CN115772555A - A method for removing ribosomal RNA sequences using dCas13 - Google Patents

Abstract

The invention discloses a method for removing ribosomal RNA sequences by using dCas13; it belongs to the technical field of biomolecules. The complex formed by dead Cas13 without RNase activity and biotin-modified crRNA groups specifically binds to the RNA sequence of ribosomes, and then performs negative selection through streptavidin-modified magnetic beads to remove ribosomes, etc. High-peak RNA sequences can specifically reduce the proportion of ribosomal RNA sequences in sequencing data, improve the sequencing efficiency of RNA libraries and the sequencing depth of target RNAs. The method of the present invention is applied to RNA-seq sequencing and RNA pathogen metagenomic sequencing to quickly remove high-peak rRNA sequences, improve the sequencing depth and efficiency of target RNA sequences, and reduce sequencing costs.

Description

A method for removing ribosomal RNA sequences using dCas13

technical field

The invention relates to a process and method for constructing an RNA next-generation sequencing library and a technology for removing high-peak ribosomal rRNA sequences in the RNA sequencing process, in particular to a method for removing ribosomal RNA sequences by using dCas13, which belongs to the field of biomolecular technology.

technical background

With the development of high-throughput DNA sequencing technology, also known as next-generation sequencing technology (Next-Generation Sequencing), its application in scientific research and clinical diagnosis is becoming more and more extensive. Next-generation sequencing technology obtains a large amount of DNA sequence data, and through the comparison and analysis of DNA sequences, the purpose of precise diagnosis and treatment of diseases can be achieved. At present, from the diagnosis of genetic diseases, the detection of tumor driver gene mutation sites, to the application of pathogenic metagenomic sequencing, next-generation sequencing NGS technology has played a role in the diagnosis of various diseases.

Cas13, also known as C2C2 protein, is a new member of the CRISPR/Cas system discovered in 2016. Cas13 was found to function to recognize and cut target RNA through specific RNA guidance. At the same time, Cas13 was found to have a "random cut" Activity—Once the target RNA is recognized, its protein conformation will undergo allosteric changes, and a non-specific RNase activity will be obtained. Taking advantage of this feature, Cas13 is currently mostly used in the molecular detection of nucleic acids. Of course, Cas13's ability to specifically recognize and cut target RNA is also used to knock down the expression level of gene RNA in cells. Recently, researchers have also used dead Cas13 (that is, Cas13 protein without RNA activity) to form a complex by binding to crRNA, specifically binding to the target RNA region, thereby inhibiting the translation of the RNA in the cell, achieving the effect of knocking down RNA and regulating genes. expression.

In the application of next-generation sequencing technology, a large part of the application requires sequencing and analysis of RNA sequences in samples, such as transcriptome sequencing and single-cell transcriptome sequencing, which can study the expression levels of all genes, and more comprehensive evaluation in The expression of genes in the body under different diseases, periods, and states provides more scientific and comprehensive support for disease diagnosis, treatment, and drug research and development; RNA pathogen sequencing in pathogen metagenomics can directly find a certain RNA in the sample The existence of pathogens provides supporting information for precise treatment. For example, the new coronavirus SARS-CoV-2 is an RNA virus, which was discovered by sequencing the RNA library through next-generation sequencing technology.

In the process of high-throughput sequencing of RNA libraries, since 80-90% of the total RNA is ribosomal rRNA sequence, if the sequence is directly reverse-transcribed, 80-90% of the sequence is ribosomal sequence, and this part of the sequence is often It is a meaningless invalid sequence, which increases the cost of sequencing. Therefore, whether it is in transcriptome RNA-seq sequencing or in the application of metagenomic sequencing of RNA pathogens, it is necessary to remove this part of the high-peak ribosomal rRNA sequence to the greatest extent to improve the efficiency of RNA sequencing and reduce the practicality of RNA sequencing. cost. At present, there are mainly two methods to eliminate the high peak ribosomal rRNA in the RNA library.

1) The method of enrichment sequencing only for mRNA: Traditional RNA-seq transcriptome sequencing often uses the characteristics of mRNA containing polyA during the RNA extraction process, and uses oligo T probes to enrich only mRNA in RNA. Ribosomal rRNA is removed because it does not contain polyA. However, there are many application defects in the method. For example, this method can only sequence RNA containing PolyA. For many non-polyA transcripts, small RNA transcripts cannot be sequenced to obtain effective information. However, they are important in the regulation of gene expression and cell Both play an important role in the course of fate, and exist in large numbers. In addition, since the RNA of many bacteria and viruses is different from the polyA of the mRNA of eukaryotic cells, and many RNA transcripts do not contain polyA, this method cannot be used for current RNA pathogenic metagenomic sequencing.

rRNA Depletion Kit和ThermoFisher公司的RiboMinus，

rRNA Depletion Kit的主要原理是通过序列特异的单链DNA探针和核糖体rRNA序列，进行杂交形成DNA-RNA杂交双链，在同过RNase H酶切割消化掉杂交双链中的RNA链后，再通过DNase 1酶切割消化掉体系中的单链DNA探针，然后进行RNA的逆转录，文库构建和二代测序。ThermoFisher-RiboMinus主要是通过标记的DNA探针和rRNA序列进行杂交，再通过特异性磁珠和标记探针的特异性结合，从而去除核糖体rRNA的序列。目前这两种方法都需要大量的探针，繁琐的人工操作，且商品化的试剂都比较昂贵，并且单纯DNA探针和RNA的分子杂交需要很长的杂交时间(一般从几个小时到十几个小时)。2) A method for removing ribosomal rRNA in total RNA: This method currently has many kits for removing rRNA in the market, mainly based on NEB’s

rRNA Depletion Kit and ThermoFisher's RiboMinus,

The main principle of the rRNA Depletion Kit is to hybridize the sequence-specific single-stranded DNA probe and the ribosomal rRNA sequence to form a DNA-RNA hybrid double-strand. After the RNA strand in the hybrid double-strand is digested by RNase H enzyme, Then, the single-stranded DNA probe in the system is digested by DNase 1 enzyme, and then reverse transcription of RNA, library construction and next-generation sequencing are performed. ThermoFisher-RiboMinus mainly uses labeled DNA probes to hybridize with rRNA sequences, and then removes ribosomal rRNA sequences through the specific binding of specific magnetic beads and labeled probes. At present, these two methods all need a large number of probes, cumbersome manual operations, and commercialized reagents are relatively expensive, and the molecular hybridization of simple DNA probes and RNA requires a long hybridization time (generally from several hours to ten a couple of hours).

Contents of the invention

The present invention aims to solve the above problems, thereby providing a method for removing ribosomal RNA sequences by using dCas13. The present invention can remove RNA sequences with high peaks such as ribosomes, specifically reduce the proportion of ribosomal RNA sequences in subsequent sequencing data, improve the sequencing efficiency of RNA libraries and the sequencing depth of target RNAs, and the method of the present invention can be used in RNA pathogen metagenomics The rRNA sequence can also be quickly removed during sequencing to improve the detection of pathogen target sequences.

The technical scheme that the present invention solves the above problems is as follows:

The method utilizes dead Cas13 without RNase activity and biotin-modified multiple crRNAs to form a complex, specifically binds to the RNA sequence of the high peak ribosome, and then binds dCas13-crRNAs through streptavidin-modified magnetic beads -rRNA, remove the ribosome-based high peak RNA sequence, specifically reduce the proportion of ribosomal RNA sequence in subsequent sequencing data, improve the sequencing efficiency of RNA library and the sequencing depth of target RNA; the crRNA corresponds to Cas13 protein The crRNA also includes variants of the crRNA structural sequence; the dCas13 is a Cas13 protein without RNase activity.

As a preferred technical solution, the dCas13 is a Cas13 protein without RNase activity, and the Cas13 protein includes Cas13a, Cas13b; Cas13a can be LwaCas13a, LbuCas13a, LbaCas13a; Cas13b can be PsmCas13b, CcaCas13b. Among them, the Cas13 protein without RNase activity, that is, the dCas13 protein is mutated from the active sites of the two HEPN domains of the Cas13 protein, including mutations to any amino acid in any HEPN domain, and also including simultaneous mutations The dCas13 protein obtained by mutating other sites in the non-HEPN region.

As a preference of the above technical solution, the crRNA is a biotin-modified crRNA of the terminal crRNA, including other improved forms of biotin modification, and also includes biotinylation modification on any nucleotide of crRNA.

As a preference of the above technical solution, the crRNA designed for ribosomal rRNA has an average spacing of 30bp to 200bp on the rRNA sequence; the complementary matching spacer sequence length of each crRNA is in the range of 22 to 32bp.

As a preferred technical solution, the molar ratio of dCas13:crRNA dosage ranges from 1:1 to 10:1, and the molar ratio range of dCas13:crRNA:RNA input dosage ranges from 1:1:1 to 10:1:0.1; The amount of RNA nucleic acid ranges from 1 ng to 5 μg.

In the present invention, after using streptavidin-modified magnetic beads to bind dCas13-crRNAs-rRNA, discard the magnetic bead part, keep other components, and then perform purification, reverse transcription, and library construction. This step is a process of negative selection .

As a preferred technical solution, the removed ribosomal rRNA sequences include human 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA, RNR1 and RNR2 sequences of mitochondrial rRNA.

In summary, the present invention has the following beneficial effects:

1), the present invention utilizes dCas13-crRNAs to bind ribosomal rRNA sequences, and then performs negative selection through streptavidin-modified magnetic beads to remove high-peak RNA sequences such as ribosomes. The method of the present invention does not require cumbersome operations, very easy;

2), the invention utilizes the characteristics that dCas13-crRNAs can quickly and specifically bind to the target RNA sequence, and the reaction time is fast. This method is much faster than the traditional method that only relies on single-stranded DNA hybridization degradation, which can save a lot of processing time.

Description of drawings

Fig. 1 is a schematic flow chart of the principle of using dCas13 to remove high peak ribosomal RNA sequences in the present invention.

Figure 2 shows the results of different crRNA spacer densities on rRNA removal efficiency.

Figure 3 shows the effect of the input ratio of dCas13a-crRNA-sample RNA on the removal efficiency of ribosomal rRNA.

Figure 4 shows the results of rRNA removal in cultured cells by dead LwaCas13a.

Figure 5 shows the application of the dCas13-crRNA method in RNA pathogen metagenomic sequencing.

Detailed ways

The method of the present invention will be described in detail below in conjunction with specific examples. These examples should be understood as a specific description and elaboration of the present invention, and should not be construed as limitations on the scope of the present invention. Any modifications or changes made by those skilled in the art on the basis of the present invention will be protected by the patent law as long as they are within the scope of the claims.

Example 1

In this example, we selected the sequence of 18S rRNA in human ribosomal RNA as the target for dCas13-crRNA removal, and the designed crRNA targeting sequence is shown in the table below. The selected dCas13 is dead LwaCas13a.

Table 1. List of ribosomal 18S rRNA crRNA targeting sequences

A total of 61 crRNAs were designed for targeting 18S rRNA sequences, and the targeted sequences are shown in Table 1.

Synthesis of biotinylated crRNA: The target sequence in Table 1 is reverse-complemented, and a common backbone sequence GATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC is added to the 5', and biotin biotin is added to the 3' end of the crRNA to synthesize crRNA, for example, The crRNA1 sequence synthesized for target sequence 1 in Table 1 is:

5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCCCGGCGCCCCGCAAGC GAGGAGGACG-3'-biotin

All crRNAs in the examples of the present invention were obtained by the above-mentioned method.

The crRNA pool 1 obtained after mixing all the crRNAs in Table 1, that is, the average interval length on the 18S rRNA is about 30bp; the crRNA with every other serial number selected in Table 1 is mixed (for example, the 1 selected in this embodiment column) , 3, 5, 7, 9.........sequence number crRNA), and finally select 31 as crRNA pool 2, that is, the average interval length on the 18S rRNA is about 60bp; The crRNAs of the sequence numbers are mixed (for example, the crRNAs of the 1, 5, 9, 13, 17... sequence numbers selected in this embodiment), and finally 16 are selected as the crRNA pool 3, that is, in the 18S rRNA The upper average interval length is about 120bp. These three crRNA pools were used to remove the target 18S rRNA and compared with the control group without adding crRNA and the untreated control group, respectively, building a library, performing mixed sequencing, and comparing the reads sequence of the sequenced data specifically to 18S rRNA Sequences of 18S rRNA depletion by different densities of spaced crRNAs were analyzed. The results show that when the average interval of crRNA on the target sequence is about 30bp, the effect of removing the rRNA target sequence is the best. (as shown in Figure 2).

In this embodiment, the steps to remove the high peak rRNA are as follows:

1) dCas13-crRNA pool-rRNA combination: Combine dCas13, crRNAslibrary and total RNA in the reaction solution according to the reaction system in the table below, and incubate at 37°C for 30 minutes.

2) Streptavidin magnetic beads cleaning preparation: Take 50 μL streptavidin magnetic beads, add 100 μL cleaning solution A: (DEPC-treated 0.1M NaOH DEPC-treated 0.05M NaC), wash twice, then add Cleaning solution B: (DEPC-treated 0.1M NaCl), wash once. Then resuspend the magnetic beads in 100 μL streptavidin magnetic bead binding and washing buffer, namely 2X B&W Buffer: (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2M NaCl).

3) Negative selection of streptavidin magnetic beads: add the reaction solution in step 1 to the magnetic bead solution in step 2, rotate gently at room temperature, incubate for 15 minutes, place on the magnetic stand for 2 minutes, and collect the solution part , for RNA purification.

4) Reverse transcription of RNA after removal, library construction and on-machine sequencing, and comparison of sequencing data.

5) For the analysis of the rRNA data from the sequencing machine, refer to the python code written below for analysis. Among them, the reference sequence of rRNA, 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA mitochondrial rRNA RNR1 and RNR2, see the sequence list, the rRNA analysis code is as follows:

Example 2

On the basis of the crRNA sequence designed in Example 1, continue to design crRNA for the rRNA sequences of human 28S rRNA, 5.8S rRNA, 5S rRNA, mitochondrial rRNA RNR1 and mitochondrial rRNA RNR2. Most of the high peak rRNA sequences in the RNA sample (accounting for about 80-90% of the total RNA) have been covered by crRNA targeting design, and the average design density is to design a crRNA with an interval of 30bp. The synthesis of all crRNAs in this example was carried out according to the method in Example 1. All crRNAs designed for 28S, 18S, 5.8S, 5S, RNR1, and RNR2 were mixed at equal concentrations of 100 μM and equal volumes to form a crRNA library that removes total rRNA . The removal of rRNA was carried out referring to the method in Example 1. In this embodiment, the following ratios of dCas13, crRNAs and RNA are used to evaluate the removal efficiency of the input ratio of dCas13-crRNAs and RNA to the overall rRNA.

(The molar concentration of RNA in all the examples of the present invention is estimated with a size of 500bp, for example, the molar concentration of RNA in a 100ng/100μL system is 5.88nM)

1) dLwaCas13a:crRNA:RNA=5:5:1=500nM:500nM:100nM

2) dLwaCas13a:crRNA:RNA=5:1:1=500nM:100nM:100nM

3) dLwaCas13a:crRNA:RNA=5:1:0.5=500nM:100nM:50nM

4) dLwaCas13a:crRNA:RNA=5:1:0.1=500nM:100nM:10nM

The steps for removing rRNA in this example are the same as those in Example 1, except that the ratio and amount of dLwaCas13a-crRNA-RNA used are different. The results of the experiment show that when the ratio of the three is: dLwaCas13a:crRNA:RNA=5:1:0.1=500nM:100nM:10nM, the rRNA removal effect is the best, and the rRNA sequencing ratio in the sample can be reduced from 85% to about 22% (as shown in Figure 3).

Table 2. List of ribosomal 28S rRNA crRNA targeting sequences

Table 3. Ribosome 5.8S rRNA crRNA targeting sequence list

serial number _cr RNA targeting sequence serial number _cr RNA targeting sequence 1 CTCTTAGCGGTGGATCACTCGGCTCGTG 4 ATTAATGTGAATTGCAGGACACATTGAT 2 CTCTTAGCGGTGGATCACTCGGCTCGTG 5 ATCGACACTTCGAACGCACTTGCGGCCC 3 GTCGATGAAGAACGCAGCTAGCTGCGAG 6 GTTCCTCCCGGGGCTACGCCTGTCTGAG

Table 4. Ribosome 5.8S rRNA crRNA targeting sequence list

serial number _cr RNA targeting sequence serial number _cr RNA targeting sequence 1 TCTACGGCCATACCACCCCTGAACGCGCC 3 GGGCCTGGTTAGTACTTGGATGGGAGAC 2 TCGTCTGATCTCGGAAGCTAAGCAGGGT 4 CGCCTGGGAATACCGGGTGCTGTAGGCT

Table 5. Mitochondrial ribosomal RNR1 crRNA targeting sequence list

serial number crRNA targeting sequence serial number crRNA targeting sequence 1 AGGTTTGGTCCTAGCCTTTCTATTAG 16 AGCTTAAAACTCAAAGGACCTGGCGGTG 2 CTTAGTAAGATTACACATGCAAGCATCC 17 ATCCCCTTAGAGGAGCCTGTTCTGTAAT 3 GTTCCAGTGAGTTCACCTCTAAATCAC 18 CGATAAACCCCGATCAACCTCACCCACCT 4 CGATCAAAAAGGAACAAGCATCAAGCACG 19 TGCTCAGCCTATATACCGCCATCTTCAG 5 GCAATGCAGCTCAAAACGCTTAGCCTAG 20 AACCCTGATGAAGGCTACAAAGTAAGCG 6 CACCCCCACGGGAAACAGCAGTGATTAA twenty one CACGTAAAGACGTTAGGTCAAGGTGTAG 7 TTTAGCAATAAACGAAAGTTTAACTAAGC twenty two TGAGGTGGCAAGAAATGGGCTACATTTT 8 ACTAACCCCAGGGTTGGTCAATTTCGTG twenty three CCAGAAAACTACGATAGCCCTTATGAAA 9 CCACCGCGGTCACACGATTAACCCAAGT twenty four AAGGGTCGAAGGTGGATTTAGCAGTAAA 10 AGAAGCCGGCGTAAAGAGTGTTTTAGAT 25 AAGAGTAGAGTGCTTAGTTGAACAGGGC 11 CCCCCTCCCCCAATAAAGCTAAAACTCAC 26 GAAGCGCGTACACACCGCCCGTCACCCT 12 TAAAAAACTCCAGTTGACACAAAATAGA 27 CCTCAAAGTATACTTCAAAGGACATTTAA 13 ACGAAAGTGGCTTTAACATATCTGAACA 28 CCTACGCATTTATATAGAGGAGACAAGT 14 GCTTAGCCCTAAACCTCAACAGTTAAAT 29 TAACATGGTAAGTGTACTGGAAAGTGCA 15 ACAAAACTGCTCGCCAGAACACTACGAG

Table 6. Mitochondrial ribosomal RNR2 crRNA targeting sequence list

Example 3

In this example, we extract RNA from the cultured jurkat cells, and then use the total rRNA to remove it. The crRNA pool used in this example is the same as that in Example 2, for 28S, 18S, 5.8S, 5S, RNR1, A crRNA pool designed for all rRNAs of RNR2. The results show that this method can greatly reduce the ribosomal rRNA sequence in the RNA sample, and significantly improve the sequencing efficiency of other target sequences.

The steps for removing rRNA in this example are the same as those in Example 1 and Example 2, except that the ratio and amount of dLwaCas13a-crRNA-RNA used are different. When the ratio of the three is: dLwaCas13a:crRNA:RNA=5:1:0.1=500nM:100nM:10nM, the rRNA removal effect is the best, and the rRNA sequencing ratio in the sample can be reduced from 81% to about 12%. The overall rRNA removal efficiency can reach 85% (as shown in Figure 4).

Example 4

In this example, we performed RNA extraction on three clinical respiratory samples, and then carried out RNA reverse transcription library construction and metagenomic sequencing on samples that had been depleted of total rRNA by dCas13a using the method of the present invention and samples that had not been correspondingly depleted of rRNA, and compared the sequencing. To analyze the improvement of the effect of sequencing after rRNA removal on the detection of respiratory RNA pathogens.

In sample 1 in this embodiment, after dCas13a removed rRNA, the proportion of rRNA was reduced from 83% to 15.2%, and the detection of influenza A virus in the sample increased from RPM 34.2 to RPM 270.9, and the abundance of detection increased 7.3 times. After rRNA was removed by dCas13a in sample 2, the proportion of rRNA decreased from 75.8% to 13.9%, and the detection of respiratory syncytial virus type A in the sample increased from RPM 71.6 to RPM 556.2, and the detected abundance increased by 7.8 times. After dCas13a removed rRNA in sample 3, the proportion of rRNA decreased from 74.8% to 16.2%. No other RNA pathogens were detected before rRNA removal, and influenza A virus RPM 4.8 was detected by sequencing after rRNA removal by dCas13a. It shows that in the sequencing of pathogenic metagenomes, the method of the present invention can greatly reduce the proportion of redundant rRNA sequences with high peak height, and improve the detection of RNA pathogens.

In this embodiment, the following process is used to remove rRNA, and the ratio of dCas13-crRNA pool-RNA is: dLwaCas13a:crRNA:RNA=5:1:0.1.

1) dCas13-crRNA pool-rRNA combination: Combine dCas13, crRNAslibrary and total RNA in the reaction solution according to the reaction system in the table below, and incubate at 37°C for 30 minutes.

2) Streptavidin magnetic beads cleaning preparation: Take 50μL streptavidin magnetic beads, add 100μL cleaning solution A: DEPC-treated 0.1M NaOH DEPC-treated 0.05M NaC, wash both sides, then add cleaning solution B : DEPC-treated 0.1M NaCl, wash once. Then resuspend the magnetic beads in 100 μL streptavidin magnetic bead binding and washing buffer, ie 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2M NaCl in 2X B&W Buffer.

3) Negative selection of streptavidin magnetic beads: add the reaction solution in step 1 to the magnetic bead solution in step 2, rotate gently at room temperature, incubate for 15 minutes, place on the magnetic stand for 2 minutes, and collect the solution part , for RNA purification.

4) Reverse transcription of RNA after removal, library construction and on-machine sequencing, and comparison of sequencing data.

Claims (10)

1. A method utilizing dCas13 to remove ribosomal RNA sequences, characterized in that: the method utilizes dead Cas13 without RNase activity and a plurality of crRNAs modified by biotin to form a complex, specifically binding to the RNA of high peak ribosomes sequence, and then bind dCas13-crRNAs-rRNA with streptavidin-modified magnetic beads to remove high-peak RNA sequences dominated by ribosomes, specifically reduce the proportion of ribosomal RNA sequences in subsequent sequencing data, and improve The sequencing efficiency of the RNA library and the sequencing depth of the target RNA; the crRNA is the crRNA corresponding to the Cas13 protein, and also includes a variant of the crRNA structural sequence; the dCas13 is the Cas13 protein without RNase activity. 2. a kind of method utilizing dCas13 to remove ribosomal RNA sequence according to claim 1, is characterized in that: Cas13 protein without RNase activity is to mutate the active sites of 2 HEPN structural domains of Cas13 protein , including mutations to any amino acid in any HEPN domain, and also include mutations to other sites in non-HEPN regions at the same time. 3. a kind of method utilizing dCas13 to remove ribosomal RNA sequence according to claim 2 is characterized in that: described Cas13 albumen comprises Cas13a, Cas13b; Described Cas13a comprises LwaCas13a, LbuCas13a, LbaCas13a; Described Cas13b Including PsmCas13b, CcaCas13b. 4. a kind of method utilizing dCas13 to remove ribosomal RNA sequence according to claim 1, is characterized in that: described crRNA is the biotin-modified crRNA of terminal crRNA, comprises the biotin modification of other forms of improvement, also Includes biotinylation on any nucleotide in crRNA. 5. a kind of method utilizing dCas13 to remove ribosomal RNA sequence according to claim 1 is characterized in that: the crRNA designed for ribosomal rRNA has an average interval of 30bp～200bp on the rRNA sequence; The matching spacer sequence length ranges from 22 to 32 bp. 6. A method for removing ribosomal RNA sequences by using dCas13 according to claim 1, wherein the molar ratio of dCas13:crRNA dosage ranges from 1:1 to 10:1. 7. A method for removing ribosomal RNA sequences by using dCas13 according to claim 1, wherein the molar ratio of dCas13:crRNA:RNA input is in the range of 1:1:1 to 10:1:0.1. 8. A method for removing ribosomal RNA sequences by using dCas13 according to claim 1, characterized in that: the amount of total RNA nucleic acid input ranges from 1 ng to 5 μg. 9. a kind of method utilizing dCas13 to remove ribosomal RNA sequence according to claim 1 is characterized in that: after the magnetic beads modified by streptavidin are used in conjunction with dCas13-crRNAs-rRNA, the magnetic beads part is discarded and retained Other components are then purified, reverse transcribed, and library built. 10. a kind of method utilizing dCas13 to remove ribosomal RNA sequence according to claim 1, is characterized in that: the ribosomal rRNA sequence that wherein removes comprises people's 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA, the mitochondrial rRNA RNR1, RNR2 sequence.

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