Cr ispr rna mutants and their use in gene editing and nucleic acid detection

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to CRISPR RNA mutants and application thereof in gene editing and nucleic acid detection. The present application provides crRNA mutants capable of regulating the activity of LbCas12a. Based on the gene expression regulation of the crRNA mutants, the regulation is not affected by leaky inhibition, is more controllable, and meanwhile, precise regulation of the gene expression level can be realized. Based on the base editing of the crRNA mutants, various deaminases are compatible, the editing window is narrower, and more precise editing can be realized. Based on the gene editing driven by homologous recombination of the crRNA mutants, higher conversion and editing efficiency can be obtained in bacteria. Based on the nucleic acid detection of the crRNA mutants, one-pot detection without modification, additional substances and target site replacement can be realized, and meanwhile, semi-quantitative results without calculation and processing and capable of direct reading can be output.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to CRISPR……RNA mutants and their applications in gene editing and nucleic acid detection. BACKGROUND

[0002] CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) is a powerful tool for nucleic acid localization and cleavage, in which Cas12a can specifically recognize and cleave target DNA under the guidance of CRISPR RNA (crRNA). After completing a specific cleavage, the collateral cleavage activity of Cas12a is activated, which will non-specifically cleave the single-stranded DNA existing around. This performance has achieved wide application in gene editing and nucleic acid detection. Cas12a-based gene editing method is easy to construct, has strong specificity and excellent effect, and can realize gene expression regulation, base editing, homologous recombination driven gene editing, etc. in various organisms, which has important significance for disease treatment, plant yield increase, and engineering strain development. Cas12a-based nucleic acid detection method can get rid of the dependence on laboratory and professional instruments in traditional qPCR-based nucleic acid detection method, realize accurate instant detection, and provide a powerful means for infectious disease prevention and diagnosis.

[0003] However, the performance of wild-type Cas12a and its crRNA is difficult to adapt to various application scenarios. For example, in gene expression regulation, wild-type Cas12a and its crRNA have the problem of leaky inhibition (i.e. once Cas12a and crRNA are introduced into an organism, whether they are constitutively expressed or inducibly expressed, whether an inducer is added or not, the gene expression at the target site will be affected), and it is difficult to realize the regulation of gene expression level. In base editing, wild-type Cas12a and its crRNA will edit multiple bases at the target site, and cannot accurately locate a specific base. In homologous recombination driven gene editing, the specific cleavage activity of wild-type Cas12a and its crRNA is too strong, which easily causes biological death when applied to prokaryotes, and the transformation and editing efficiency is low. In nucleic acid detection, wild-type Cas12a and its crRNA are difficult to be compatible with nucleic acid amplification, and when they are combined into one step, it will cause low sensitivity of the detection system, and at the same time cannot output quantitative results. Therefore, it is necessary to regulate the activity of Cas12a to solve the problems it faces in gene editing and nucleic acid detection.

[0004] Current methods for regulating Cas12a activity include Cas12a and crRNA engineering. Cas12a engineering usually involves mutating residues in the protein, and crRNA engineering usually involves extending, truncating, chemically modifying, etc. the nucleic acid strand. These methods, although successful in specific applications, still have problems such as high cost, cumbersome process, and lack of universality. Therefore, it is necessary to develop a simple and effective method for regulating Cas12a performance, so as to optimize its performance in expression regulation, base editing, homologous recombination-driven gene editing, and nucleic acid detection. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a CRISPR RNA mutant and its application in gene editing and nucleic acid detection.

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

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

[0008] the U at the 1st position is mutated to C or G;

[0009] the U at the 5th position is mutated to G;

[0010] the U at the 11th position is mutated to A, C, or G;

[0011] the A at the 12th position is mutated to C, G, or U;

[0012] the A at the 13th position is mutated to C, G, or U;

[0013] the G at the 14th position is mutated to C, G, or U;

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

[0015] In some embodiments, the number of mutation sites of the mutant is 2, 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, U01G and U11A, U01G and U11C, U01G and U11G, U01G and A12C, U01G and A12G, U01G and A12U, U01G 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 A13U, 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, U11G 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, A12G 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, A13U 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, A13U, 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, 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 comprises U01G, A12C, U15G, or comprises U01G, A12G, G14C, or comprises U01G, A12G, G14U, or comprises U01G, A12G, U15C, or comprises U01G, A12G, U15A, or comprises U01G, A12G, U15G, or comprises U01G, A12U, G14C, or comprises U01G, A12U, G14U, or comprises U01G, A12U, U15C, or comprises U01G, A12U, U15A, or comprises U01G, A12U, U15G, or comprises U01G, A13U, U15C, or comprises U01G, A13U, U15A, or comprises U01G, A13U, U15G, or comprises U01G, A13G, G14U, or comprises U01G, A13G, U15C, or comprises U01G, A13G, U15A, or comprises U01G, A13G, U15G, or comprises U01G, A13C, U15C, or comprises U01G, A13C, U15A, or comprises U01G, A13C, U15G, or comprises U05G, U11C, A12C, or comprises U05G, U11C, A12G, or comprises U05G, U11C, A12U, or comprises U05G, U11C, A13U) U05G, U11G, A12U, or comprises U05G, U11G, A13U, or comprises U05G, U11G, A13G, or comprises 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, U11C, 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, U05G, 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 U05G, 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, U15C, 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, A12G, G14C, or including U01C, U05G, U11A, A12G, G14U, or including U01C, U05G, U11A, A12U, G14A, or including U01C, U05G, U11A, A12U, G14C, 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, U11C, 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 comprises U01C, U05G, U11G, A13C, G14U, or comprises U01C, U05G, U11G, U15C, G14A, or comprises U01C, U05G, U11G, U15C, G14C, or comprises U01C, U05G, U11G, U15C, G14U, or comprises U01C, U05G, U11G, U15A, G14A, or comprises U01C, U05G, U11G, U15A, G14C, or comprises U01C, U05G, U11G, U15A, G14U, or comprises U01C, U05G, U11G, U15G, G14A, or comprises U01C, U05G, U11G, U15G, G14C, or comprises U01C, U05G, U11G, U15G, G14U, or comprises U01G, U05G, U11A, A12C, G14A, or comprises U01G, U05G, U11A, A12C, G14C, or comprises U01G, U05G, U11A, A12C, G14U, or comprises U01G, U05G, U11A, A12G, G14A, or comprises U01G, U05G, U11A, A12G, G14C, or comprises U01G, U05G, U11A, A12G, G14U, or comprises U01G, U05G, U11A, A12U, G14A, or comprises U01G, U05G, U11A, A12U, G14C, or comprises U01G, U05G, U11A, A12U, G14U, or comprises U01G, U05G, U11A, A13U, G14A, or comprises U01G, U05G, U11A, A13U, G14C, or comprises U01G, U05G, U11A, A13U, G14U, or comprises U01G, U05G, U11A, A13G, G14A.

[0020] In some embodiments, the number of mutation sites of the mutant is 6, comprising:

[0021] U01C, U05G, U11A, A12C, G14A, U15C, or comprising U01C, U05G, U11A, A12C, G14A, U15A, or comprising U01C, U05G, U11A, A12C, G14A, U15G, or comprising U01C, U05G, U11A, A12C, G14C, U15C, or comprising U01C, U05G, U11A, A12C, G14C, U15A, or comprising U01C, U05G, U11A, A12C, G14C, U15G, or comprising U01C, U05G, U11A, A12C, G14U, U15C, or comprising U01C, U05G, U11A, A12C, G14U, U15A, or comprising U01C, U05G, U11A, A12C, G14U, U15G, or comprising U01C, U05G, U11A, A12G, G14A, U15C, or comprising U01C, U05G, U11A, A12G, G14A, U15A, or comprising U01C, U05G, U11A, A12G, G14A, U15G, or comprising U01C, U05G, U11A, A12G, G14C, U15C, or comprising U01C, U05G, U11A, A12G, G14C, U15A, or comprising U01C, U05G, U11A, A12G, G14C, U15G, or comprising U01C, U05G, U11A, A12G, G14U, U15C, or comprising U01C, U05G, U11A, A12G, G14U, U15A, or comprising U01C, U05G, U11A, A12G, G14U, U15G, or comprising U01C, U05G, U11A, A12U, G14A, U15C, or comprising U01C, U05G, U11A, A12U, G14A, U15A, or comprising U01C, U05G, U11A, A12U, G14A, U15G, or comprising U01C, U05G, U11A, A12U, G14C, U15C, or comprising U01C, U05G, U11A, A12U, G14C, U15A, or comprising U01C, U05G, U11A, A12U, G14C, U15G, or comprising U01C, U05G, U11A, A12U, G14U, U15C, or comprising U01C, U05G, U11A, A12U, G14U, U15A, or comprising U01C, U05G, U11A, A12U, G14U, U15G, or comprising U01C, U05G, U11A, A13U, G14A, U15C, or comprising U01C, U05G, U11A, A13U, G14A, U15A, or comprising U01C, U05G, U11A, A13U, G14A, U15G,or comprises U01C, U05G, U11A, A13U, G14C, U15C, or comprises U01C, U05G, U11A, A13U, G14C, U15A, or comprises U01C, U05G, U11A, A13U, G14C, U15G, or comprises U01C, U05G, U11A, A13U, G14U, U15C, or comprises U01C, U05G, U11A, A13U, G14U, U15A, or comprises U01C, U05G, U11A, A13U, G14U, U15G, or comprises U01C, U05G, U11A, A13C, G14A, U15C, or comprises U01C, U05G, U11A, A13C, G14A, U15A, or comprises U01C, U05G, U11A, A13C, G14A, U15G, or comprises U01C, U05G, U11A, A13C, G14U, U15C, or comprises U01C, U05G, U11A, A13C, G14U, U15A, or comprises U01C, U05G, U11A, A13C, G14U, U15G, or comprises U01G, U05G, U11A, A12C, G14A, U15C.

[0022] comprises U01C, U05G, U11A, A13G, G14U, U15G, or comprises U01C, U05G, U11A, A13C, G14A, U15C, or comprises U01C, U05G, U11A, A13C, G14A, U15A, or comprises U01C, U05G, U11A, A13C, G14A, U15G, or comprises U01C, U05G, U11A, A13C, G14C, U15C, or comprises U01C, U05G, U11A, A13C, G14C, U15A, or comprises U01C, U05G, U11A, A13C, G14C, U15G, or comprises U01C, U05G, U11A, A13C, G14U, U15C, or comprises U01C, U05G, U11A, A13C, G14U, U15A, or comprises U01C, U05G, U11A, A13C, G14U, U15G, or comprises U01G, U05G, U11C, A12C, G14A, U15C.

[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, U15G, or including U01C, U05G, U11A, A12C, A13U, G14C, U15C, or including U01C, U05G, U11A, A12C, A13U, G14C, U15A, or including U01C, U05G, U11A, A12C, A13U, G14C, 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, A13G, 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, 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, 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, U15C 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, U15G, or including U01C, U05G, U11A, A12G, A13U, G14A, U15C, or including U01C, U05G, U11A, A12G 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, U15G, or including U01C, U05G, U11A, A12G, A13C, G14C, U15C, or including U01C, U05G, U11A, A12G, A13C, G14C, U15C. ,

[0024] The present application provides several crRNA mutants, which are obtained by mutating the classic crRNA of LbCas12a. The above-mentioned crRNA mutants endow LbCas12a with different activities. Experimental results show that the binding ability of the mutants described in Table 1 to LbCas12a is relatively small.

[0025] The mutation sites of the mutants described 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 comprises U11A, A12C, A13G, G14A and U15G;

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

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

[0047] or comprises A12G, A13U and G14A;

[0048] or comprises A13U, G14C and U15A;

[0049] or comprises G14U and U15G;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0067] or comprises U1G, U5G, A12G and G14U.

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

[0069] The mutants described in Table 1 all have diversity in activity of specifically cleaving target DNA after forming a binary complex with LbCas12a, and for the six typical representatives L1, L2, FL1, FL2, F1, F2, their specific cleavage activity generally follows the order F2>F1>L2>FL2>FL1>L1.

[0070] The mutants described in Table 1 all have diversity in collateral cleavage activity of LbCas12a guided by the mutants after completing specific cleavage, and for the six typical representatives L1, L2, FL1, FL2, F1, F2, their collateral cleavage activity generally follows the order F2>F1>L2>FL1≈FL2>L1.

[0071] The present application also provides a crRNA comprising a spacer sequence and a repeat sequence connected in sequence, the repeat sequence being a mutant as described above, and the spacer sequence being complementary to a target nucleic acid to be targeted.

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

[0073] In the present invention, the target nucleic acid includes a gene editing site, a characteristic sequence site of the nucleic acid to be detected, etc. In some specific embodiments, the exogenous nucleic acid includes gfp-1, gfp-3, NCR1, Pseudo1, Pseudo2, zapE, and the 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 cassette, 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 cassette as described above.

[0078] In some embodiments, the plasmid vector further includes an expression cassette for 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] 1), the expression cassette as described above;

[0082] II), 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 the evoCDA-dLbCas12a-UGI is shown as SEQ ID NO: 57.

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

[0088] Further, the application also provides a preparation method of the crRNA, which comprises reacting the DNA template of the crRNA in a system containing T7 RNA polymerase, removing the DNA, and then purifying to obtain the crRNA.

[0089] Further, the application also provides application of the mutant, the crRNA and / or the corresponding DNA fragment, the expression frame, and the plasmid vector in the preparation of a gene editing and / or nucleic acid detection reagent.

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

[0091] Further, the application also provides a reagent for regulating the gene expression level, which comprises: a plasmid vector and reagents required for plasmid transformation.

[0092] The plasmid vector comprises the expression frame and the expression frame of dLbCas12a.

[0093] In this embodiment, the backbone of the plasmid vector adopts pYYDT-CRISPRi plasmid, wherein the replication origin and the antibiotic resistance gene can be replaced, and the key elements related to the function of regulating the gene expression level include the crRNA and dLbCas12a involved in the application. The spacer sequence part of the crRNA should be complementary to the protospacer base adjacent to the PAM in the gene to be regulated. In the embodiment, the crRNA is expressed under the manipulation of J23119 promoter, and the dLbCas12a is expressed under the manipulation of P BAD The promoters can be replaced.

[0094] The application also provides a method for regulating the gene expression level, which comprises transforming the plasmid vector into cells.

[0095] The plasmid vector comprises the expression frame and the expression frame of dLbCas12a.

[0096] The specific steps include: transforming the pYYDT plasmid into a strain to be regulated in expression, adding an inducer (adopting P BADWhen dLbCas12a expression is manipulated, L-arabinose is used, and the final concentration is preferably selected according to the expected expression inhibition effect, that is, gene expression inhibition can be performed. If it is necessary to verify the gene expression inhibition effect, RNA in the strain can be extracted, and RT-qPCR detection is performed on the transcript of the inhibited gene. If the expression inhibition is successful, the transcript of the inhibited gene should decrease compared with the control group.

[0097] The present application provides a gene expression regulation tool based on a crRNA mutant, which comprises a binary complex of dLbCas12a and a crRNA mutant, can specifically bind to a target site in an organism, and inhibit gene expression at the target site. Experimental results show that the gene expression regulation tool comprising the crRNA mutant L1 can avoid the influence of leaky inhibition, that is, after introducing the inducible expression dLbCas12a and the constitutive expression crRNA into an organism, the gene expression at the target site is not affected without adding an inducer. The gene expression regulation tools comprising different crRNA mutants can inhibit the gene expression level in an organism to different degrees, thereby realizing the precise regulation of the gene expression level in an organism.

[0098] Furthermore, the present application also provides a genome base editing reagent, which comprises a plasmid vector and a transformation reagent required for the plasmid.

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

[0100] In the present application, the backbone of the plasmid vector is pYYDT-BE plasmid, wherein the replication origin and the antibiotic resistance gene can be replaced, and the key elements related to base editing function include the crRNA and LbCas12-BE involved in the present application. The spacer sequence part of the crRNA should be complementary to the adjacent PAM in the gene to be edited. LbCas12a-BE comprises dLbCas12a, generally also comprises various sources of deaminase and other enzymes that can catalyze the editing of bases in DNA, and can also comprise a uracil glycosylase inhibitor (UGI) and other elements that can assist in improving editing efficiency. In the embodiment, the crRNA is expressed under the control of the J23119 promoter, and the LbCas12a-BE is expressed under the control of the P BAD The promoters can be replaced.

[0101] The present application also provides a genome base editing method, which comprises transforming the plasmid vector into cells.

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

[0103] The steps specifically include: transforming the pYYDT plasmid into a strain to be base edited, adding an inducer to the culture (using P BAD When the LbCas12a-BE expression is manipulated, L-arabinose is used, and the final concentration is preferably 10 mM), and the base editing starts. After a certain period of culture, the base editing is completed. If necessary, the base editing effect can be verified by extracting nucleic acid from the strain, amplifying the fragment in the base editing region, and performing Sanger sequencing on the amplification product.

[0104] The present application provides a base editor based on a crRNA mutant. The base editor comprises a crRNA mutant and an LbCas12a-BE. The LbCas12a-BE comprises a dLbCas12a, generally also comprises a deaminase from various sources and other enzymes that can catalyze the editing of bases in DNA, and can also comprise a uracil glycosylase inhibitor (UGI) and other auxiliary elements to improve editing efficiency. The above-mentioned base editor can specifically bind to the target site in vivo, and edit the bases in the region. In the present application, the LbCas12a-BE used is APOBEC-dLbCas12a-UGI and evoCDA-dLbCas12a-UGI. Experimental results show that the above-mentioned crRNA mutant can be compatible with these LbCas12a-BE, and when each LbCas12a-BE is used, the above-mentioned crRNA mutant can control the editing window (i.e. the range of bases that can be edited at the target site) of the base editor. Among them, the mutant FL2 can achieve precise editing while maintaining high editing efficiency when used with evoCDA-dLbCas12a-UGI.

[0105] Further, the present application also provides a gene editing reagent driven by homologous recombination, which comprises a plasmid vector 1, a plasmid vector 2 and reagents required for plasmid transformation;

[0106] The plasmid vector 1 comprises an expression frame as described above and a repair template comprising upstream and downstream homologous arms;

[0107] The plasmid vector 2 comprises an expression frame of LbCas12a.

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

[0109] The present application also provides a homologous recombination driven gene editing method, which comprises transforming plasmid vector 1 and plasmid vector 2 into cells by twice transformation;

[0110] The plasmid vector 1 comprises the expression frame and the repair template containing upstream and downstream homologous arms as described above.

[0111] The plasmid vector 2 comprises the expression frame of LbCas12a.

[0112] The specific steps of the method are as follows: pYYDT plasmid and p15A plasmid are transformed into the strain to be gene edited by twice transformation, and an inducer (L-arabinose for LbCas12a expression, and the final concentration is preferably 5mM) is added to the culture, and gene editing starts. After a certain period of culture, gene editing is completed. If necessary, the gene editing effect can be verified by extracting nucleic acid from the strain, amplifying the fragment in the gene editing region, and performing Sanger sequencing on the amplification product. BAD The inducer for LbCas12a expression is L-arabinose, and the final concentration is preferably 5mM), and gene editing starts. After a certain period of culture, gene editing is completed. If necessary, the gene editing effect can be verified by extracting nucleic acid from the strain, amplifying the fragment in the gene editing region, and performing Sanger sequencing on the amplification product.

[0113] The present application also provides a homologous recombination driven gene editing based on crRNA mutants. The above editing tool comprises crRNA mutants, LbCas12a and repair templates containing upstream and downstream homologous arms. The above editing tool can cause DNA double-strand break at a specific target site in an organism, and guide the organism to repair the DNA at this site according to the repair template with a certain probability, thereby introducing gene editing. Experimental results show that in E. coli, the expression vector containing the above editing tool causes different transformation and editing efficiencies, wherein the transformation efficiency of crRNA mutant L1 is about 10000 times that of classical crRNA, and the editing efficiency is several times that of classical crRNA.

[0114] The gene expression regulation tool, the base editing tool, and the homologous recombination driven gene editing tool based on the crRNA mutant improve and enrich the use effect of LbCas12a in organisms, and provide a powerful supplement to the current gene editing method in organisms.

[0115] Furthermore, the application also provides a nucleic acid detection reagent, which comprises a polymerase, dNTP, an LbCas12a protein, and a crRNA as described above.

[0116] The reagent described in the application also comprises primers for amplifying the target nucleic acid and / or a single-stranded oligonucleotide probe labeled with a fluorescent group and a quencher at two ends.

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

[0118] The application provides a semi-quantitative one-pot nucleic acid detection system based on a crRNA mutant. When used for DNA detection, the detection system comprises a polymerase, deoxyribonucleotide triphosphate (dNTP), primers 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 two ends. When used for RNA detection, the detection system further comprises a reverse transcriptase and primers for reverse transcribing the target nucleic acid on the basis of the above-mentioned elements. Experimental results show that the use of a crRNA mutant can realize rapid one-pot nucleic acid detection, and the result output time is about 20-30 min. The detection limit of the detection system comprising the L1 mutant is 100 aM (60 cp / μL); the detection limit of the detection system comprising the FL2 mutant is 10 aM (6 cp / μL); and the detection limit of the detection system comprising the F1 mutant is 1 aM (0.6 cp / μL), which is comparable to qPCR and is sufficient to provide accurate detection results.

[0119] For the same sample, three detection systems comprising the above-mentioned crRNA mutants are used for detection respectively, and the content of the target nucleic acid in the sample can be judged according to the number of detection systems outputting positive results. If all the three systems output positive results, it indicates that the content of the target nucleic acid is more than 100 aM; if two systems output positive results and one system outputs negative results, it indicates that the content of the target nucleic acid is between 10 aM and 100 aM; if one system outputs positive results and two systems output negative results, it indicates that the content of the target nucleic acid is between 1 aM and 10 aM; and if all the three systems output negative results, it indicates that the content of the target nucleic acid is less than 1 aM.

[0120] The semi-quantitative one-pot nucleic acid detection system based on the crRNA mutant solves the problems of difficult quantification and low sensitivity in the one-pot CRISPR detection, and provides an unprecedented simple semi-quantitative one-pot detection strategy, which is suitable for scenes requiring instant detection such as township hospitals, home detection, bedside detection and the like.

[0121] The present application obtains a series of crRNA mutant sets capable of regulating the activity of LbCas12a by modifying the crRNA repeat sequence part of LbCas12a in the CRISPR family. Compared with existing methods, the gene expression regulation based on the crRNA mutant is not affected by leaky inhibition, is more controllable, and can realize precise regulation of gene expression level. The base editing based on the crRNA mutant is compatible with various deaminases, and compared with existing methods, the editing window is narrower, and more precise editing can be realized. The gene editing driven by homologous recombination based on the crRNA mutant can obtain higher conversion and editing efficiency in bacteria compared with existing methods. The nucleic acid detection based on the crRNA mutant can realize one-pot detection without modification, additional substances and target site replacement, and output 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 DRAWINGS

[0122] Figure 1 Effect of repeat sequence mutation on the affinity between crRNA and LbCas12a;

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

[0124] Figure 3 Specific cleavage activity of LbCas12a guided by crRNA mutant;

[0125] Figure 4 Accessory cleavage activity of LbCas12a guided by crRNA mutant;

[0126] Figure 5 Specificity of LbCas12a guided by crRNA mutant;

[0127] Figure 6 Principle of gene expression regulation by dLbCas12a guided by crRNA;

[0128] Figure 7 Effect of gene expression level regulation by dLbCas12a guided by crRNA mutant;

[0129] Figure 8 Principle of crRNA mutant-guided LbCas12a-BE for base editing;

[0130] Figure 9 Efficiency of crRNA mutant-guided APOBEC-dLbCas12a-UGI for base editing;

[0131] Figure 10 Efficiency of crRNA mutant-guided evoCDA-dLbCas12a-UGI for base editing;

[0132] Figure 11 Editing window change of crRNA mutant-guided APOBEC-dLbCas12a-UGI for base editing;

[0133] Figure 12 Editing window change of crRNA mutant-guided evoCDA-dLbCas12a-UGI for base editing;

[0134] Figure 13 Basic flow of crRNA mutant-guided LbCas12a for homologous recombination-mediated gene editing in E. coli;

[0135] Figure 14 Effect of crRNA mutant-guided LbCas12a for homologous recombination-mediated fragment knockout editing in E. coli, wherein A is transformation efficiency and B is editing efficiency;

[0136] Figure 15 Effect of crRNA mutant-guided LbCas12a for homologous recombination-mediated fragment knock-in editing in E. coli, wherein A is transformation efficiency and B is editing efficiency;

[0137] Figure 16 Detection limit of crRNA mutant-guided LbCas12a for one-pot Mycoplasma pneumoniae nucleic acid detection, wherein A is the detection limit of the detection system containing mutant L1, B is the detection limit of the detection system containing mutant FL2, and C is the detection limit of the detection system containing mutant F1;

[0138] Figure 17 Specificity of crRNA mutant-guided LbCas12a for one-pot Mycoplasma pneumoniae nucleic acid detection, wherein A is the specificity of the detection system containing mutant L1, B is the specificity of the detection system containing mutant FL2, and C is the specificity of the detection system containing mutant F1;

[0139] Figure 18Reading mode of results of semi-quantitative one-pot Mycoplasma pneumoniae nucleic acid detection guided by crRNA mutant LbCas12a. DETAILED DESCRIPTION

[0140] The present application provides CRISPR RNA mutants and their applications in gene editing and nucleic acid detection. Those skilled in the art can refer to the content herein and appropriately improve the process parameters to achieve. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present application to achieve and apply the present application technology.

[0141] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have meanings that are commonly understood by those of ordinary skill in the art.

[0142] In addition, unless otherwise stated herein, the singular form of the terms used herein shall include the plural form, and the plural form of the terms shall include the singular form. More specifically, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless specifically stated otherwise.

[0143] In the present application, "comprise", "include" and "have" are used interchangeably and are intended to mean the inclusive nature of the scheme, meaning that the scheme can have other elements in addition to those listed. It should also be understood that the use of "comprise", "include" and "have" in the text also provides a "consisting of" scheme.

[0144] In the present application, "and / or" is used herein to include the meaning of "and", "or" and "all or any other combination of elements linked by the term".

[0145] In the present application, "at least one" means one or more, and "a plurality of" means two or more. "At least one of the following" or similar expressions means any combination of these items, including any combination of single or multiple items.

[0146] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market.

[0147] The sequences involved in the text include:

[0148] Table 1 crRNA repeat sequence mutants

[0149]

[0150]

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

[0152] Table 2 crRNA spacer sequence

[0153]

[0154] *This table is only the part of crRNA spacer sequence, in the examples, each repeat sequence is connected with CN, L1, L2, FL1, FL2, F1 and F2 7 kinds of repeat sequences (SEQ ID NO: 1-7) to constitute 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] 16 Amino acid sequence of the 16 amino acid linker: SGSETPGTSESATPES (SEQ ID NO: 58)

[0170] 4 Amino acid sequence of the 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 a fluorescent group and a quencher at both ends: FAM-5’-CCCCCCCC-3’-BHQ1 (SEQ ID NO: 62)

[0174] The application will be further described below in conjunction with examples:

[0175] Example 1 crRNA mutants for E. coli gene expression level regulation

[0176] The main elements of the plasmid constructed in this example include: (1) pYYDT-CRISPRi plasmid; (2) crRNA mutant expression cassette driven by J23119 promoter, i.e. inserting the crRNA mutant targeting the corresponding target site into the crRNA expression cassette driven by J23119 promoter; (3) dLbCas12a expression cassette driven by P BAD Promoter. Figure 6 ).

[0177] To evaluate the activity of each crRNA mutant guiding dLbCas12a to express the level of regulation, the crRNA spacer sequence part (SEQ ID NO: 44) was designed to target the gfp gene (pre-inserted) on the E. coli NEB 10-beta genome, and the plasmid was transformed into the strain. The resulting transformants were cultured in 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 show that different crRNA mutants can provide different levels of expression inhibition, thereby achieving fine regulation of gene expression levels Figure 7). In addition, mutant L1 does not have expression inhibition without the addition of inducers, overcoming the problem of leaky inhibition often faced by CRISPR-based expression regulation Figure 7

[0179] Example 2 crRNA Mutants for E. coli Genome Base Editing

[0180] The main elements of the plasmid constructed in this example include: (1) pYYDT-BE, (2) a crRNA mutant expression cassette driven by J23119 promoter, i.e. inserting the crRNA mutant targeting the corresponding target site into the crRNA expression cassette driven by the J23119 promoter; (3) a evoCDA-dLbCas12a-UGI expression cassette driven by the P BAD The evoCDA is connected to the N-terminus of dLbCas12a through a 16aa XTEN linker peptide, and the UGI is connected to the C-terminus of dLbCas12a through a 4aa linker peptide, together constituting the LbCas12a-CBE base editor Figure 8

[0181] To evaluate the activity of crRNA mutants in guiding evoCDA-dLbCas12a-UGI to perform base editing, the crRNA spacer sequence part (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, and after 24h of culture, the colonies were collected for Sanger sequencing of the target region to verify the C-to-T editing efficiency at different sites.

[0182] By sequencing the first 20 sites of the E. coli NEB 10-beta protospacer, the editing efficiency of each cytosine was evaluated. The results show that evoCDA-dLbCas12a-UGI guided by different crRNA mutants has different editing windows. By combining the results of each target site, the editing window of mutant FL2 is C8-C10, which is narrower than that of the classic crRNA C8-11 and 16, and can achieve more precise base editing (e.g. Figure 10 、 Figure 12 ).

[0183] Example 3 crRNA Mutants for E. coli Genome Fragment Knockout Driven by Homologous Recombination

[0184] ​​This example constructs two plasmids (p15A and pYYDT plasmid), wherein the first plasmid mainly includes the following elements: (1) a crRNA mutant expression cassette driven by the J23119 promoter, i.e., a crRNA mutant targeting the corresponding target site is inserted into a crRNA expression cassette driven by the J23119 promoter; (2) a repair template containing 1000 bp homologous arms upstream and downstream of the knockout fragment. The main elements of the second plasmid include a P BAD a promoter-driven LbCas12a expression cassette Figure 13 ).

[0185] To evaluate the activity of the crRNA mutant in guiding LbCas12a to drive homologous recombination for fragment knockout on the E. coli genome, the crRNA spacer sequence part (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 solid medium containing 5mM l-arabinose inducer for 20h, followed by counting the number of transformants, collecting colonies, amplifying around the target site, determining the length of the amplified fragments by agarose gel electrophoresis, and judging the knockout efficiency by combining Sanger sequencing results Figure 14 ).

[0186] The transformation efficiency of the editing tool containing different crRNA mutants was determined by counting the number of transformants, and the editing efficiency of different crRNA mutants in driving gene knockout on the E. coli NEB 10-beta genome by homologous recombination was determined by the length and sequence of the amplified fragments. The results show that the number of transformants obtained by mutant L1 is 4 orders of magnitude higher than that of the classic crRNA, and the editing efficiency (58.3%) is more than 10 times that of the classic crRNA, and the overall editing effect is significantly improved compared with the classic crRNA Figure 16 ).

[0187] Example 4 crRNA mutant for semi-quantitative one-tube Mycoplasma pneumoniae nucleic acid detection

[0188] The nucleic acid detection system in this example includes a polymerase, deoxynucleotide triphosphates (dNTPs), primers for amplifying target nucleic acids, LbCas12a, crRNA mutants, single-stranded oligonucleotide probes labeled with fluorescent groups at both ends and quenchers.

[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 6xHis tag. The E. coli strain Rosetta transformed with the recombinant plasmid was cultured to OD600=0.8, 0.2 mM isopropyl β-D-l-thiogalactopyranoside (IPTG) was added for incubation, and cultured at 16 °C for 16 h. The protein was separated from the cell lysate using Ni-NTA resin, and eluted with a buffer (50 mM Tris-HCl, 1.5 M NaCl, 5% glycerol and 600 mM imidazole, pH=8.0).

[0190] In detail, crRNA was prepared by the following method: the crRNA spacer sequence part (SEQ ID NO: 50) targeting M. pneumoniae P1 was designed, an oligonucleotide containing a T7 promoter sequence and an oligonucleotide containing a spacer sequence were polymerized to obtain a DNA template for in vitro transcription. The template was incubated with T7 RNA polymerase (Vazyme, Nanjing, China) at 37 °C for 2 h for in vitro transcription (IVT). The IVT product was incubated with DNAse I (Vazyme, Nanjing, China) at 37 °C for 20 min to remove the template DNA, and then incubated at 65 °C for 20 min 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 related reagent is from Weifang Amp-Future Biotech (Weifang, China). However, the semi-quantitative one-tube nucleic acid detection method based on the crRNA mutant can be used with other nucleic acid amplification methods, and is not limited to RPA. The RPA amplification primer sequences used are SEQ ID NOs: 60-61.

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

[0193] Nucleic acid detection was performed according to the following steps: according to the manufacturer’s instructions, a tube of RPA reagent lyophilized powder was resuspended in 29.4 μL buffer A, and 2 μL of each 10 μM RPA amplification primer and 14.1 L DEPC-treated water were added to form an RPA mixture. Each detection reaction system contained 18 μL RPA mixture, 50 nM LbCas12a, 50 nM crRNA mutant, 400 nM FQ ssDNA reporter molecule (FAM-CCCCCCCC-BHQ) and 2 μL RPA amplification kit in buffer B. 3 μL of the sample to be tested was added to the obtained reaction system, and the reaction system was then 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 a real-time fluorescence quantitative PCR instrument was used to read the test results in this example, the method of 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 to the detection system, the present invention can also be used for RNA detection.

[0194] The above detection system was used to detect gradient dilutions of 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 the L1 mutant is 100aM (60cp / μL); the detection limit of the detection system containing the FL2 mutant is 10aM (6cp / μL); the detection limit of the detection system containing the F1 mutant is 1aM (0.6cp / μL), which is comparable to qPCR and sufficient to provide accurate detection results ( Figure 17 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, and the results were all negative, indicating that the detection method has high specificity ( Figure 18 ).

[0195] When the target sample is subjected to qualitative detection, only the detection system comprising mutant F1 is used. When the target sample is subjected to semi-quantitative detection, the detection systems comprising mutants L1, FL2 and F1 are used simultaneously, and the concentration range of the Mycoplasma pneumoniae nucleic acid in the sample is determined according to the number of detection systems outputting positive results. If all the three systems output positive results, it indicates that the content of the target nucleic acid is more than 100 aM, if two systems output positive results and one system outputs negative results, it indicates that the content of the target nucleic acid is between 10 aM and 100 aM, if one system outputs positive results and two systems output negative results, it indicates that the content of the target nucleic acid is between 1 aM and 10 aM, and if all the three systems output negative results, it indicates that the content of the target nucleic acid is less than 1 aM. ​

[0196] The above merely describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A mutant of a CRISPR RNA repeat sequence, characterized in that The nucleic acid sequence of the mutant is shown in SEQ ID NO: 2, 5 or 6.

2. crRNA, comprising a sequentially connected spacer sequence and a repeat sequence, wherein the repeat sequence is the mutant according to claim 1, and the spacer sequence is complementary to the targeted target nucleic acid.

3. The crRNA according to claim 2, wherein The length of the spacer sequence is 12 to 30 bp; The targeted nucleic acid includes a gene editing site and a characteristic sequence site of the nucleic acid to be detected.

4. The crRNA according to claim 3, wherein The targeted nucleic acids include gfp-1, gfp-3, NCR1, Pseudo1, Pseudo2, zapE, and the P1 adhesin gene of Mycoplasma pneumoniae.

5. The crRNA according to any one of claims 2 to 4, wherein The spacer sequence has a nucleic acid sequence as described in any one of SEQ ID NOs: 44 to 50.

6. An expression cassette comprising a promoter and a DNA sequence corresponding to the crRNA according to any one of claims 2 to 5.

7. The expression frame according to claim 6, characterized in that The expression cassette includes a J23119 promoter, a DNA sequence corresponding to crRNA, and a terminator.

8. Plasmid vector, It comprises a DNA sequence corresponding to the crRNA according to any one of claims 2 to 5, Or comprising the expression cassette of claim 6 or 7.

9. The plasmid vector according to claim 8, characterized in that The expression cassette of Cas12a is also included.

10. The plasmid vector according to claim 9, characterized in that The Cas12a includes LbCas12a or dLbCas12a.

11. The plasmid vector according to claim 10, characterized in that The plasmid vector also includes a base editor, which includes APOBEC, evoCDA or UGI.

12. The plasmid vector according to any one of claims 8 to 11, characterized in that It includes I) and II): 1), the expression cassette according to claim 6 or 7; 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.

13. The plasmid vector according to claim 12, characterized in that The backbone of the plasmid vector is p15A or pYYDT.

14. The method for preparing the crRNA according to any one of claims 2 to 4, comprising reacting the DNA template of the crRNA according to any one of claims 2 to 4 in a system containing T7 RNA polymerase, removing the DNA, and purifying to obtain the crRNA.

15. Use of the mutant according to claim 1, the crRNA and / or its corresponding DNA sequence according to any one of claims 2 to 3, the expression cassette according to claim 6 or 7, and the plasmid vector according to any one of claims 8 to 11 in the preparation of gene editing and / or nucleic acid detection reagents.

16. The use according to claim 15, characterized in that The gene editing includes genome base editing or genome fragment knockout.

17. A reagent for regulating gene expression levels, comprising: Plasmid vectors and reagents required for plasmid transformation; The plasmid vector includes the expression cassette described in claim 6 or 7, and the expression cassette of dLbCas12a.

18. 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 6 or 7, and the expression cassette of dLbCas12a.

19. A reagent for genome base editing, comprising: Plasmid vectors and reagents required for plasmid transformation; The plasmid vector includes the expression cassette described in claim 6 or 7, and the expression cassette of evoCDA-dLbCas12a-UGI.

20. A method for genome base editing, comprising transforming a plasmid vector into a cell; The plasmid vector includes the expression cassette described in claim 6 or 7, and the expression cassette of evoCDA-dLbCas12a-UGI.

21. A homologous recombination-driven gene editing reagent comprising plasmid vector 1, plasmid vector 2, and reagents required for plasmid transformation; The plasmid vector 1 comprises the expression cassette according to claim 6 or 7 and a repair template comprising upstream and downstream homology arms; The plasmid vector 2 includes an expression cassette of LbCas12a.

22. 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 6 or 7 and a repair template comprising upstream and downstream homology arms; The plasmid vector 2 includes an expression cassette of LbCas12a.

23. A nucleic acid detection reagent comprising: Polymerase, dNTP, LbCas12a protein and the crRNA according to any one of claims 2 to 4.

24. The detection reagent according to claim 23, 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.

25. A nucleic acid detection method, comprising: The sample to be tested is mixed with the detection reagent according to claim 23 or 24, and the detection is performed after the reaction.

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