Thermal-stability Cas9-based nucleic acid targeted enrichment system construction method
Through a nucleic acid targeted enrichment system based on thermally stable Cas9, the nucleic acid complexity problem is solved by using gRNA-CAS complex cleavage and exonuclease digestion, and the efficient purification of target nucleic acid fragments is achieved and the complexity of nucleic acid samples is reduced.
Patent Information
- Application Number
- CN202410069143.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-17
- Publication Date
- 2025-07-18
AI Technical Summary
The prior art lacks precise methods to reduce nucleic acid complexity, especially when enriching target nucleic acid fragment samples for genetic research or subsequent analysis, it is difficult to achieve efficient nucleic acid fragment isolation and purification.
Using a nucleic acid targeted enrichment system based on thermally stable Cas9, nucleic acid molecules were cleaved at 37°C by the first and second gRNA-CAS complexes, target nucleic acid fragments were generated and non-target nucleic acid fragments were digested using exonuclease, and then target nucleic acid fragments containing the sequence of interest were purified.
The precise reduction of nucleic acid complexity is achieved, the purification efficiency and quality of target nucleic acid fragments is improved, and it is suitable for subsequent genetic research and analysis.
Abstract
Description
Technical Field
[0001] This application belongs to the field of biomedical technology, and more particularly, relates to a method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9. Background Art
[0002] The CAS protein Cas9 is a major component of the type II CRISPR-CAS system from Streptococcus pyogenes, and forms an endonuclease when combined with crRNA and a second RNA called trans-activating crRNA (tracrRNA). The endonuclease targets invading pathogenic DNA and degrades it by introducing DNA double-strand breaks (DSBs) at the genomic positions defined by the crRNA. This type II CRISPR-Cas9 system has proven to be a convenient and effective tool in biochemistry, enabling the introduction of modifications at sites of interest in the eukaryotic genome by targeting the introduction of double-strand nicks and subsequent activation of endogenous repair mechanisms. In addition to the CRISPR-CAS system that uses RNA guidance to direct the endonuclease to specific positions of nucleic acid molecules, other endonucleases using DNA or RNA guidance are known in the art.
[0003] However, there is still a need in the art for an accurate method to reduce nucleic acid complexity, and there is a particular need in the art for a general method for enriching samples for one or more target nucleic acid fragments, for example for subsequent analysis or processing in genetic research. Summary of the Invention
[0004] The purpose of the embodiments of this application is to provide a method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 to solve the problem of nucleic acid complexity existing in related technologies.
[0005] To achieve the above purpose, the technical solution adopted in the embodiments of this application is: Provide a method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9, including the following steps: Provide a sample containing nucleic acid molecules, the nucleic acid molecules containing sequences of interest; Cut the nucleic acid molecules with the first and second gRNA-CAS complexes to generate target nucleic acid fragments and non-target nucleic acid fragments containing the sequences of interest, the target nucleic acid fragments being immune to exonuclease cleavage; wherein, the first and second gRNA-CAS complexes are incubated with the nucleic acid molecules at a temperature of 37°C for 60 minutes; Contact the cut nucleic acid molecules with the exonuclease and allow the exonuclease to digest the non-target nucleic acid fragments; wherein, the cut nucleic acid molecules are incubated with the exonuclease at a temperature of 37°C for 30 minutes; The resulting digest purifies target nucleic acid fragments containing the sequence of interest.
[0006] In one embodiment, at least one of the first and second gRNA-CAS complexes comprises a Cas9 protein.
[0007] In one embodiment, at least one of the first and second gRNA-CAS complexes comprises an sgRNA.
[0008] In one embodiment, at least one of the first and second gRNA-CAS complexes comprises a crRNA and a tracrRNA as distinct molecules.
[0009] In one embodiment, at least one of the first and second gRNA-CAS complexes is capable of inducing DSB.
[0010] In one embodiment, it further comprises the steps of: ligating the target nucleic acid fragment to an adaptor; The step of ligating the target nucleic acid fragment to an adaptor is located after the step of purifying the resulting digest to obtain target nucleic acid fragments containing the sequence of interest.
[0011] In one embodiment, the adaptor is a sequencing adaptor.
[0012] In one embodiment, it further comprises the step of: sequencing the target nucleic acid fragment; The step of sequencing the target nucleic acid fragment is located after the step of ligating the target nucleic acid fragment to an adaptor.
[0013] In one embodiment, the nucleic acid molecule is genomic DNA.
[0014] In one embodiment, the nucleic acid molecule is obtained from nucleic acid molecules in plants, animals, humans or microorganisms.
[0015] The method for constructing a nucleic acid target enrichment system based on thermostable Cas9 provided by the embodiments of the present application has at least the following beneficial effects: By providing a nucleic acid molecule containing the sequence of interest, the nucleic acid molecule is cleaved with the first and second gRNA-CAS complexes to generate target nucleic acid fragments and non-target nucleic acid fragments of the sequence of interest, and the cleaved nucleic acid molecule is contacted with an exonuclease to purify the resulting digest to obtain target nucleic acid fragments containing the sequence of interest. Therefore, through this method for constructing a nucleic acid target enrichment system based on thermostable Cas9, the purpose of precisely reducing the complexity of the nucleic acid can be achieved. Detailed implementation manners
[0016] It should be noted that when an element is referred to as "fixed to" or "disposed on" another element, it can be directly on the other element or indirectly on the other element. When an element is referred to as "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element.
[0017] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of these features. In the description of the present application, "a plurality" means two or more, unless otherwise specifically defined. "Several" means one or more, unless otherwise specifically defined.
[0018] In the description of the present application, it should be noted that unless otherwise clearly specified and limited, the terms "installed", "connected", and "coupled" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the internal communication of two elements or the interaction relationship between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present application can be understood according to specific circumstances.
[0019] Reference throughout the specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present application. Thus, the phrases "in one embodiment" or "in some embodiments" appearing throughout the specification are not necessarily all referring to the same embodiment. In addition, in one or more embodiments, the specific features, structures, or characteristics may be combined in any suitable manner.
[0020] The method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 provided by the embodiments of the present application will now be described. The method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 includes the following steps: 1. Provide a sample containing nucleic acid molecules, the nucleic acid molecules containing the sequence of interest; 2. Cut the nucleic acid molecules with the first and second gRNA-CAS complexes to produce target nucleic acid fragments and non-target nucleic acid fragments containing the sequence of interest, the target nucleic acid fragments being immune to exonuclease cleavage; wherein, the first and second gRNA-CAS complexes are incubated with the nucleic acid molecules at a temperature of 37 °C for 60 minutes; 3. The cleaved nucleic acid molecule is contacted with an exonuclease, and the exonuclease is allowed to digest non-target nucleic acid fragments; wherein, the cleaved nucleic acid molecule is incubated with the exonuclease at a temperature of 37 °C for 30 minutes; 4. The resulting digest is purified to obtain the target nucleic acid fragment containing the sequence of interest.
[0021] In this structure, the present application provides a nucleic acid molecule containing a sequence of interest, cuts the nucleic acid molecule with the first and second gRNA-CAS complexes to generate target nucleic acid fragments and non-target nucleic acid fragments of the sequence of interest, and contacts the cleaved nucleic acid molecule with an exonuclease so that the resulting digest is purified to obtain the target nucleic acid fragment containing the sequence of interest. Therefore, through this method for constructing a thermostable Cas9-based nucleic acid targeting enrichment system, the purpose of precisely reducing the complexity of nucleic acids can be achieved.
[0022] In one embodiment, as a specific implementation manner of the method for constructing a thermostable Cas9-based nucleic acid targeting enrichment system provided in the embodiments of the present application, at least one of the first and second gRNA-CAS complexes contains a Cas9 protein. Specifically, the first gRNA-CAS complex contains a Cas9 protein; or, the second gRNA-CAS complex contains a Cas9 protein; or, both the first and second gRNA-CAS complexes contain a Cas9 protein.
[0023] In one embodiment, as a specific implementation manner of the method for constructing a thermostable Cas9-based nucleic acid targeting enrichment system provided in the embodiments of the present application, at least one of the first and second gRNA-CAS complexes contains an sgRNA. Specifically, the first gRNA-CAS complex contains an sgRNA; or, the second gRNA-CAS complex contains an sgRNA; or, both the first and second gRNA-CAS complexes contain an sgRNA.
[0024] In one embodiment, as a specific implementation manner of the method for constructing a thermostable Cas9-based nucleic acid targeting enrichment system provided in the embodiments of the present application, at least one of the first and second gRNA-CAS complexes contains crRNA and tracrRNA as different molecules. Specifically, the first gRNA-CAS complex contains crRNA and tracrRNA; or, the second gRNA-CAS complex contains crRNA and tracrRNA; or, both the first and second gRNA-CAS complexes contain crRNA and tracrRNA.
[0025] In one embodiment, as a specific implementation of the method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 provided in the embodiments of the present application, at least one of the first and second gRNA-CAS complexes is capable of inducing DSB. Specifically, the first gRNA-CAS complex is capable of inducing DSB; or, the second gRNA-CAS complex is capable of inducing DSB; or, both the first and second gRNA-CAS complexes are capable of inducing DSB.
[0026] In one embodiment, as a specific implementation of the method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 provided in the embodiments of the present application, the method comprises the following steps: 1. Provide a sample containing a nucleic acid molecule, the nucleic acid molecule containing a sequence of interest; 2. Cut the nucleic acid molecule with the first and second gRNA-CAS complexes to generate target nucleic acid fragments and non-target nucleic acid fragments containing the sequence of interest, the target nucleic acid fragments being immune to exonuclease cleavage; wherein, the first and second gRNA-CAS complexes are incubated with the nucleic acid molecule at a temperature of 37 °C for 60 minutes; 3. Contact the cut nucleic acid molecule with an exonuclease and allow the exonuclease to digest the non-target nucleic acid fragments; wherein, the cut nucleic acid molecule is incubated with the exonuclease at a temperature of 37 °C for 30 minutes; 4. Purify the target nucleic acid fragments containing the sequence of interest from the resulting digest; 5. Ligate the target nucleic acid fragments to an adaptor.
[0027] In this structure, the adaptor may contain functional domains, preferably selected from restriction site domains, capture domains, sequencing primer binding sites, amplification primer binding sites, detection domains, barcode sequences, transcription promoter domains, and PAM sequences or any combination thereof.
[0028] In one embodiment, as a specific implementation of the method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 provided in the embodiments of the present application, the adaptor is a sequencing adaptor. In this structure, the functional domains contained in the sequencing adaptor allow Roche 454A and 454B sequencing, Pacific Biosciences' SMRTTM sequencing, Oxford Nanopore Technology, or whole genome sequencing, etc. Among them, the adaptor can be single-stranded, double-stranded, partially double-stranded, Y-shaped, hairpin, or circularizable adaptor.
[0029] In one embodiment, as a specific implementation of the method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 provided in the embodiments of the present application, the method comprises the following steps: 1. Provide a sample containing a nucleic acid molecule, the nucleic acid molecule containing a sequence of interest; 2. Cut the nucleic acid molecule with the first and second gRNA-CAS complexes to generate a target nucleic acid fragment containing the sequence of interest and a non-target nucleic acid fragment, where the target nucleic acid fragment is protected from exonuclease cleavage; wherein, the first and second gRNA-CAS complexes are incubated with the nucleic acid molecule at a temperature of 37 °C for 60 minutes; 3. Contact the cleaved nucleic acid molecule with an exonuclease and allow the exonuclease to digest the non-target nucleic acid fragment; wherein, the cleaved nucleic acid molecule is incubated with the exonuclease at a temperature of 37 °C for 30 minutes; 4. Purify the target nucleic acid fragment containing the sequence of interest from the resulting digest; 5. Ligate the target nucleic acid fragment to an adaptor; 6. Sequence the target nucleic acid fragment.
[0030] In one embodiment, as a specific implementation of the method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 provided in the embodiments of the present application, the nucleic acid molecule is obtained from nucleic acid molecules in plants, animals, humans, or microorganisms. For example, the nucleic acid molecule is genomic DNA, chromosomal DNA, artificial chromosomes, plasmid DNA or episomal DNA, cDNA, RNA, mitochondria, or artificial libraries such as BAC or YAC, etc. DNA can be nuclear or organelle DNA. DNA is preferably chromosomal DNA, preferably endogenous to the cell.
[0031] It should be understood that the magnitudes of the sequence numbers of the steps in the above embodiments do not mean the order of execution. The order of execution of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.
[0032] The above are only optional embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present application shall be included within the protection scope of the present application.
Claims
1. Method for constructing nucleic acid targeting and enrichment system based on thermostable Cas9, characterized in that, Comprising the following steps: Providing a sample containing a nucleic acid molecule, the nucleic acid molecule containing a sequence of interest; Cutting the nucleic acid molecule with first and second gRNA-CAS complexes to produce a target nucleic acid fragment containing the sequence of interest and a non-target nucleic acid fragment, the target nucleic acid fragment being resistant to exonuclease cleavage; wherein, the first and second gRNA-CAS complexes are incubated with the nucleic acid molecule at a temperature of 37 °C for 60 minutes; Contacting the cut nucleic acid molecule with the exonuclease and allowing the exonuclease to digest the non-target nucleic acid fragment; wherein, the cut nucleic acid molecule is incubated with the exonuclease at a temperature of 37 °C for 30 minutes; Purifying the target nucleic acid fragment containing the sequence of interest from the resulting digest.
2. The method for constructing a nucleic acid targeting and enrichment system based on thermostable Cas9 according to claim 1, wherein: At least one of the first and second gRNA-CAS complexes contains a Cas9 protein.
3. The method for constructing a nucleic acid targeting and enrichment system based on thermostable Cas9 according to claim 1, wherein: At least one of the first and second gRNA-CAS complexes contains an sgRNA.
4. The method for constructing a nucleic acid targeting and enrichment system based on thermostable Cas9 according to claim 1, wherein: At least one of the first and second gRNA-CAS complexes contains a crRNA and a tracrRNA as different molecules.
5. The method for constructing a nucleic acid targeting and enrichment system based on thermostable Cas9 according to claim 1, wherein: At least one of the first and second gRNA-CAS complexes is capable of inducing DSB.
6. The method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 according to claim 1, wherein, Further comprising the step of: Ligating the target nucleic acid fragment to an adaptor; The step of ligating the target nucleic acid fragment to an adaptor is located after the step of purifying the target nucleic acid fragment containing the sequence of interest from the resulting digest.
7. The method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 according to claim 6, wherein: The adaptor is a sequencing adaptor.
8. The method for constructing a nucleic acid targeting and enrichment system based on thermostable Cas9 according to claim 6, wherein, Further comprising the step of: Sequencing the target nucleic acid fragment; The step of sequencing the target nucleic acid fragment is located after the step of ligating the target nucleic acid fragment to an adaptor.
9. The method for constructing a nucleic acid targeting enrichment system based on thermostable Cas9 according to any one of claims 1-8, characterized in that: The nucleic acid molecule is genomic DNA.
10. The method for constructing a nucleic acid targeting and enrichment system based on thermostable Cas9 according to any one of claims 1-8, characterized in that: The nucleic acid molecule is obtained from a nucleic acid molecule in a plant, an animal, a human or a microorganism.