CN117947027B - Method for removing host DNA in metagenome by using high-specificity guide RNA and CRISPR-dCAS9 - Google Patents
Method for removing host DNA in metagenome by using high-specificity guide RNA and CRISPR-dCAS9 Download PDFInfo
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Abstract
本发明公开了利用高特异性向导RNA和CRISPR‑dCas9去除宏基因组中宿主DNA的方法，本发明提供的方法不仅能够提高去宿主DNA的特异性和效率，而且保证了该方法的低成本性，此外，本发明提供了一组高特异性sgRNA，将其和CRISPR‑dCas9结合能够显著提高去除宏基因组中宿主DNA的效率，具有良好的应用前景。
The present invention discloses a method for removing host DNA from a metagenome using a highly specific guide RNA and CRISPR-dCas9. The method provided by the present invention can not only improve the specificity and efficiency of removing host DNA, but also ensure the low cost of the method. In addition, the present invention provides a group of highly specific sgRNAs, which, combined with CRISPR-dCas9, can significantly improve the efficiency of removing host DNA from a metagenome, and have good application prospects.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a method for removing host DNA in a metagenome by utilizing high-specificity guide RNA and CRISPR-dCAs 9.
Background
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated protein 9 (Cas 9) system is a defense mechanism of bacteria against phage infection, and its principle of action is that Cas9 binds to and double-strand cleaves target DNA under the mediation of guide RNAs (grnas). Binding of Cas9 to target DNA occurs only when PAM (protospacer adjacent motif) sequences are present and 17-20 bases on the complementary strand immediately adjacent to PAM are complementary to gRNA. For applications where DNA cleavage is not required, one mutates the endonuclease active site of Cas9 to give dCas9 (read Cas 9), dCas9 still has the ability to bind DNA. The CRISPR-dCAS9 system has been widely used in the research of transcription regulation, genome imaging, DNA capture and the like.
Metagenomic sequencing (mNGS) is a technology for detecting pathogenic microorganisms in clinical samples without deviation, and one major challenge faced by the technology is that a large amount of host nucleic acids exist in metagenomic samples to be detected, and the host nucleic acids not only occupy most of sequencing data amount in the sequencing process to cause waste, but also reduce detection sensitivity of microorganisms, so that removing host DNA in metagenomic samples becomes a key technology for improving metagenomic detection sensitivity and reducing sequencing cost. The existing common method for removing host is differential cracking method, most bacteria and fungi have cell wall structure and are firmer than human cell membrane, so that the method can realize the cracking of human cells by using milder cracking buffer solution while keeping the integrity of most microorganisms, and after the released human genome DNA is digested by DNase, the microbial genome can be extracted by using cracking solution with higher cracking strength, thereby realizing the purpose of removing host DNA.
Different from the differential lysis method, the extracted metagenome sample is subjected to removal of human DNA by using a CRISPR-dmas 9 method, so that the loss of microbial DNA can be effectively avoided, and the method is simple to operate, has high specificity, does not have microbial preference and is basically not influenced by the sample state. However, the length of the sgRNA used in the existing CRISPR-dCAS9 host removal method is 20nt, and the tolerance of two bases at the 5' end of the crRNA of 20nt is higher, so that the existing method has higher non-specificity and is easy to cause microbial loss; in addition, the target DNA of the sgRNA of the current method is a sequence with a certain interval on the whole genome or Alu elements, the former has wide coverage range on the genome but needs more sgRNA, so that the design, synthesis, preparation and quality inspection of the sgRNA are more complicated and have higher cost, and the latter has limited coverage range on the genome, thereby affecting the removal efficiency of host DNA, namely, the sgRNA used by the current method can not meet the control cost and simultaneously has higher human-source-removing efficiency.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a method for removing host DNA in a metagenome using highly specific guide RNAs and CRISPR-dCas9 in order to overcome the above technical problems in the prior art. In order to improve the specificity of removing host DNA by the existing CRISPR-dCAs9 method, the invention designs a crRNA sequence with high specificity and length of 18nt (two bases less than the 5' -end of 20nt crRNA); in addition, to compromise the cost and efficiency of the CRISPR-dCas9 decoating method, the present invention designs sgrnas for conserved sequences of primate-specific Alu and L1 (L1 PA) elements that are widely distributed across the human genome and have complementary properties at the distribution sites.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The first aspect of the invention provides a set of sgrnas that are highly specific and highly efficient in host DNA removal.
Further, the sgrnas include alu_ trusgRNA targeted to Alu and/or l1_ trusgRNA targeted to L1;
The alu_ trusgRNA is Alu/u trusgRNA _1 to Alu trusgRNA _8;
The L1\u trusgRNA is L1\u trusgRNA _1 to L1_ trusgRNA _8;
The sequences of alu_ trusgRNA _1 to alu_ trusgRNA _8 are shown in SEQ ID NO 17 to 24;
The sequences of the L1_ trusgRNA _1-L1_ trusgRNA _8 are shown in SEQ ID NO 25-32.
In the present invention, sgRNA (single guide RNA) refers to one-way guide RNA, which is a molecular tool commonly used in genetic engineering. The sgRNA is obtained by engineering crRNA (CRISPR RNA) and tracrRNA (transactivating CRISPR RNA). The sgrnas can be complementary to a target DNA sequence, forming RNA-DNA hybridization, and then activate Cas9 (CRISPR-associated protein) enzyme.
In a specific embodiment of the invention, the primer sequences required for preparing the sgrnas of the first aspect of the invention are shown in SEQ ID NOs 34 to 66, respectively, and the template sequences required for preparing the sgrnas of the first aspect of the invention are shown in SEQ ID NO 33.
In a second aspect the invention provides a sgRNA-dCAS9 complex.
Further, the sgRNA-dCas9 complex comprises the sgrnas of the first aspect of the invention;
Preferably, the sgRNA-dCas9 complex further comprises dCas9, 10X NEB buffer, RNase inhibitor;
more preferably, the alu_trusgRNA, L1_ trusgRNA, dCas, 10 XNEB buffer, RNase inhibitor are used in an amount of (0.1-0.5) μg, (0.1-1) μg, (0.5-1.5) μL, (0.1-1) μL, (5-15) μL, respectively;
most preferably, the alu_trusgRNA, L1_ trusgRNA, dCas, 10 XNEB buffer, RNase inhibitor are used in an amount of 0.25. Mu.g, 0.5. Mu.g, 1. Mu.L, 0.5. Mu.L, 10. Mu.L, respectively;
Most preferably, the sgRNA-dCAS9 complex is supplemented to 10. Mu.L by NFW.
In the present invention, dCas9 (dead Cas 9) is a mutant of Cas9 protein, and two nuclease domains of RuvC (D10A) and NHN (H840A) are subjected to amino acid mutation, respectively, to obtain a Cas9 protein without nuclease activity, which loses the function of cleaving DNA, but retains the ability to bind DNA, and such Cas9 is called dCas9. At present, CRISPR-dCAS9 system is commonly used for genome transcription regulation and control to activate or inhibit the expression of specific genes, thereby achieving the purpose of researching the gene functions.
In a specific embodiment of the present invention, in order to improve the specificity of removing human DNA by CRISPR-dCAS9 system, the present invention designed crRNAs of 18nt and 20nt in length for Alu, and synthesized by in vitro transcription sgsn RNAs of trusgRNA (truncated single-guide RNA) and 100nt in length, and assembled the two lengths of sgRNAs with dCAS9 protein into RNP complex, respectively, and after the two complexes were used for the decommissioning experiment, the decommissioning efficiency of the two complexes was examined by qPCR, and the results showed that 98nt sgRNAs were substantially free from microbial loss compared to 100nt sgRNAs.
In a specific embodiment of the present invention, to further achieve effective removal of human genome, the present invention designs trusgRNA and sgrnas of 98nt and 100nt in length for the conserved sequences of Alu and L1 (L1 PA), respectively, synthesizes Alu-sgRNA, L1-sgRNA, alu-trusgRNA, L1-trusgRNA, and assembles them with dCas9 as RNP complex, respectively. After separate decommissioning experiments with different combinations of RNP complexes, the different sgrnas combinations were tested for decommissioning efficiency by qPCR, which showed that trusgRNA using simultaneous targeting Alu and L1 had higher decommissioning efficiency than the single use of the targeted Alu or L1.
In a third aspect, the invention provides a highly specific, highly efficient agent for removing host DNA from a metagenome.
Further, the agent comprises the sgRNA-dCas9 complex according to the second aspect of the invention.
In some embodiments, any other agent that facilitates removal of host DNA from the metagenome may also be included in the agents described herein.
In a fourth aspect, the invention provides a highly specific, highly efficient product for removing host DNA from the metagenome.
Further, the product comprises an agent according to the third aspect of the invention.
Further, the product includes a kit.
In some embodiments, the kit is used for high specificity, high efficiency removal of host DNA in the metagenome.
In a fifth aspect, the invention provides a method for removing host DNA from the metagenome using high specificity sgRNA and CRISPR-dCAs 9.
Further, the method comprises the following steps:
(1) Extracting metagenomic total nucleic acid from a sample derived from a host to obtain a metagenomic nucleic acid sample comprising host genomic DNA;
(2) Formulating an sgRNA-dCas9 complex comprising alu_ trusgRNA and/or l1_ trusgRNA, dCas9 as described in the first aspect of the invention;
(3) Preparing a reaction system comprising the nucleic acid sample described in step (1), the sgRNA-dCas9 complex described in step (2), and reacting in a buffer;
(4) Capturing the sgRNA-dCas9 complex and the bound host DNA with streptavidin-coated magnetic beads to form a magnetic bead-protein-RNA-DNA complex;
(5) Magnetic separation is carried out to remove the magnetic bead-protein-RNA-DNA complex, thereby achieving the purpose of removing host DNA in the metagenome.
Further, the sgRNA-dCAS9 complex in the step (2) further comprises dCAS9, 10 XNEB buffer and RNase inhibitor;
preferably, the alu_trusgRNA, L1_ trusgRNA, dCas9, 10 XNEB buffer, RNase inhibitor are used in an amount of (0.1-0.5) μg, (0.1-1) μg, (0.5-1.5) μL, (0.1-1) μL, (5-15) μL, respectively;
More preferably, the alu_trusgRNA, L1_ trusgRNA, dCas, 10 XNEB buffer, RNase inhibitor are used in an amount of 0.25. Mu.g, 0.5. Mu.g, 1. Mu.L, 0.5. Mu.L, 10. Mu.L, respectively;
Most preferably, the sgRNA-dCAS9 complex is supplemented to 10. Mu.L by NFW.
Further, the composition of the reaction system in step (3) is as follows: nucleic acid sample, sgRNA-dCas9 complex, 10XNEB buffer;
preferably, the nucleic acid sample, the sgRNA-dCAs9 complex and the 10 XNEB buffer are used in an amount of (5-15) ng, (0.5-1.5) mu L and (0.5-3.5) mu L respectively;
more preferably, the nucleic acid sample, sgRNA-dCAs9 complex, 10 XNEB buffer are used in an amount of 10ng, 1. Mu.L, 2. Mu.L, respectively;
most preferably, the reaction system is supplemented to 20. Mu.L by NFW.
Further, in the step (4), the streptavidin-coated magnetic beads are M-270 streptavidin magnetic beads.
In some embodiments, the host of the invention is a human, and the nucleic acid sample is extracted from human whole blood, plasma, serum, tears, saliva, alveolar lavage fluid, hydrothorax, ascites fluid, drainage fluid, pus, mucus, cerebrospinal fluid, other puncture fluids, teeth, bone, nails, stool, urine, other tissue or biopsy samples, and the like. The host DNA is one of host nucleic acids, contains all human chromosome DNA, is not interrupted or fragmented by an artificial mechanical method, and is double-stranded cell nucleus DNA with good integrity.
The sixth aspect of the invention provides the use of an sgRNA according to the first aspect of the invention, an sgRNA-dCas9 complex according to the second aspect of the invention, an agent according to the third aspect of the invention or a product according to the fourth aspect of the invention for removing host DNA in the metagenome using CRISPR-dCas 9.
Compared with the prior art, the invention has the following advantages and beneficial effects:
In order to improve the specificity of the CRISPR-dCAS9 system for removing the human DNA, the invention designs crRNAs with the length of 18nt and 20nt for Alu, synthesizes trusgRNA (alu_ trusgRNA and L1_ trusgRNA with the length of 98nt through in vitro transcription, if the first base of the 5 'end of the sgRNA is not G, one G needs to be added at the 5' end, the sgRNAs with the length of 99 nt) and the sgRNAs with the length of 100nt (alu_sgRNA and L1_sgRNA, if the first base of the 5 'end of the sgRNAs is not G, one G needs to be added at the 5' end, the length of the sgRNAs is 101 nt), assembles the sgRNAs with the length of two lengths and dCAS9 proteins into RNP complexes respectively, and then carries out a host removal experiment on the two complexes through qPCR, so that compared with 100 sgRNAs, the result shows that the removal of the sgRNAs with the length of 100nt is basically free from microorganism loss, and unexpected technical effects are obtained. The method provided by the invention improves the specificity and efficiency of the CRISPR-dCAS9 host DNA removal method and ensures the low cost of the method.
Drawings
Fig. 1: the effect of removing hosts of sgrnas of different lengths, wherein the complementary sequence length of alu_sgrnas and DNA is 20nt, the complementary sequence length of alu_trusgrnas and DNA is 18nt, representing that t-test p <0.05, representing that t-test p <0.01; fig. 2: trusgRNA targeting Alu and L1 compared to the effect of the sgrnas targeting Alu or L1 were decommissioned, where x represents t-test p <0.05 and x represents t-test p <0.01.
Detailed Description
The invention is further illustrated below in conjunction with specific examples, which are provided solely to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The experimental procedure, in which no specific conditions are noted in the examples below, is generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.
EXAMPLE 1 comparison of the effect of different lengths of sgRNA on the removal of host
1. Experimental materials
High-Fidelity 2X PCR Master Mix,NEB；
HiScribeTMT7 Quick High Yield RNA Synthesis Kit,NEB；
dCas9-3xFLAG-Biotin Protein,Sigma-Aldrich；
Dynabeads TM M-270 streptavidin, thermoFisher;
RNA Clean&Concentrator-5,ZYMO RESEARCH；
RNase Inhibitor,NEB；
NEBuffer,NEB；
Probe qPCR Mix,Takara。
2. Experimental method
(1) Alu_sgrna mixtures (including alu_sgrna_1 to alu_sgrna_8, see table 1) and alu_ trusgRNA mixtures (including alu_ trusgRNA _1 to alu_ trusgRNA _8, see table 1) were synthesized by in vitro transcription. The primers and templates required for the preparation of the sgrnas are shown in table 2.
TABLE 1sgRNA sequences
TABLE 2 primers and templates required for the preparation of sgRNA
(2) Two sgRNA-dCas9 complexes were formulated: the composition of the first complex is shown in Table 3, and the composition of the second complex is shown in Table 4.
TABLE 3 Complex one
TABLE 4 Complex II
(3) Preparing nucleic acid standard substances with human source accounting for 90 percent and Acinetobacter baumannii and Klebsiella pneumoniae accounting for 5 percent respectively.
(4) The 3 reaction systems in table 5 below were formulated: the sgRNA-dCas9 complexes used in the two experimental groups were the two complexes assembled in tables 3 and 4, respectively.
Table 53 reaction systems
(5) For three sets of reaction products, 10. Mu. L M-270 streptavidin beads were taken and the beads were washed twice with 50. Mu.L of 1 XNEB buffer.
(6) The beads were resuspended with 20. Mu.L of product and spin-bound at room temperature for 5-10min, the supernatant recovered, and 3 techniques repeated.
(7) QPCR was performed using the recovered product, and the qPCR reaction system and the procedure were set as shown in Table 6 below.
TABLE 6 reaction System for qPCR and program set-up
3. Experimental results
As shown in FIG. 1, the complementary sequence length of alu_sgRNA and DNA is 20nt, and the complementary sequence length of alu_trusgRNA and DNA is 18nt, which shows that trusgRNA has higher specificity (less loss of Klebsiella pneumoniae) and higher host removal efficiency than sgRNA, indicating that trusgRNA adopted by the invention has unexpected technical effects.
Example 2 comparison of the effect of different combinations of sgrnas on the removal of hosts
1. Experimental materials
High-Fidelity 2X PCR Master Mix,NEB；
HiScribeTMT7 Quick High Yield RNA Synthesis Kit,NEB；
dCas9-3xFLAG-Biotin Protein,Sigma-Aldrich；
Dynabeads TM M-270 streptavidin, thermoFisher;
RNA Clean&Concentrator-5,ZYMO RESEARCH；
RNase Inhibitor,NEB；
NEBuffer,NEB；
Probe qPCR Mix,Takara。
2. Experimental method
(1) Alu_sgrna mixtures (including alu_sgrna_1 to alu_sgrna_8, see table 1), l1_sgrna mixtures (including l1_sgrna_1 to l1_sgrna_8, see table 1), alu_ trusgRNA mixtures (including alu_ trusgRNA _1 to alu_ trusgRNA _8, see table 1), l1_ trusgRNA mixtures (including l1_ trusgRNA _1 to l1_ trusgRNA _8, see table 1) were synthesized by in vitro transcription.
(2) Three sgRNA-dCas9 complexes were formulated: the composition of the first complex is shown in Table 7, the composition of the second complex is shown in Table 8, and the composition of the third complex is shown in Table 9.
TABLE 7 Complex one
Table 8 Complex II
Table 9 Complex III
(3) Preparing nucleic acid standard substances with human source accounting for 90 percent and Acinetobacter baumannii and Klebsiella pneumoniae accounting for 5 percent respectively.
(4) 4 Reaction systems were formulated as in table 10 below: the sgRNA-dCas9 complexes used in the three experimental groups were three complexes assembled in tables 7-9, respectively.
TABLE 104 reaction systems
(5) The corresponding 4 groups of reaction products were each 10 mu L M-270 streptavidin beads, and the beads were washed twice with 50. Mu.L of 1 XNEB buffer.
(6) The beads were resuspended with 20. Mu.L of product and spin-bound at room temperature for 5-10min, the supernatant recovered, and 3 techniques repeated.
(7) The qPCR detection was performed using the recovered product, and the qPCR reaction system and the procedure were set as shown in Table 11 below.
TABLE 11 reaction System and program settings for qPCR
3. Experimental results
The results are shown in FIG. 2, which shows that using trusgRNA targeting Alu and L1 can effectively increase the efficiency of decommissioning compared to using only the sgRNA targeting Alu or L1.
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