G14A, U11 G and G14C, U11G and G14U, U11G and U15C, U11G and U15A, U11G and U15G, A12C and A13U, A12C and A13G, A12C and A13C, A12C and G14A, A12C and G14C, A12C and G14U, A12C and U15C, A12C and U15A, A12C and U15G, A12G and A13U, A12G and A13G, A12G and A13C, A1 2G and G14A, A12G and G14C, A12G and G14U, A12G and U15C, A12G and U15A, A12G and U15G, A12U and A13U, A12U and A13G, A12U and A13C, A12U and G14A, A12U and G14C, A12U and G14U, A12U and U15C, A12U and U15A, A12U and U15G, A 13U and G14A, A13U and G14C, A13U and G14U, A13U and U15C, A13U and U15A, A13U and U15G, A13G and G14A, A13G and G14C, A13G and G14U, A13G and U15C, A13G and U15A, A13G and U15G, A13C and G14A, A13C and G14C, A13C and G14U, A13C and U15C, A13C and U15A, A13C and U15G, G14A and U15C, G14A and U15A, G14A and U15G, G14C and U15C, G14C and U15A, G14C and U15G, G14U and U15C, G14U and U15A, G14U and U15G, U15C and U15A, U15C and U15G, or U15A and U15G.

[0017] In some embodiments, the number of mutation sites of the mutant is 3, including: U01C, U05G, U11A, or including U01C, U05G, U11C, or including U01C, U05G, U11G, or including U01C, U05G, A12C, or including U01C, U05G, A12G, or including U01C, U11A, A12C, or including U01C, U11A, A12G, or including U01C, U11A, A12U, or including U01C, A12U, G14U, or including U01C, A12U, U15C, or including U01C, A12U, U15A, or including U01C, A12U, U15G, or including U01C, A12C, A12U, U15G. 13U, U15A, or including U01C, A13U, U15G, or including U01C, A13G, G14A, or including U01C, A13G, G14C, or including U01C, A13G, G14U, or including U01C, A13G, U15C, or including U01C, A13G, U15A, or including U01C, A13G, U15G, or including U01C, A13C, G14A, or including U01C, A13C, G14C, or including U01C, A13C, G14U, or including U01C, A13C, U15C, or including U01C, A13C, U15A, or including U01C, A13C, U15G, or including U01C, A13C, G14 C, G14A, U15C, or including U01C, G14A, U15A, or including U01C, G14A, U15G, or including U01C, G14C, U15C, or including U01C, G14C, U15A, or including U01C, G14C, U15G, or including U01C, G14U, U15C, or including U01C, G14U, U15A, or including U01C, G14U, U15G, or including U01G, U05G, A13C, or including U01G, U05G, G14A, or including U01G, U05G, G14C, or including U01G, U05G, G14U, or including U01G, U05G, U15C, or including U01G, U05G, U15A, or including U01G, U05G, U15G, or including U01G, U11G, A12C, or including U01G, U11G, A12G, or including U01G, U11G, G14A, or including U01G, U11G, G14C, or including U01G, U11G, G14U, or including U01G, U11G, U15C, or including U01G, U11G, U15A, or including U01G, U11G, U15G, or including U01G, A12C, G14C, or including U01G, A12C, G14U, or including U01G, A12C, U15C, or including U01G, A12C, U15A,or including U01G, A12C, U15G, or including U01G, A12G, G14C, or including U01G, A12G, G14U, or including U01G, A12G, U15C, or including U01G, A12G, U15A, or including U01G, A12G, U15G, or including U01G, A12U, G14C, or including U01G, A12U, G14U, or including U01G, A12U, U15C, or including U01G, A12U, U15A, or including U01G, A12U, U15G, or including U01G, A13U, U15C, or including U01G, A13U, U15A, or including U01G, A13U, U15G, or including U01G , A13G, G14U, or including U01G, A13G, U15C, or including U01G, A13G, U15A, or including U01G, A13G, U15G, or including U01G, A13C, U15C, or including U01G, A13C, U15A, or including U01G, A13C, U15G, or including U05G, U11 C, A12C, or including U05G, U11C, A12G, or including U05G, U11C, A12U, or including U05G, U11C, A13U)U05G, U11G, A12U, or including U05G, U11G, A13U, or including U05G, U11G, A13G, or including U05G, U11G, A13C. ,

[0018] In some embodiments, the number of mutation sites of the mutant is 4, including: U01C, U05G, U11A, A12C, or including U01C, U05G, U11A, A12G, or including U01G, U05G, U11C, A13U, or including U01G, U05G, U11C, A13G, or including U01G, U05G, U11C, A13C, or including U01G, U05G, U11C, G14A, or including U01G, U05G, U11C, G14C, or including U01G, U05G, U11C, G14U, or including U01G, U05G, U11C, U15C, or including U01G, U05G, U11 C, U15A, or including U01G, U05G, U11C, U15G, or including U01G, U05G, U11G, A12C, or including U01G, U05G, U11G, A12G, or including U01G, U05G, U11G, A12U, or including U01G, U05G, U11G, A13U, or including U01G, U05G, U11G, A13G, or including U01G, U05G, U11G, A13C, or including U01G, U05G, U11G, G14A, or including U01G, U05G, U11G, G14C, or including U01G, U05G, U11G, G14U, or including U01G, U05 G, U11G, U15C, or including U01G, U05G, U11G, U15A, or including U01G, U05G, U11G, U15G, or including U05G, U11A, A12C, G14A, or including U05G, U11A, A12C, G14C, or including U05G, U11A, A12C, G14U, or including U05G, U11A, A12G, G14A, or including U05G, U11A, A12G, G14C, or including U05G, U11A, A12G, G14U, or including U05G, U11A, A12U, G14A, or including U05G, U11A, A12U, G14C, or including U0 5G, U11A, A12U, G14U, or including U05G, U11A, A13U, G14A, or including U05G, U11A, A13U, G14C, or including U05G, U11A, A13U, G14U, or including U05G, U11A, A13G, G14A, or including U05G, U11A, A13G, G14C, or including U05G, U11A, A13G, G14U, or including U05G, U11A, A13C, G14A, or including U05G, U11A, A13C, G14C, or including U05G, U11A, A13C, G14U, or including U05G, U11A, A13C, G14A,or including U05G, U11A, U15C, G14C, or including U05G, U11A, U15C, G14U, or including U05G, U11A, U15A, G14A, or including U05G, U11A, U15A, G14C, or including U05G, U11A, U15A, G14U, or including U05G, U11A, U15G, G14A, or including U05G, U11A, U15G, G14C, or including U05G, U11A, U15G, G14U, or including U05G, U11C, A12C, G14A. ,

[0019] In some embodiments, the number of mutation sites of the mutant is 5, including: U01C, U05G, U11A, A12C, G14A, or including U01C, U05G, U11A, A12C, G14C, or including U01C, U05G, U11A, A12C, G14U, or including U01C, U05G, U11A, A12G, G14A, or including U01C, U05G, U11A, A12C, G14C, or including U01C, U05G, U11A, A12G, G14U, or including U01C, U05G, U11A, A12U, G14A. 14C, or including U01C, U05G, U11A, A12U, G14U, or including U01C, U05G, U11A, A13U, G14A, or including U01C, U05G, U11A, A13U, G14C, or including U01C, U05G, U11A, A13U, G14U, or including U01C, U05G, U11A, A13G, G14A, or including U01C, U05G, U11C, U15A, G14U, or including U01C, U05G, U11C, U15G, G14A, or including U01C, U05G, U11C, U15G, G14C, or including U01C, U05G, U11 C, U15G, G14U, or including U01C, U05G, U11G, A12C, G14A, or including U01C, U05G, U11G, A12C, G14C, or including U01C, U05G, U11G, A12C, G14U, or including U01C, U05G, U11G, A12G, G14A, or including U01C, U05G, U11G, A12G, G14C, or including U01C, U05G, U11G, A12G, G14U, or including U01C, U05G, U11G, A12U, G14A, or including U01C, U05G, U11G, A12U, G14C, or including U01C, U05G, U11G, A12U, G14U, or including U01C, U05G, U11G, A13U, G14A, or including U01C, U05G, U11G, A13U, G14C, or including U01C, U05G, U11G, A13U, G14U, or including U01C, U05G, U11G, A13G, G14A, or including U01C, U05G, U11G, A13G, G14C, or including U01C, U05G, U11G, A13G, G14U, or including U01C, U05G, U11G, A13C, G14A, or including U01C, U05G, U11G, A13C, G14C,or including U01C, U05G, U11G, A13C, G14U, or including U01C, U05G, U11G, U15C, G14A, or including U01C, U05G, U11G, U15C, G14C, or including U01C, U05G, U11G, U15C, G14U, or including U01C, U05G, U11G, U15A, G14A, or including U01C, U05G, U11G, U15 A, G14C, or including U01C, U05G, U11G, U15A, G14U, or including U01C, U05G, U11G, U15G, G14A, or including U01C, U05G, U11G, U15G, G14C, or including U01C, U05G, U11G, U15G, G14U, or including U01G, U05G, U11A, A12C, G14A, or including U01G, U05G, U11G, U15G, G14 11A, A12C, G14C, or including U01G, U05G, U11A, A12C, G14U, or including U01G, U05G, U11A, A12G, G14A, or including U01G, U05G, U11A, A12G, G14C, or including U01G, U05G, U11A, A12G, G14U, or including U01G, U05G, U11A, A12U, G14A, or including U01G , U05G, U11A, A12U, G14C, or including U01G, U05G, U11A, A12U, G14U, or including U01G, U05G, U11A, A13U, G14A, or including U01G, U05G, U11A, A13U, G14C, or including U01G, U05G, U11A, A13U, G14U, or including U01G, U05G, U11A, A13G, G14A. ,

[0020] In some embodiments, the mutant has 6 mutation sites, including:

[0021] U01C, U05G, U11A, A12C, G14A, U15C, or including U01C, U05G, U11A, A12C, G14A, U15A, or including U01C, U05G, U11A, A12C, G14A, U15G, or including U01C, U05G, U11A, A12C, G14C, U15C, or including U01C, U05G, U11A, A12C, G14C, U15A, or including U01C, U05G, U11A, A12C, G14C, U15G, or including U01C, U05G, U11A, A12C, G14U, U15C, or including U01C, U05G, U11A, A12C, G14U, U15C A12C, G14U, U15A, or including U01C, U05G, U11A, A12C, G14U, U15G, or including U01C, U05G, U11A, A12G, G14A, U15C, or including U01C, U05G, U11A, A12G, G14A, U15A, or including U01C, U05G, U11A, A12G, G14A, U15G, or including U01C, U05G, U11A, A12G, G14A, U15G, or including U01C, U05G, U11A, A12G, G14C, U15C, or including U01C, U05G, U11A, A12G, G14C, U15A, or including U01C, U05G, U11A, A12G, G14C, U15G, or Includes U01C, U05G, U11A, A12G, G14U, U15C, or includes U01C, U05G, U11A, A12G, G14U, U15A, or includes U01C, U05G, U11A, A12G, G14U, U15G, or includes U01C, U05G, U11A, A12U, G14A, U15C, or includes U01C, U05G, U11A, A12U, G14A, U15A, or includes U01C, U05G, U11A, A12U, G14A, U15C, or includes U01C, U05G, U11A, A12U, G14A, U15G, or includes U01C, U05G, U11A, A12U, G14C, U15C, or includes U01C, U05G, U11A , A12U, G14C, U15A, or including U01C, U05G, U11A, A12U, G14C, U15G, or including U01C, U05G, U11A, A12U, G14U, U15C, or including U01C, U05G, U11A, A12U, G14U, U15A, or including U01C, U05G, U11A, A12U, G14U, U15A, or including U01C, U05G, U11A, A12U, G14U, U15G, or including U01C, U05G, U11A, A13U, G14A, U15C, or including U01C, U05G, U11A, A13U, G14A, U15A,or including U01C, U05G, U11A, A13U, G14C, U15C, or including U01C, U05G, U11A, A13U, G14C, U15A, or including U01C, U05G, U11A, A13U, G14C, U15G, or including U01C, U05G,

[0022] Includes U01C, U05G, U11A, A13G, G14U, U15G, or includes U01C, U05G, U11A, A13C, G14A, U15C, or includes U01C, U05G, U11A, A13C, G14A, U15A, or includes U01C, U05G, U11A, A13C, G14A, U15G, or includes U01C, U05G, U11A, A13C, G14A, U15C, or includes U01C, U05G, U11A, A13C, G14C, U15C, or includes U01C, U05G, U11A , A13C, G14C, U15A, or including U01C, U05G, U11A, A13C, G14C, U15G, or including U01C, U05G, U11A, A13C, G14U, U15C, or including U01C, U05G, U11A, A13C, G14U, U15A, or including U01C, U05G, U11A, A13C, G14U, U15G, or including U01C, U05G, U11A, A13C, G14U, U15G

[0023] In some embodiments, the number of mutation sites of the mutant is 7, including: U01C, U05G, U11A, A12C, A13U, G14A, U15C, or including: U01C, U05G, U11A, A12C, A13U, G14A, U15A, or including: U01C, U05G, U11A, A12C, A13U, G14A, U15A, or including: U01C, U05G, U11A, A12C, A13U, G14A, U15C, or including: U01C, U05G, U11A, A12C, A13U, G14A, U15A, or including: 14C, U15G, or including U01C, U05G, U11A, A12C, A13U, G14U, U15C, or including U01C, U05G, U11A, A12C, A13U, G14U, U15A, or including U01C, U05G, U11A, A12C, A13U, G14U, U15G, or including U01C, U05G, U11A, A12C, A13U, G14A, U15C, or including U01C, U05G, U11A, A12C, A13G, G14A, U15A, or including U01C, U05G, U11A, A12C, A13G, G14A, U15G, or including U01C, U05G, U11A, A12C, A13G, G14A, U15G , U05G, U11A, A12C, A13G, G14C, U15C, or including U01C, U05G, U11A, A12C, A13G, G14C, U15A, or including U01C, U05G, U11A, A12C, A13G, G14C, U15G, or including U01C, U05G, U11A, A12C, A13G, G14U, U15C, or including U01C, U05G, U11A, A12C, A13G, G14U, U15C, or including U01C, U05G, U11A, A12C, A13G, G14U, U15A, or including U01C, U05G, U11A, A12C, A13G, G14U, U15G, or including U01C, U05G, U11A, A12C , A13C, G14A, U15C, or including U01C, U05G, U11A, A12C, A13C, G14A, U15A, or including U01C, U05G, U11A, A12C, A13C, G14A, U15G, or including U01C, U05G, U11A, A12C, A13C, G14C, U15C, or including U01C, U05G, U11A, A12C, A13C, G14C, U15A, or including U01C, U05G, U11A, A12C, A13C, G14C, U15G, or including U01C, U05G, U11A, A12C, A13C, G14U, U15C,or including U01C, U05G, U11A, A12C, A13C, G14U, U15A, or including U01C, U05G, U11A, A12C, A13C, G14U, U15G, or including U01C, U05G, U11A, A12G, A13U, G14A, U15C, or including U01C, U05G, U11A, A12G, A13U, G14A, U15A, or including U01C, U05G, U11A, A12G, A13U, G14A, U15G, or including U01C, U05G, U11A, A12G, A13U, G14A, U15C, or including U01C, U05G, U11A, A12G, A13U, G14A, U15 05G, U11A, A12G, A13U, G14C, U15A, or including U01C, U05G, U11A, A12G, A13U, G14C, U15G, or including U01C, U05G, U11A, A12G, A13U, G14U, U15C, or including U01C, U05G, U11A, A12G, A13U, G14U, U15A, or including U01C, U05G, U11A, A12G, A13U, G14U, U15A, or including U01C, U05G, U11A, A12G, A13U, G14U, U15C, or including U01C, U05G, U11A, A12G, A13U, G14U, U15 12G, A13G, G14A, U15A, or including U01C, U05G, U11A, A12G, A13G, G14A, U15G, or including U01C, U05G, U11A, A12G, A13G, G14C, U15C, or including U01C, U05G, U11A, A12G, A13G, G14C, U15A, or including U01C, U05G, U11A, A12G, A13G, G14C, U15G, or including U01C, U05G, U11A, A12G, A13G, G14U, U15C, or including U01C, U05G, U11A, A12G, A13G, G14U, U15A, or including U01C, U05G, U11A, A12G, A13G, G14U, U15G, or including U01C, U05G, U11A, A12G, A13C, G14A, U15C, or including U01C, U05G, U11A, A12G, A13C, G14A, U15A, or including U01C, U05G, U11A, A12G, A13C, G14A, U15A, or including U01C, U05G, U11A, A12G, A13C, G14A, U15G, or including U01C, U05G, U11A, A12G, A13C, G14C, U15C, or including U01C, U05G, U11A, A12G, A13C, G14C, U15A. ,

[0024] The present invention provides several crRNA mutants, which are obtained by mutating the classical crRNA of LbCas12a. The above crRNA mutants give LbCas12a different activities. The experimental results show that the binding ability of the mutants described in Table 1 to LbCas12a is slightly different.

[0025] The mutation sites of the mutants recorded in Table 1 include:

[0026] U11A, G14A, and U15C;

[0027] or including A12C and U15C;

[0028] or including U1C, U5G, A12C and U15C;

[0029] Or including U1G, U5G and A12C;

[0030] Or including U1C and U5G;

[0031] or including U1G and U5G;

[0032] or including A12C, G14U and U15A;

[0033] or including U11C, A12G, A13U, G14A and U15A;

[0034] or including A12U, G14A and U15G;

[0035] or including U11C, A12G, A13U, G14U and U15C;

[0036] or including U11C, G14U and U15G;

[0037] or including A12G, A13G and U15C;

[0038] or including A12U, A13U, G14C and U15C;

[0039] or including A13G and U15A;

[0040] or including A12U and U15A;

[0041] or including A13U, G14C and U15A;

[0042] or including U11A, A12U, A13G and G14U;

[0043] or including U11C, A12U, A13C, G14U and U15C;

[0044] or including U11A, A12C, A13G, G14A and U15G;

[0045] or including U11C, A13U, G14A and U15C;

[0046] or including A12G, A13C, G14C and U15C;

[0047] or including A12G, A13U and G14A;

[0048] or including A13U, G14C and U15A;

[0049] or including G14U and U15G;

[0050] or including U1C, U5G, A12G, A13U, G14C and U15A;

[0051] or including U1C, U5G, A12G, A13G and G14U;

[0052] or including U1C, U5G, A12C, A13U and G14U;

[0053] or including U1C, U5G, A12G, A13G and G14A;

[0054] or including U1C, U5G, U11C, A12U, A13U and G14C;

[0055] or including U1C, U5G, A12C, A13G, G14C and U15C;

[0056] or including U1C, U5G, U11C, A13U, G14U and U15C;

[0057] or including U1C, U5G, A12C, G14C and U15C;

[0058] or including U1C, U5G, U11C, A12C, A13C, G14C and U15C;

[0059] or including U1G, U5G, U11G, A12U, A13C and G14C;

[0060] or including U1G, U5G, A13C, G14A and U15C;

[0061] or including U1G, U5G, A12C, A13G and G14C;

[0062] or including U1G, U5G, U11A, A12U and A13G;

[0063] or including U1G, U5G, U11C, A13C and G14U;

[0064] or including U1G, U5G, U11G, G14A and U15C;

[0065] or including U1G, U5G, U11A, A12G and A13G;

[0066] or including U1G, U5G, A12U, A13C, G14C and U15A;

[0067] Or include U1G, U5G, A12G and G14U.

[0068] The mutants described in Table 1 have diverse affinities with the target DNA after forming a binary complex with LbCas12a. For the six typical representatives L1, L2, FL1, FL2, F1, and F2 (the nucleic acid sequences are shown in any one of SEQ ID NOs: 2 to 7), their target DNA affinities generally follow the order of F1≈F2>L2>L1>FL1≈FL2.

[0069] The mutants described in Table 1 all have diverse activities in specifically cutting target DNA after forming a binary complex with LbCas12a. For the six typical representatives L1, L2, FL1, FL2, F1, and F2, their specific cutting activities roughly follow the order of F2>F1>L2>FL2>FL1>L1.

[0070] The mutant-guided LbCas12a described in Table 1 has diverse accessory cleavage activities after completing specific cleavage. For the six typical representatives L1, L2, FL1, FL2, F1, and F2, their accessory cleavage activities roughly follow the order of F2>F1>L2>FL1≈FL2>L1.

[0071] The present invention also provides a crRNA, which comprises a sequentially connected spacer sequence and a repeat sequence, wherein the repeat sequence is a mutant as described above, and the spacer sequence is complementary to the targeted target nucleic acid.

[0072] In an embodiment of the present invention, the length of the spacer sequence in the crRNA is 12 to 30 bp; for example, the fragment length is 12bp, 13bp, 14bp, 15bp, 16bp, 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, 25bp, 26bp, 27bp, 28bp, 29bp or 30bp.

[0073] In the present invention, the target nucleic acid includes gene editing sites, characteristic sequence sites of nucleic acid to be detected, etc. In some specific embodiments, the exogenous nucleic acid includes gfp-1, gfp-3, NCR1, Pseudo1, Pseudo2, zapE, and P1 adhesin gene of Mycoplasma pneumoniae.

[0074] More preferably, as a feasible example, the spacer sequence has a nucleic acid sequence as described in any one of SEQ ID NOs: 44 to 50.

[0075] Furthermore, the present invention also provides an expression frame, which includes a promoter and a DNA fragment corresponding to the crRNA as described above.

[0076] In some embodiments, the promoter includes but is not limited to the J23119 promoter, a DNA fragment corresponding to crRNA, and a terminator.

[0077] Furthermore, the present invention also provides a plasmid vector, which includes a DNA fragment corresponding to the crRNA as described above, or includes an expression frame as described above.

[0078] In some embodiments, the plasmid vector further includes an expression cassette of Cas12a. In the present invention, the Cas12a includes LbCas12a or dLbCas12a; or is a fusion fragment of LbCas12a or dLbCas12a and a base editor.

[0079] In some embodiments, the base editor comprises APOBEC, evoCDA or UGI.

[0080] In some specific embodiments, the plasmid vector comprises I) and II):

[0081] I), the expression cassette as described above;

[0082] II), the expression cassette of LbCas12a, dLbCas12a, APOBEC-dLbCas12a-UGI or evoCDA-dLbCas12a-UGI.

[0083] More specifically, the amino acid sequence of LbCas12a is shown in SEQ ID NO:51,

[0084] The amino acid sequence of dLbCas12a is shown in SEQ ID NO:52,

[0085] The amino acid sequence of APOBEC-dLbCas12a-UGI is shown in SEQ ID NO:56,

[0086] The amino acid sequence of evoCDA-dLbCas12a-UGI is shown in SEQ ID NO:57.

[0087] As a feasibility case, the backbone of the plasmid vector is p15A or pYYDT.

[0088] Furthermore, the present invention also provides a method for preparing crRNA as described above, which comprises reacting the DNA template of the crRNA as described above in a system containing T7 RNA polymerase, removing DNA, and purifying to obtain the crRNA.

[0089] Furthermore, the present invention also provides the use of the mutant as described above, the crRNA as described above and / or its corresponding DNA fragment, the expression frame as described above, and the plasmid vector as described above in the preparation of gene editing and / or nucleic acid detection reagents.

[0090] In the present invention, the gene editing includes gene expression level regulation, genome base editing or homologous recombination-driven gene editing.

[0091] Furthermore, the present invention also provides a reagent for regulating gene expression level, which includes: a plasmid vector and a reagent required for plasmid transformation;

[0092] The plasmid vector includes the expression cassette and the expression cassette of dLbCas12a.

[0093] In this embodiment, the backbone of the plasmid vector adopts pYYDT-CRISPRi plasmid, in which the replication origin and antibiotic resistance gene can be replaced, and the key elements related to the gene expression level regulation function include crRNA and dLbCas12a involved in the present invention. The spacer sequence portion of crRNA should be complementary to the pre-spacer sequence base adjacent to PAM in the gene to be regulated. In the embodiment, crRNA is expressed under the control of J23119 promoter, and dLbCas12a is expressed under the control of PAM. BAD The expression is controlled by promoters, which can be replaced.

[0094] The present invention also provides a method for regulating gene expression level, which comprises transforming a plasmid vector into a cell;

[0095] The plasmid vector includes an expression cassette as described above and an expression cassette of dLbCas12a.

[0096] The specific steps include: transforming the pYYDT plasmid into the strain to be regulated for expression, adding an inducer (using P BADWhen manipulating the expression of dLbCas12a, L-arabinose is used, and the final concentration should be selected according to the expected expression inhibition effect), and gene expression inhibition can be performed. If the gene expression inhibition effect needs to be verified, RNA in the strain can be extracted, and the transcript of the inhibited gene can be detected by RT-qPCR. If the expression inhibition is successful, the transcript of the inhibited gene should be decreased compared with the control group.

[0097] The present invention provides a gene expression regulation tool based on crRNA mutants, which comprises a binary complex formed by dLbCas12a and crRNA mutants, and can specifically bind to the target site in vivo to inhibit gene expression there. Experimental results show that the gene expression regulation tool comprising crRNA mutant L1 can avoid the influence of leakage inhibition, that is, after the genes of inducible expression dLbCas12a and constitutive expression crRNA are introduced into the organism, without adding an inducer, the gene expression at the target site is not affected. The gene expression regulation tool comprising different crRNA mutants can form different degrees of inhibition on the gene expression level in the organism, so as to achieve accurate regulation of the gene expression level in the organism.

[0098] Furthermore, the present invention also provides a reagent for genome base editing, which includes: a plasmid vector and a transformation reagent required for the plasmid;

[0099] The plasmid vector includes the expression cassette as described above and the expression cassette of evoCDA-dLbCas12a-UGI.

[0100] In the present invention, the backbone of the plasmid vector is a pYYDT-BE plasmid, in which the replication origin and the antibiotic resistance gene can be replaced, and the key elements related to the base editing function include the crRNA involved in the present invention and LbCas12-BE. The spacer sequence portion of the crRNA should be complementary to the pre-spacer sequence base adjacent to the PAM in the gene to be edited. LbCas12a-BE contains dLbCas12a, and generally also contains enzymes such as deaminases from various sources that can catalyze the editing of bases in DNA, and can also contain uracil glycosylase inhibitors (UGI) and other elements that assist in improving editing efficiency. In the embodiment, crRNA is expressed under the control of the J23119 promoter, and LbCas12a-BE is expressed in P BAD The expression is controlled by promoters, which can be replaced.

[0101] The present invention also provides a method for genome base editing, which comprises transforming a plasmid vector into a cell;

[0102] The plasmid vector includes the expression cassette as described above and the expression cassette of evoCDA-dLbCas12a-UGI.

[0103] The specific steps include: transforming the pYYDT plasmid into the strain to be base edited, adding an inducer (using P BAD When LbCas12a-BE is expressed, L-arabinose is used, and the final concentration is preferably 10mM), and base editing begins. After a certain period of culture, base editing is completed. If the base editing effect needs to be verified, the nucleic acid in the strain can be extracted, the fragment of the base editing region can be amplified, and the amplified product can be Sanger sequenced.

[0104] The present invention provides a base editor based on a crRNA mutant. The base editor includes a crRNA mutant and LbCas12a-BE. LbCas12a-BE includes dLbCas12a, and generally also includes enzymes such as deaminases from various sources that can catalyze the editing of bases in DNA, and can also include uracil glycosylase inhibitors (UGI) and other elements that assist in improving editing efficiency. The above-mentioned base editor can specifically bind to the target site in vivo, so that the bases in the region are edited. In the present invention, the LbCas12a-BE used is APOBEC-dLbCas12a-UGI and evoCDA-dLbCas12a-UGI. Experimental results show that the above-mentioned crRNA mutants can be compatible with these LbCas12a-BE, and when using each LbCas12a-BE, the above-mentioned crRNA mutants can regulate the editing window of the base editor (i.e., the base range that may be edited at the target site). Among them, the FL2 mutant, when used in combination with evoCDA-dLbCas12a-UGI, can achieve precise editing while maintaining high editing efficiency.

[0105] Furthermore, the present invention also provides a homologous recombination-driven gene editing reagent, which includes a plasmid vector 1, a plasmid vector 2, and reagents required for plasmid transformation;

[0106] The plasmid vector 1 comprises the expression cassette as described above and a repair template comprising upstream and downstream homology arms;

[0107] The plasmid vector 2 includes an expression cassette of LbCas12a.

[0108] In the present invention, the backbone of plasmid vector 1 is p15A, and the backbone of plasmid vector 2 is pYYDT plasmid. The replication origin and antibiotic resistance gene of both plasmids can be replaced, but in order to ensure the editing effect, it is advisable to use a plasmid backbone with a medium to low copy number to replace p15A, and a plasmid backbone with a medium to high copy number to replace pYYDT. The key elements related to the gene editing function on p15A are crRNA and a repair template containing upstream and downstream homologous arms. The spacer sequence portion of crRNA should be complementary to the appropriate pre-spacer sequence base adjacent to PAM in the gene to be edited, while ensuring that the edited gene does not contain a sequence that can pair with crRNA. The sequence of the repair template should be consistent with the expected result of gene editing. In the embodiment, crRNA is expressed under the control of the J23119 promoter, and LbCas12a is expressed under the control of the PAM promoter. BAD The expression is controlled by promoters, which can be replaced.

[0109] The present invention also provides a homologous recombination-driven gene editing method, which comprises transforming plasmid vector 1 and plasmid vector 2 into cells through two transformations;

[0110] The plasmid vector 1 comprises the expression cassette as described above and a repair template comprising upstream and downstream homology arms;

[0111] The plasmid vector 2 includes an expression cassette of LbCas12a.

[0112] The specific steps of the method are: through two transformations, the pYYDT plasmid and the p15A plasmid are sequentially transformed into the strain to be gene-edited, and an inducer (using P BAD When LbCas12a expression is manipulated, L-arabinose is used, and the final concentration is preferably 5mM), and gene editing begins. After a certain period of culture, gene editing is completed. If the effect of gene editing needs to be verified, the nucleic acid in the strain can be extracted, the fragment of the gene editing region can be amplified, and the amplified product can be Sanger sequenced.

[0113] The present invention also provides gene editing driven by homologous recombination based on crRNA mutants. The above-mentioned editing tool comprises a crRNA mutant, LbCas12a and a repair template comprising upstream and downstream homologous arms. The above-mentioned editing tool can cause DNA double-strand breaks at specific target sites in the organism, and guide the organism to repair the DNA there according to the repair template with a certain probability, thereby introducing gene editing. The experimental results show that in Escherichia coli, the expression vector containing the above-mentioned editing tool causes different conversion and editing efficiencies, among which the conversion efficiency produced by the crRNA mutant L1 is about 10,000 times that of the classic crRNA, and the editing efficiency is several times that of the classic crRNA.

[0114] The above-mentioned crRNA mutant-based gene expression regulation tools, base editing tools, and homologous recombination-driven gene editing tools have improved and enriched the use effect of LbCas12a in vivo, providing a powerful supplement to the current gene editing methods in vivo.

[0115] Furthermore, the present invention also provides a nucleic acid detection reagent, which includes: a polymerase, dNTP, a LbCas12a protein and a crRNA as described above.

[0116] The reagents of the present invention also include primers for amplifying target nucleic acids and / or single-stranded oligonucleotide probes with fluorescent groups and quenchers labeled at both ends, respectively.

[0117] The present invention also provides a nucleic acid detection method, which comprises: mixing a sample to be detected with the detection reagent as described above, and performing detection after reaction.

[0118] The semi-quantitative one-pot nucleic acid detection system based on crRNA mutants provided by the present invention. When used for DNA detection, the detection system comprises a polymerase, deoxynucleoside triphosphates (dNTPs), primers for amplifying target nucleic acids, LbCas12a, crRNA mutants, single-stranded oligonucleotide probes labeled with fluorescent groups and quenchers at both ends. When used for RNA detection, the detection system further comprises a reverse transcriptase and a primer for reverse transcription of the target nucleic acid on the basis of the above elements. The experimental results show that rapid one-pot nucleic acid detection can be achieved using crRNA mutants, and the result output time is about 20-30min. The detection limit of the detection system containing L1 mutants is 100aM (60cp / μL); the detection limit of the detection system containing FL2 mutants is 10aM (6cp / μL); the detection limit of the detection system containing F1 mutants is 1aM (0.6cp / μL), which is comparable to qPCR and is sufficient to provide accurate detection results.

[0119] For the same sample, three detection systems containing the above crRNA mutants were used for detection, and the content of the target nucleic acid in the sample could be judged according to the number of detection systems that output positive results. All three systems output positive results, indicating that the target nucleic acid content exceeds 100aM, 2 systems output positive results, and 1 system outputs negative results, indicating that the target nucleic acid content is between 10 and 100aM, 1 system outputs positive results, and 2 systems output negative results, indicating that the target nucleic acid content is between 1 and 10aM; all three systems output negative results, indicating that the target nucleic acid content is less than 1aM.

[0120] The above-mentioned semi-quantitative one-pot nucleic acid detection system based on crRNA mutants solves the problems of difficulty in quantification and low sensitivity in one-pot CRISPR detection, and provides an unprecedentedly simple semi-quantitative one-pot detection strategy, which is suitable for scenarios requiring immediate detection, such as township hospitals, home testing, and bedside testing.

[0121] The present invention obtains a series of crRNA mutant sets that can regulate the activity of LbCas12a by modifying the crRNA repeat sequence part of LbCas12a in the CRISPR family. Compared with the existing methods, the gene expression regulation based on the crRNA mutant is not affected by leakage inhibition, is more controllable, and can achieve precise regulation of gene expression levels. The base editing based on the crRNA mutant is compatible with a variety of deaminases, and the editing window is narrower than the existing methods, which can achieve more accurate editing. Compared with the existing methods, the homologous recombination-driven gene editing based on the crRNA mutant can obtain higher conversion and editing efficiency in bacteria. Compared with the existing methods, the nucleic acid detection based on the crRNA mutant can achieve one-pot detection without modification, without the addition of substances, and without changing the target site, and outputs semi-quantitative results that can be directly read without calculation and processing. The above functions supplement powerful tools for gene editing and nucleic acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 Shows the effect of repeated sequence mutation on the affinity between crRNA and LbCas12a;

[0123] Figure 2 Shows the effect of repeat sequence mutation on the affinity between the dLbCas12a-crRNA binary complex and the target DNA;

[0124] Figure 3 It shows the specific cutting activity of LbCas12a under the guidance of crRNA mutants;

[0125] Figure 4 The figure shows the accessory cleavage activity of LbCas12a guided by crRNA mutants;

[0126] Figure 5 Shows the specificity of LbCas12a under the guidance of crRNA mutants;

[0127] Figure 6 It shows the principle that crRNA guides dLbCas12a to regulate gene expression;

[0128] Figure 7 It shows the effect of crRNA mutants guiding dLbCas12a to regulate gene expression levels;

[0129] Figure 8 The figure shows the principle of crRNA mutants guiding LbCas12a-BE to perform base editing;

[0130] Fig. 9 The efficiency of base editing guided by crRNA mutants in APOBEC-dLbCas12a-UGI is shown;

[0131] Fig.10 The results show the efficiency of base editing guided by crRNA mutants in evoCDA-dLbCas12a-UGI.

[0132] Fig.11 The figure shows the changes in the editing window of APOBEC-dLbCas12a-UGI for base editing guided by crRNA mutants;

[0133] Fig.12 The figure shows the changes in the editing window of base editing guided by crRNA mutants evoCDA-dLbCas12a-UGI;

[0134] Fig.13 The basic process of crRNA mutants guiding LbCas12a to perform homologous recombination-mediated gene editing in Escherichia coli is shown;

[0135] Fig.14 The figure shows the effect of crRNA mutants guiding LbCas12a to perform homologous recombination-mediated fragment knockout editing in Escherichia coli, where A is the transformation efficiency and B is the editing efficiency;

[0136] Fig.15 The figure shows the effect of crRNA mutants guiding LbCas12a to perform homologous recombination-mediated fragment knock-in editing in Escherichia coli, where A is the transformation efficiency and B is the editing efficiency;

[0137] Fig.16 The detection limits of crRNA mutants guiding LbCas12a for one-pot Mycoplasma pneumoniae nucleic acid detection are shown, wherein A is the detection limit of the detection system comprising mutant L1, B is the detection limit of the detection system comprising mutant FL2, and C is the detection limit of the detection system comprising mutant F1;

[0138] Fig.17 The specificity of crRNA mutants guiding LbCas12a for one-pot Mycoplasma pneumoniae nucleic acid detection is shown, wherein A is the specificity of the detection system comprising mutant L1, B is the specificity of the detection system comprising mutant FL2, and C is the specificity of the detection system comprising mutant F1;

[0139] Fig.18The result reading method of semi-quantitative one-pot Mycoplasma pneumoniae nucleic acid detection using crRNA mutants to guide LbCas12a is shown. DETAILED DESCRIPTION

[0140] The present invention provides CRISPR RNA mutants and their applications in gene editing and nucleic acid detection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted in particular that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0141] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0142] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0143] In this application, "include", "comprising" and "having" are used interchangeably, and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements besides the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" in this article also provides a "consisting of..." solution.

[0144] In the present application, “and / or” when used herein includes the meanings of “and”, “or” and “all or any other combinations of elements linked by the corresponding terms”.

[0145] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0146] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0147] The sequences involved in this article include:

[0148] Table 1 crRNA repeat sequence mutants

[0149]

[0150]

[0151] *This table only shows the crRNA repeat sequence part. The crRNA spacer sequence part can be designed according to the target site.

[0152] Table 2 crRNA spacer sequences

[0153]

[0154] *This table only shows the crRNA spacer sequence. In the examples, each repeat sequence is connected to 7 repeat sequences (SEQ ID NO: 1-7) of CN, L1, L2, FL1, FL2, F1 and F2 to form a complete crRNA.

[0155] Amino acid sequence of LbCas12a:

[0156]

[0157] Amino acid sequence of dLbCas12a:

[0158]

[0159] Amino acid sequence of APOBEC:

[0160] MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK(SEQ ID NO:53)

[0161] Amino acid sequence of evoCDA:

[0162] MTDAEYVRIHEKLDIYTFKKQFSNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWVCKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMFQVKILHTTKSPAV(SEQ ID NO:54)

[0163] Amino acid sequence of UGI:

[0164] TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML(SEQ ID NO:55)

[0165] Amino acid sequence of APOBEC-dLbCas12a-UGI:

[0166]

[0167] Amino acid sequence of evoCDA-dLbCas12a-UGI:

[0168]

[0169] Amino acid sequence of 16 amino acid linker: SGSETPGTSESATPES (SEQ ID NO: 58)

[0170] Amino acid sequence of 4 amino acid linker: SGGS (SEQ ID NO: 59)

[0171] RPA amplification F primer sequence 5'-CACCAACGAACAGAGCTTAGGTCTCCGCTT-3' (SEQ ID NO: 60)

[0172] RPA amplification R primer sequence 5'-CTAAACAAGGTTTGGGGACCTTGACTGGAG-3' (SEQ ID NO: 61)

[0173] Single-stranded oligonucleotide probe sequence labeled with fluorescent groups and quenchers at both ends: FAM-5'-CCCCCCCC-3'-BHQ1 (SEQ ID NO: 62)

[0174] The present invention will be further described below in conjunction with embodiments:

[0175] Example 1: CrRNA mutants are used to regulate gene expression levels in Escherichia coli

[0176] The main components of the plasmid constructed in this example include: (1) pYYDT-CRISPRi plasmid; (2) crRNA mutant expression cassette driven by J23119 promoter, that is, crRNA mutant targeting the corresponding target site is inserted into the crRNA expression cassette driven by J23119 promoter; (3) pYYDT-CRISPRi plasmid; (4) pYYDT-CRISPRi plasmid; (5) pYYDT-CRISPRi plasmid; (6) pYYDT-CRISPRi plasmid; (7) pYYDT-CRISPRi plasmid; (8) pYYDT-CRISPRi plasmid; (9) pYYDT-CRISPRi plasmid; (10) pYYDT-CRISPRi plasmid; (11) pYYDT-CRISPRi plasmid; (12) pYYDT-CRISPRi plasmid; (13) pYYDT-CRISPRi plasmid; (14) BAD Promoter-driven dLbCas12a expression cassette ( Figure 6 ).

[0177] In order to evaluate the activity of each crRNA mutant guiding dLbCas12a to regulate the expression level, the crRNA spacer sequence portion (SEQ ID NO: 44) was designed to target the gfp gene (pre-inserted) on the Escherichia coli NEB 10-beta genome, and the plasmid was transformed into the strain. The resulting transformants were cultured in a medium containing different concentrations of l-arabinose inducer for 30 h, and the gfp expression level was determined by a microplate reader.

[0178] The results showed that different crRNA mutants can provide different expression inhibition levels, thereby achieving fine regulation of gene expression levels ( Figure 7In addition, mutant L1 showed no expression inhibition in the absence of an inducer, overcoming the problem of leaky inhibition commonly faced by CRISPR-based expression regulation ( Figure 7 ).

[0179] Example 2: Use of crRNA mutants for base editing of the Escherichia coli genome

[0180] The main components of the plasmid constructed in this example include: (1) pYYDT-BE, (2) a crRNA mutant expression cassette driven by the J23119 promoter, that is, the crRNA mutant targeting the corresponding target site is inserted into the crRNA expression cassette driven by the J23119 promoter; (3) a crRNA mutant expression cassette driven by the P BAD Promoter-driven evoCDA-dLbCas12a-UGI expression cassette (but the available deaminases are not limited to evoCDA). evoCDA is linked to the N-terminus of dLbCas12a via a 16aa XTEN linker peptide, and UGI is linked to the C-terminus of dLbCas12a via a 4aa linker peptide, together forming the LbCas12a-CBE base editor ( Figure 8 ).

[0181] In order to evaluate the activity of crRNA mutants in guiding evoCDA-dLbCas12a-UGI for base editing, the crRNA spacer sequence portion (SEQ ID NO: 45-49) was designed to target multiple sites on the E. coli NEB 10-beta genome, and the plasmid was transformed into the strain. After 24 h of culture, the colonies were collected and the target region was subjected to Sanger sequencing to verify the C-to-T editing efficiency at different sites.

[0182] By sequencing the 1-20 positions of the pre-spacer sequence of E. coli NEB 10-beta, the editing efficiency of each cytosine was evaluated. The results showed that evoCDA-dLbCas12a-UGI guided by different crRNA mutants had different editing windows. Comprehensive statistics of the results of each target site showed that the editing window of mutant FL2 was C8-C10, which was narrower than C8-11 and 16 of the classic crRNA, and could achieve more precise base editing (such as Fig.10 , Fig.12 ).

[0183] Example 3: Use of crRNA mutants for homologous recombination-driven knockout of Escherichia coli genome fragments

[0184] In this example, two plasmids (p15A and pYYDT plasmids) were constructed, wherein the main components of the first plasmid included: (1) a crRNA mutant expression cassette driven by the J23119 promoter, i.e., the crRNA mutant targeting the corresponding target site was inserted into the crRNA expression cassette driven by the J23119 promoter; (2) a repair template containing 1000 bp homology arms upstream and downstream of the knockout fragment. The main components of the second plasmid included P BAD Promoter-driven LbCas12a expression cassette ( Fig.13 ).

[0185] In order to evaluate the activity of crRNA mutants in guiding LbCas12a to perform homologous recombination-driven fragment knockout on the E. coli genome, the crRNA spacer sequence portion (SEQ ID NO: 43) was designed to target the gfp gene (pre-inserted) on the E. coli NEB 10-beta genome, and the double plasmid was transformed into the strain. The resulting transformants were cultured in a solid medium containing 5mM l-arabinose inducer for 20h, and the number of transformants was then counted, and the colonies were collected, and the target site was amplified. The length of the amplified fragment was determined by agarose gel electrophoresis, and the knockout efficiency was judged in combination with the Sanger sequencing results ( Fig.13 ).

[0186] The transformation efficiency of the editing tools containing different crRNA mutants was determined by counting the number of transformants, and the editing efficiency of different crRNA mutants in homologous recombination-driven gene knockout on the E. coli NEB 10-beta genome was determined by the length and sequence of the amplified fragments. The results showed that the number of transformants obtained by mutant L1 was 4 orders of magnitude higher than that of classical crRNA, and the editing efficiency (58.3%) was more than 10 times that of classical crRNA, and the overall editing effect was significantly improved compared with classical crRNA ( Fig.14 ).

[0187] Example 4: Use of crRNA mutants for semi-quantitative one-tube Mycoplasma pneumoniae nucleic acid detection

[0188] The nucleic acid detection system in this embodiment includes a polymerase, deoxynucleoside triphosphates (dNTPs), a primer for amplifying the target nucleic acid, LbCas12a, a crRNA mutant, and a single-stranded oligonucleotide probe labeled with a fluorescent group and a quencher at both ends.

[0189] In detail, LbCas12a was expressed and purified by the following method: the gene fragment encoding LbCas12a was cloned into a pET-based expression vector containing a C-terminal 6×His tag. The Escherichia coli strain Rosetta transformed with the recombinant plasmid was cultured to OD600=0.8, 0.2mM isopropyl β-D-1-thiogalactoside (IPTG) was added and incubated together, and cultured at 16°C for 16h. Proteins were separated from cell lysates using Ni-NTA resin and eluted with buffer (50mMTris-HCl, 1.5M NaCl, 5% glycerol and 600mM imidazole, pH=8.0).

[0190] In detail, crRNA was prepared by the following method: Designing the crRNA spacer sequence portion (SEQ ID NO: 50) to target Mycoplasma pneumoniae P1, polymerizing the oligonucleotide containing the T7 promoter sequence and the oligonucleotide containing the spacer sequence to obtain a DNA template for in vitro transcription. The template was incubated with T7 RNA polymerase (Vazyme, Nanjing, China) at 37°C for 2h for in vitro transcription (IVT). The IVT product was incubated with DNase I (Vazyme, Nanjing, China) at 37°C for 20min to remove the template DNA, and then incubated at 65°C for 20min to inactivate DNase I. The resulting crRNA was purified using an RNA purification kit (Genstone Biotech, Beijing, China).

[0191] In detail, the nucleic acid amplification method used in this embodiment is RPA, and the source of the relevant reagents is from Weifang Amp-Future Biotech (Weifang Amp-Future Biotech, Shandong, China). However, the semi-quantitative one-tube nucleic acid detection method based on crRNA mutants can be used in conjunction with other nucleic acid amplification methods, not limited to RPA. The RPA amplification primer sequences used are shown in SEQ ID NO: 60-61.

[0192] In detail, the single-stranded oligonucleotide probe labeled with fluorescent groups and quenchers at both ends used in this embodiment is a single-stranded DNA with a sequence of 5'-CCCCCCCC-3', wherein the 5' end is labeled with fluorescent group FAM, and the 3' end is labeled with fluorescent group BHQ1.

[0193] Nucleic acid detection is carried out according to the following steps: According to the manufacturer's instructions, a tube of RPA reagent lyophilized powder is resuspended in 29.4 μL buffer A, and 2 μL of 10 μM RPA amplification primers and 14.1L DEPC-treated water are added to form an RPA mixture. Each detection reaction system contains 18 μL RPA mixture, 50nM LbCas12a, 50nM crRNA mutant, 400nM FQ ssDNA reporter molecule (FAM-CCCCCCCC-BHQ) and 2 μL RPA amplification kit in buffer B. 3 μL of the sample to be tested is added to the obtained reaction system, and then the reaction system is placed in a StepOnePlus real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific, USA) and read at 37 ° C. It is worth noting that although the real-time fluorescence quantitative PCR instrument is used to read the test results in this example, the method in the present invention is also compatible with other signal output methods, such as flow chromatography test paper, small fluorescence reading equipment, etc. In addition, although the nucleic acid detection in this example is for Mycoplasma pneumoniae nucleic acid (DNA), after adding reverse transcriptase and primers for reverse transcription of target nucleic acid into the detection system, the present invention can also be used for RNA detection.

[0194] The above detection system was used to detect gradient-diluted Mycoplasma pneumoniae nucleic acid. The experimental results showed that the use of crRNA mutants can achieve rapid one-pot nucleic acid detection, and the result output time is about 20-30 minutes. The detection limit of the detection system containing L1 mutants is 100aM (60cp / μL); the detection limit of the detection system containing FL2 mutants is 10aM (6cp / μL); the detection limit of the detection system containing F1 mutants is 1aM (0.6cp / μL), which is comparable to qPCR and sufficient to provide accurate detection results ( Fig.16 The above detection system was used to detect nucleic acids from monkeypox virus, severe acute respiratory syndrome coronavirus type 2, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Shewanella putrefaciens, and Helicobacter pylori. The results were all negative, indicating that the detection method has high specificity ( Fig.17 ).

[0195] When performing qualitative detection on the target sample, only the detection system containing mutant F1 needs to be used. When performing semi-quantitative detection on the target sample, the detection systems containing mutants L1, FL2 and F1 need to be used at the same time, and the range of Mycoplasma pneumoniae nucleic acid concentration in the sample is judged according to the number of detection systems that output positive results. All three systems output positive results, indicating that the target nucleic acid content exceeds 100aM, 2 systems output positive results, and 1 system outputs negative results, indicating that the target nucleic acid content is between 10 and 100aM, 1 system outputs positive results, and 2 systems output negative results, indicating that the target nucleic acid content is between 1 and 10aM; all three systems output negative results, indicating that the target nucleic acid content is lower than 1aM ( Fig.18 ).

[0196] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutant of a CRISPR RNA repeat fragment repeat sequence, the nucleic acid sequence of the CRISPR RNA repeat sequence is shown in SEQ ID NO: 1, and the mutant is mutated at at least one site in the nucleic acid at positions 1, 5, 11, 12, 13, 14, and 15.

2. The mutant according to claim 1, characterized in that The mutation includes at least one of the following: The U at position 1 mutates to C or G; The U at position 5 mutated to G; The U at position 11 mutated to A, C, or G; The A at position 12 mutates to C, G, or U; The A at position 13 mutated to C, G, or U; The G at position 14 mutated to C, G, or U; The U at position 15 is mutated to C, G or A.

3. The mutant according to claim 1, characterized in that The mutant includes the following mutation sites: U11A, G14A, and U15C; or including A12C and U15C; or including U1C, U5G, A12C and U15C; Or including U1G, U5G and A12C; Or including U1C and U5G; or including U1G and U5G; or including A12C, G14U and U15A; or including U11C, A12G, A13U, G14A and U15A; or including A12U, G14A and U15G; or including U11C, A12G, A13U, G14U and U15C; or including U11C, G14U and U15G; or including A12G, A13G and U15C; or including A12U, A13U, G14C and U15C; or including A13G and U15A; or including A12U and U15A; or including A13U, G14C and U15A; or including U11A, A12U, A13G and G14U; or including U11C, A12U, A13C, G14U and U15C; or including U11A, A12C, A13G, G14A and U15G; or including U11C, A13U, G14A and U15C; or including A12G, A13C, G14C and U15C; or including A12G, A13U and G14A; or including A13U, G14C and U15A; or including G14U and U15G; or including U1C, U5G, A12G, A13U, G14C and U15A; or including U1C, U5G, A12G, A13G and G14U; or including U1C, U5G, A12C, A13U and G14U; or including U1C, U5G, A12G, A13G and G14A; or including U1C, U5G, U11C, A12U, A13U and G14C; or including U1C, U5G, A12C, A13G, G14C and U15C; or including U1C, U5G, U11C, A13U, G14U and U15C; or including U1C, U5G, A12C, G14C and U15C; or including U1C, U5G, U11C, A12C, A13C, G14C and U15C; or including U1G, U5G, U11G, A12U, A13C and G14C; or including U1G, U5G, A13C, G14A and U15C; or including U1G, U5G, A12C, A13G and G14C; or including U1G, U5G, U11A, A12U and A13G; or including U1G, U5G, U11C, A13C and G14U; or including U1G, U5G, U11G, G14A and U15C; or including U1G, U5G, U11A, A12G and A13G; or including U1G, U5G, A12U, A13C, G14C and U15A; Or include U1G, U5G, A12G and G14U.

4. The mutant according to any one of claims 1 to 3, characterized in that The nucleic acid sequence of the mutant is shown in any one of SEQ ID NOs: 2-7.

5. crRNA, comprising a sequentially connected spacer sequence and a repeat sequence, wherein the repeat sequence is a mutant according to any one of claims 1 to 4, and the spacer sequence is complementary to a targeted target nucleic acid.

6. The crRNA according to claim 5, characterized in that The length of the spacer sequence is 12 to 30 bp; The targeted nucleic acid includes gene editing sites, characteristic sequence sites of the nucleic acid to be detected, etc.

7. The crRNA according to claim 5, characterized in that The targeted nucleic acids include gfp-1, gfp-3, NCR1, Pseudo1, Pseudo2, zapE, and P1 adhesin genes of Mycoplasma pneumoniae.

8. The crRNA according to any one of claims 5 to 7, characterized in that The spacer sequence has a nucleic acid sequence as described in any one of SEQ ID NOs: 44 to 50.

9. An expression cassette comprising a promoter and a DNA fragment corresponding to the crRNA according to any one of claims 5 to 8.

10. The expression cassette according to claim 9, characterized in that: The promoter includes a J23119 promoter, a DNA fragment corresponding to crRNA, and a terminator.

11. Plasmid vector, It comprises a DNA fragment corresponding to the crRNA according to any one of claims 5 to 8, Or comprising the expression cassette of claim 9 or 10.

12. The plasmid vector according to claim 11, characterized in that The expression cassette of Cas12a is also included.

13. The plasmid vector according to claim 12, characterized in that The Cas12a includes LbCas12a or dLbCas12a; or is a fusion fragment of LbCas12a or dLbCas12a and a base editor.

14. The plasmid vector according to claim 13, characterized in that The base editors include APOBEC, evoCDA or UGI.

15. The plasmid vector according to any one of claims 11 to 14, characterized in that It includes I) and II): 1), the expression cassette according to claim 9 or 10; II), expression cassette of LbCas12a, dLbCas12a, APOBEC-dLbCas12a-UGI or evoCDA-dLbCas12a-UGI; The amino acid sequence of LbCas12a is shown in SEQ ID NO:

51. The amino acid sequence of dLbCas12a is shown in SEQ ID NO:52, The amino acid sequence of APOBEC-dLbCas12a-UGI is shown in SEQ ID NO:56, The amino acid sequence of evoCDA-dLbCas12a-UGI is shown in SEQ ID NO:

57.

16. The plasmid vector according to any one of claims 11 to 15, characterized in that The backbone of the plasmid vector is p15A or pYYDT.

17. A method for preparing the crRNA according to any one of claims 5 to 7, comprising reacting the DNA template of the crRNA according to any one of claims 5 to 7 in a system containing T7 RNA polymerase, removing the DNA, and obtaining the crRNA by purification.

18. Use of the mutant described in any one of claims 1 to 4, the crRNA and / or its corresponding DNA fragment described in any one of claims 5 to 8, the expression frame described in claim 9 or 10, and the plasmid vector described in any one of claims 11 to 14 in the preparation of gene editing and / or nucleic acid detection reagents.

19. The use according to claim 18, characterized in that The gene editing includes gene expression level regulation, genome base editing or genome fragment knockout.

20. A reagent for regulating gene expression level, comprising: Plasmid vectors and reagents required for plasmid transformation; The plasmid vector includes the expression cassette described in claim 9 or 10, and the expression cassette of dLbCas12a.

21. A method for regulating gene expression levels, comprising transforming a plasmid vector into a cell; The plasmid vector includes the expression cassette described in claim 9 or 10, and the expression cassette of dLbCas12a.

22. A reagent for genome base editing, comprising: Plasmid vectors and reagents required for plasmid transformation; The plasmid vector includes the expression frame described in claim 9 or 10, and the expression frame of evoCDA-dLbCas12a-UGI.

23. A method for genome base editing, comprising transforming a plasmid vector into a cell; The plasmid vector includes the expression frame described in claim 9 or 10, and the expression frame of evoCDA-dLbCas12a-UGI.

24. A homologous recombination-driven gene editing reagent, comprising a plasmid vector 1, a plasmid vector 2, and reagents required for plasmid transformation; The plasmid vector 1 comprises the expression cassette according to claim 9 or 10 and a repair template comprising upstream and downstream homology arms; The plasmid vector 2 includes an expression cassette of LbCas12a.

25. A homologous recombination-driven gene editing method, comprising transforming plasmid vector 1 and plasmid vector 2 into cells through two transformations; The plasmid vector 1 comprises the expression cassette according to claim 9 or 10 and a repair template comprising upstream and downstream homology arms; The plasmid vector 2 includes an expression cassette of LbCas12a.

26. A nucleic acid detection reagent comprising: Polymerase, dNTP, LbCas12a protein and the crRNA described in any one of claims 5 to 7.

27. The detection reagent according to claim 26, characterized in that Also included are primers for amplifying target nucleic acids and / or single-stranded oligonucleotide probes with fluorescent groups and quenchers labeled at both ends, respectively.

28. A nucleic acid detection method, comprising: The sample to be tested is mixed with the detection reagent described in claim 26 or 27, and the detection is performed after the reaction.

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