Intron retention level control method

Abstract

The invention relates to the field of biomedicine, in particular to an intron retention level control method which comprises the following steps: S101, obtaining transcriptome sequencing data of a biological sample; s102, obtaining gene expression matrix data; s103, obtaining the intron retention level of the whole transcriptome of the biological sample; s104, obtaining the transcriptome expression level of the potential RNA binding protein in the biological sample; s105, taking a potential RNA binding protein related to the intron retention level of the whole transcriptome as a candidate regulatory factor; s106, determining a cis-regulatory element region of the candidate regulatory factors acting on the regulatory target introns; s107, obtaining an effective single nucleotide polymorphic site and / or an effective degenerate base; and S108, realizing directional control on the target introne retention event. According to the invention, excavation and positioning of regulatory factors and cis-regulatory elements of the specific IR are realized, and then directional control of the specific IR is realized.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a method for controlling intron retention level. Background Art

[0002] The spatiotemporal specific expression of genes is the basis of biodiversity and the prerequisite for cells to perform specific functions normally. The accurate removal of introns from pre-mRNA and splicing to form the correct open reading frame are the key links to ensure the spatiotemporal specific expression of genes. Intron retention (IR) refers to the fact that some specific introns of pre-mRNA are not "successfully" removed and retained in mature mRNA. These transcripts (intron retaining transcripts, IRTs) that retain specific introns are often cleared by the nonsense mediated decay (NMD) pathway in cells. Therefore, IR has long been considered to be the noise generated by cellular mis-splicing.

[0003] With the rapid development of sequencing technology and the gradual deepening of IR research, people have gradually realized that IR is an "active" post-transcriptional regulatory mechanism and an independent mechanism to control and enhance the complexity of gene expression. IRT can be "stored" in the cell nucleus and quickly processed (cytoplasmic export, splicing) and expressed proteins under specific stimuli, thereby shortening the time for cells to respond to extracellular signal stimuli. Some IRTs can also be translated into new protein isomers. IR helps to form new non-coding RNAs and can also act as an adsorbent of miRNA to compete with endogenous miRNAs. Although IR has gradually become a consensus as an important functional regulatory mechanism, there is still a serious lack of understanding of the occurrence and regulatory mechanism of IR.

[0004] Abnormal IR expression patterns are considered to be important markers of many diseases such as cancer, and this pattern has been studied in depth as a biomarker and therapeutic target. For example, abnormal IR is considered to be a widespread mechanism for the inactivation of tumor suppressor genes, and tumor-specific neoantigenic peptides derived from IR are important targets for personalized cancer vaccine research. Although existing transcriptome and pan-cancer studies have revealed the possible role of IR in tumorigenesis, development and treatment, there are few reports on how specific IR affects tumor progression. One of the main reasons for the above situation is that in traditional molecular genetics research, people pay more attention to the effects of mutations or changes in expression levels of specific genes on cells or organisms, and have developed genetic tools such as RNAi, shRNA, CRISPR and overexpression to interfere with gene expression. However, these technologies are difficult to effectively knock down / overexpress specific IR, which makes research on IR much more difficult than traditional molecular biology targeting specific genes. Summary of the invention

[0005] The present invention provides a method for controlling intron retention level, characterized in that it comprises the following steps: S101 Obtain transcriptome sequencing data of biological samples; In some embodiments, the biological sample includes one or more of a blood sample, an organ sample, and a tumor sample.

[0006] S102 obtaining gene expression matrix data corresponding to the transcriptome sequencing data; S103: processing the transcriptome sequencing data to obtain the levels of all intron retention events in the biological sample and the proportion of intron retention in the introns of all expressed genes, that is, the intron retention level of the whole transcriptome of the biological sample; In some embodiments, the processing method of the S103 step specifically includes: calculating the coverage at the single base level of each intron region and taking the median as the sequencing depth of the intron region, requiring the overall coverage of the intron region of the intron retention event to be 100%; taking the median of the single base level coverage of the exon regions upstream and downstream of the intron region as a reference, calculating the level of the intron retention event; and then calculating the proportion of intron retention events occurring in the introns of all expressed genes in the whole transcriptome based on the quantitative results of intron retention, that is, obtaining the intron retention level of the whole transcriptome of the biological sample.

[0007] S104: obtaining a potential RNA binding protein of the biological sample, and obtaining a transcriptome expression level of the potential RNA binding protein in the biological sample based on the gene expression matrix data; S105 calculating the correlation between the potential RNA binding protein and the intron retention level of the whole transcriptome, and taking the potential RNA binding protein correlated with the intron retention level of the whole transcriptome as a candidate regulatory factor; In some embodiments, the correlation comprises a positive correlation or a negative correlation.

[0008] In some embodiments, the correlation is a statistically significant correlation.

[0009] S106 determining that the candidate regulatory factor acts on a cis-regulatory element region of the regulatory target intron; In some embodiments, the S106 step specifically includes: using CLIP chromatin immunoprecipitation technology to enrich and sequence the fragments that are physically bound to the candidate regulatory factor and the target RNA molecule, and determining the cis-regulatory element region based on the enrichment of the binding signal of the candidate regulatory factor at the location where the target intron retention event occurs.

[0010] In some embodiments, the step S105 and the step S106 further include: screening the RNA-binding proteins among the potential RNA-binding proteins that have a strong correlation with the intron retention level of the whole transcriptome as the candidate regulatory factors.

[0011] S107: Based on the determined cis-regulatory element region, analyzing the candidate regulatory factors and the regulatory modes of the cis-regulatory element on the target intron retention event, thereby obtaining effective single nucleotide polymorphism sites and / or effective degenerate bases in the sequence of the cis-regulatory element region that can affect the target intron retention event; In some embodiments, the step S107 specifically includes a first analysis method and / or a second analysis method.

[0012] In some embodiments, the first analysis method comprises: S201: obtaining a single nucleotide polymorphism site in the cis-regulatory element region, and using molecular quantitative trait genetic loci to analyze whether the base change of the single nucleotide polymorphism site will affect the level of the target intron retention event, thereby obtaining a potentially effective single nucleotide polymorphism site; S202 constructing a mini-gene plasmid containing all genotypes of the potentially effective single nucleotide polymorphism sites in the cis-regulatory element region, and expressing the mini-gene plasmid in a tool cell; S203 verifies whether the base change of the potentially effective single nucleotide polymorphism site will affect the level of the target intron retention event; if so, the single nucleotide polymorphism site is determined to be an effective single nucleotide polymorphism site; if not, the single nucleotide polymorphism site is determined to be unable to affect the regulation of the target intron retention event by the candidate regulatory factor.

[0013] In some embodiments, the second analysis method comprises: S301: Obtaining all degenerate bases in the cis-regulatory element region; S302 constructing a mini-gene plasmid containing all degenerate codon genotypes present in the cis-regulatory element region, and expressing it in a tool cell; S303 Analyze whether the base change of the degenerate codon will affect the level of the target intron retention event, and then obtain the effective degenerate base in the sequence of the cis-regulatory element region, that is, the effective degenerate base can affect the regulation of the candidate regulatory factor on the target intron retention event.

[0014] S108 Using base editing technology to edit the effective single nucleotide polymorphism sites and / or effective degenerate bases in the cell genome, thereby achieving directional control of the target intron retention event.

[0015] In some embodiments, the base editing technology can be any base editing technology that can achieve the intended editing purpose, such as PE2 editing.

[0016] In some embodiments, the S108 step also includes: analyzing whether the affinity of the candidate regulatory factor for the target RNA molecule changes after the effective single nucleotide polymorphism site and / or the effective degenerate base change that can affect the target intron retention event in the cis-regulatory element region of the target intron retention event that interacts with the candidate regulatory factor; if so, determining whether the effective single nucleotide polymorphism site and / or the effective degenerate base change can affect the binding of the target RNA molecule to the candidate regulatory factor, thereby achieving directional control of the target intron retention event.

[0017] In some embodiments, the method further comprises S109 verifying whether the level of the target intron retention event in the cell genome reaches the expected directional control.

[0018] In some embodiments, the directional control comprises up-regulation or down-regulation.

[0019] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: The prior art is usually limited to the quantitative analysis of intron retention in sequencing data, and there is no prior art report on how to achieve directional control of specific IR (e.g., knockdown / overexpression). The present invention creatively proposes a method for controlling the intron retention level based on the "regulatory factor-cis-regulatory element-regulatory target" strategy. The present invention analyzes the transcriptome sequencing data of the biological sample to achieve the mining of potential regulators (e.g., RNA binding proteins) for specific IR in the biological sample. Based on the candidate regulators, the present invention locates the cis-regulatory element (the binding region of the RNA molecule and the specific regulator) and the specific target IR for the specific IR. The present invention achieves directional control of IR by PE2 editing the effective single nucleotide polymorphism site and / or the effective degenerate base in the cis-regulatory element region. Specifically, the present invention increases / decreases the affinity of the regulator and the cis-regulatory element after mutating the effective single nucleotide polymorphism site and / or the effective degenerate base, so that the level of the specific IR is reduced / increased, thereby achieving the purpose of directional manipulation of IR without affecting protein coding.

[0020] As described above, the present invention constructs a method for knocking down / overexpressing specific IR with a clear mechanism by exploring key IR regulatory factors and cis-regulatory elements. The method is suitable for functional research on specific IR and its role in various fields such as pathogenesis and drug action mechanism, such as targeted treatment of diseases such as cancer (for example, analyzing and regulating the effects of specific IR on the differentiation, invasion and metastasis of tumor cells).

[0021] Although CRISPR Screening and other technologies can be used to screen regulatory factors of IR on a large scale, this technology has considerable difficulty and barriers in terms of operation and cost. The method provided by the present invention can integrate multi-omics data of different dimensions (including a large amount of published public data), reducing the cost of IR analysis while achieving directional control of IR of target introns without changing other genes of the cell, so that base editing technology can be applied to the field of intron retention, and also helps to achieve disease treatment by interfering with IR.

[0022] In addition, the inventors found that the existing IR quantification method is too sensitive to noise (such as noise from non-coding RNA and antisense RNA), and is basically not suitable for high-noise NGS data such as total RNA-Seq. The present invention provides an IR quantification method, which not only effectively avoids the influence caused by repeated sequences and non-coding RNA in the intron region, but also is suitable for the identification of unannotated IRs, and is more conducive to obtaining more accurate information on the intron retention level of the whole transcriptome. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0024] Figure 1 This is the result diagram of the IR expression profile of the mouse in Example 2; Figure 2 This is the result diagram of Example 3 showing that RBP1 may be a regulatory factor of IR; Figure 3 This is the result diagram of the cis-regulatory element region of Example 3; Figure 4 This is a result diagram showing that the SNP in the RBP1 binding region of Example 4 affects the retention level of downstream introns; Figure 5 This is a result diagram showing that the SNP in the RBP1 binding region of Example 5 affects the intron retention level and leads to changes in protein expression; Figure 6 This is a result diagram showing that specific degenerate base changes in the RBP1 binding region of Example 6 affect the intron retention level; Figure 7 A flow chart of the method for controlling intron retention level of the present invention; Figure 8 is a flow chart of the first analysis method of the present invention; Fig. 9 Flow chart of the second analysis method of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0026] In this document, the directions or positional relationships indicated by the terms "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0027] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0028] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0029] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0030] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0031] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0032] Embodiment 1 1. RNA-Seq Data Alignment The input of this step is the original fastq file downloaded from the sequencer, which can be aligned using any third-party alignment software (such as STAR) to generate a BAM file.

[0033] 2. Quantification of Gene Expression Use third-party gene expression quantification software (such as RSEM, featurecount, etc.) to obtain transcriptome gene expression matrix data.

[0034] 3. Quantification of intron retention levels All IRs of the whole transcriptome of the sample are quantified. The present embodiment provides an analytical strategy for IR quantification, which abandons the strategy of directly counting the total number of sequences in the intron region, but adopts: the coverage of each intron region at the single base level is calculated to process the sequencing data of the intron region, and then the median is calculated for the coverage of the single base level of the intron region as the sequencing depth of the intron region, and the overall coverage of the intron region is required to be 100% for the intron retention event, that is, every base in the intron region is covered. Then, the level of intron retention events (intron retention level, IRL) of the intron region is calculated with reference to the median of the coverage of the single base level of the exon region upstream and downstream of the intron region. Then, according to the quantitative results of intron retention, the level of intron retention events in the introns of all expressed genes in the whole transcriptome is counted, and then the intron retention level of the whole transcriptome of the biological sample is obtained. This strategy effectively avoids the effects caused by repeated sequences in the intron region and non-coding RNA. In addition, single-base coverage can also be used to evaluate the sequence distribution of intronic regions, thereby excluding the interference of noise such as non-coding RNA and antisense RNA. In short, this strategy can largely avoid the interference of sequencing noise and can be used for the identification of unannotated IRs.

[0035] 4. Correlation analysis between gene expression level of RNA binding protein and intron retention level of whole transcriptome All potential RNA-binding proteins (RBPs) were searched from public databases, and their expression levels in all samples were extracted from the results of step 2. The proportion of IR occurring in the introns of all expressed genes in each sample was calculated from the results of step 3 as the whole transcriptome IR level of the sample. The correlation coefficient and P value between each RBP and the whole transcriptome IR level were calculated through correlation analysis.

[0036] 5. Use gene knockdown or overexpression experiments to verify the relationship between RBP and whole transcriptome IR levels The candidate RBP genes were knocked down in tool cell lines such as K562 and HepG2 using shRNA, RNAi and other technologies to study whether there is a causal (strong correlation) relationship between RBP and the IR level of the whole transcriptome.

[0037] 6. Verify the co-localization relationship between RBP and IR using CLIP experimental data The RBPs with significant positive or negative correlation in the correlation analysis results were selected as candidate regulatory factors, and the CLIP chromatin immunoprecipitation technique was used to enrich and sequence the fragments where the RBP physically binds to the RNA molecule. The third-party alignment software and quantitative software such as featurecount were used to analyze the enrichment of the binding signal of the RBP in the IR and its surrounding areas, and to clarify the binding position of the RBP regulating the IR.

[0038] 7. Use QTL and other technical means to study the effect of motif changes in RBP binding regions on IR levels Molecular quantitative trait loci (QTL) analysis technology can be used to study the relationship between genetic variation (SNP) and molecular traits such as gene expression and IR levels. The purpose is to study whether the motif changes in the RBP binding region caused by SNP will lead to changes in the levels of target IRs regulated by RBP, thereby clarifying how RBP regulates IR.

[0039] 8. Use mini-gene reporter system to verify the effect of SNP in RBP binding region on IR level By artificially constructing cis-regulatory element sequences containing different SNP genotypes and mini-gene plasmids of target IR, and expressing them in tool cells such as K562 and HepG2, the RIP technology was used to study the changes in the affinity of RBP and mRNA after the RBP regulatory motif was changed, and the RT-PCR technology was used to study the changes in the level of target IR after the RBP regulatory motif was changed.

[0040] 9. Using codon degeneracy to achieve targeted control of target IR levels without affecting amino acid coding A mini-gene plasmid with all degenerate codon genotypes of the RBP binding region of the target IR was constructed, and the changes in the levels of the target IR after the RBP regulatory motif was changed were studied in the tool cells to discover the degenerate sequences that can directionally up-regulate or down-regulate the target IR.

[0041] 10. Use base editing technology to achieve targeted control of target IR levels Use third-party base editing technology (such as PE2 technology) to edit the directed regulatory degenerate sequence of the RBP regulatory region of the target IR in the cell line genome, use RT-PCR to test whether the level of endogenous target IR in the edited cell line has reached the expected upregulation or downregulation, and use WB to study whether the protein level of the parent gene of the target IR has undergone the expected upregulation or downregulation compared with the wild type.

[0042] Other methods: Mini-gene: The upstream and downstream exons of the retained intron and the nearby introns within about 150 bp were selected and inserted into the RHCglo vector. The constructed mini-gene plasmid was transferred into 293T cells using liposome transfection reagent. RNA was extracted after 48 hours, and RT-qPCR was performed to verify the intron retention level.

[0043] RIP: The mini-gene plasmid and RBP-flag plasmid were co-transfected into 293T cells. 48 hours after transfection, RNA-protein pull-down was performed using flag magnetic beads. The success of RBP protein pull-down was verified by Western blotting, and the amount of mini-gene RNA binding was determined by RT-qPCR experiment.

[0044] shRNA interference: miR30-based MLS retroviral vector (MSCV-shRNA-SV40-GFP) will be used for silencing of target genes and expression of control shRNA.

[0045] Primer editor (PE): PE2-based base editors will be used for mutation and downstream functional analysis of SNP sites in candidate genes.

[0046] Embodiment 2 This example uses the method of Example 1, based on ONT's third-generation long-read RNA-Seq technology, to study the IR expression profiles of 28 organs / tissues of two different strains of mice (C57 and BALBc), and predict key IR regulatory factors.

[0047] The results are as follows Figure 1 As shown, different tissues / cells of mice have their own specific IR expression profiles ( Figure 1 A), the abundance of IR in various tissues / cells of mice is significantly correlated with the expression level of RBP1 (RBP1 in this example is EIF4A3) ( Figure 1 B).

[0048] Embodiment 3 This example combines shRNA knockdown experiments, CLIP enrichment analysis, and QTL positioning analysis to discover cis-regulatory element regions and downstream regulatory targets.

[0049] The results are as follows Figure 2 As shown in Figure 2, knockdown of RBP1 in the K562 cell line resulted in significant changes in 1122 IRs, of which more than 95% of IRLs (intron retention levels) were increased. These results suggest that RBP1 may be a regulatory factor for IR.

[0050] The results are as follows Figure 3 As shown in A, it can be seen from the identified CLIP signal that RBP1 binds to the region about 50 nt upstream of the 5' splice site and participates in the removal of introns. Figure 3 As shown in B, the density distribution map of irQTLs shows that there is an enrichment of IR-related SNPs at the binding site of RBP1 (i.e., upstream of the 5' splicing site) ( Figure 3 B).

[0051] Embodiment 4 Based on the experimental results of Example 3, this example screened out IR-related SNPs. NSMAF , NCAPD2 , HCLS1 , NUP85 The effective SNPs in the RBP1 binding region of the four genes are located 26nt, 37nt, 16nt and 28nt upstream of the 5' splice site, respectively. Figure 4 shown.

[0052] Further, from Figure 4 AIt can be seen that NSMAF , NCAPD2 , HCLS1 , NUP85 Base changes in the RBP1 target sites (e.g., changing a certain base) will affect the level of intron retention. Allele-specific analysis ( Figure 4 B) and mini-gene reporter system ( Figure 4 C and Figure 4 D) The results indicate that specific SNP genotypes in the RBP1 binding region affect the level of downstream intron retention.

[0053] Embodiment 5 This embodiment uses NUP85 Targets are used as examples, such as Figure 5 As shown in the figure, RNA immunoprecipitation (RIP) and Western Blot (WB) experiments showed that the T>C mutation in the RBP1 binding region (28 nt from the 5' splice site) would lead to the binding of RBP1 to NUP85 The affinity of mRNA is increased, thus making NUP85 The IR of intron 17 (the SNP is located 28 nt upstream of intron 17) was reduced, which ultimately led to increased NUP85 protein expression.

[0054] Embodiment 6 Different from the IR-related SNP idea mentioned above, this example uses PE2 (Primer editor 2) base editing technology to NUP85 All degenerate bases in the RBP1 binding region upstream of the IR event in intron 17 were point mutated. Figure 6 The results showed that the directional control of intron retention can be achieved not only by regulating IR-related SNPs, but also by changing the specific degenerate bases in the RBP1 binding region, so as to achieve the purpose of targeted knockdown / overexpression of IR levels without affecting protein coding.

[0055] Embodiment 7 like Figure 7 As shown, the present invention provides a method for controlling intron retention level, characterized in that it comprises the following steps: S101 Obtain transcriptome sequencing data of biological samples; In some embodiments, the biological sample includes one or more of a blood sample, an organ sample, and a tumor sample.

[0056] S102 obtaining gene expression matrix data corresponding to the transcriptome sequencing data; S103: processing the transcriptome sequencing data to obtain the levels of all intron retention events in the biological sample and the proportion of intron retention in the introns of all expressed genes, that is, the intron retention level of the whole transcriptome of the biological sample; In some embodiments, the processing method of the S103 step specifically includes: calculating the coverage at the single base level of each intron region and taking the median as the sequencing depth of the intron region, requiring the overall coverage of the intron region of the intron retention event to be 100%; taking the median of the single base level coverage of the exon regions upstream and downstream of the intron region as a reference, calculating the level of the intron retention event; and then calculating the proportion of intron retention events occurring in the introns of all expressed genes in the whole transcriptome based on the quantitative results of intron retention, that is, obtaining the intron retention level of the whole transcriptome of the biological sample.

[0057] S104 obtaining a potential RNA binding protein (RBP) of the biological sample, and obtaining a transcriptome expression level of the potential RNA binding protein in the biological sample based on the gene expression matrix data; S105 calculating the correlation between the potential RNA binding protein and the intron retention level of the whole transcriptome, and taking the potential RNA binding protein correlated with the intron retention level of the whole transcriptome as a candidate regulatory factor; In some embodiments, the correlation comprises a positive correlation or a negative correlation.

[0058] In some embodiments, the correlation is a statistically significant correlation.

[0059] S106 determining that the candidate regulatory factor acts on a cis-regulatory element region of the regulatory target intron; In some embodiments, the step S106 specifically includes: using CLIP chromatin immunoprecipitation technology to enrich and sequence the fragments physically bound to the candidate regulatory factor and the target RNA molecule, and determining the cis-regulatory element region based on the enrichment of the binding signal of the candidate regulatory factor at the location where the target intron retention event occurs. Taking RBP1 (EIF4A3) as an example, the cis-regulatory element region is approximately within the region 50nt upstream of the 5' splice site.

[0060] In some embodiments, the cis-regulatory element region refers to a binding site of a regulatory factor (RNA binding protein) on a target RNA molecule (eg, mRNA).

[0061] In some embodiments, the step S105 and the step S106 further include: screening the RNA-binding proteins among the potential RNA-binding proteins that have a strong correlation with the intron retention level of the whole transcriptome as the candidate regulatory factors.

[0062] S107: Based on the determined cis-regulatory element region, analyzing the candidate regulatory factors and the regulatory modes of the cis-regulatory element on the target intron retention event, thereby obtaining effective single nucleotide polymorphism sites and / or effective degenerate bases in the sequence of the cis-regulatory element region that can affect the target intron retention event; In some embodiments, the step S107 specifically includes a first analysis method and / or a second analysis method.

[0063] In some embodiments, Figure 8 As shown, the first analysis method includes: S201: obtaining a single nucleotide polymorphism site in the cis-regulatory element region, and using molecular quantitative trait genetic loci to analyze whether the base change of the single nucleotide polymorphism site will affect the level of the target intron retention event, thereby obtaining a potentially effective single nucleotide polymorphism site; S202 constructing a mini-gene plasmid containing all genotypes of the potentially effective single nucleotide polymorphism sites in the cis-regulatory element region, and expressing the mini-gene plasmid in a tool cell; S203 verifies whether the base change of the potentially effective single nucleotide polymorphism site will affect the level of the target intron retention event; if so, the single nucleotide polymorphism site is determined to be an effective single nucleotide polymorphism site; if not, the single nucleotide polymorphism site is determined to be unable to affect the regulation of the target intron retention event by the candidate regulatory factor.

[0064] In some embodiments, "effective" means that changes in single nucleotide polymorphism sites and / or degenerate base sequences in the sequence of the cis-regulatory element region can affect the regulation of the target intron retention event by the regulatory factor, thereby changing the level of the target intron retention event.

[0065] In some embodiments, Fig. 9 As shown, the second analysis method includes: S301: Obtaining all degenerate bases in the cis-regulatory element region; S302 constructing a mini-gene plasmid containing all degenerate codon genotypes present in the cis-regulatory element region, and expressing it in a tool cell; S303 Analyze whether the base change of the degenerate codon will affect the level of the target intron retention event, and then obtain the effective degenerate base in the sequence of the cis-regulatory element region, that is, the effective degenerate base can affect the regulation of the candidate regulatory factor on the target intron retention event.

[0066] S108 Using base editing technology to edit the effective single nucleotide polymorphism sites and / or effective degenerate bases in the cell genome, thereby achieving directional control of the target intron retention event.

[0067] In some embodiments, the base editing technique comprises PE2 editing.

[0068] In some embodiments, the S108 step also includes: analyzing whether the affinity of the candidate regulatory factor for the target RNA molecule changes after the effective single nucleotide polymorphism site and / or the effective degenerate base change that can affect the target intron retention event in the cis-regulatory element region of the target intron retention event that interacts with the candidate regulatory factor; if so, determining whether the effective single nucleotide polymorphism site and / or the effective degenerate base change can affect the binding of the target RNA molecule to the candidate regulatory factor, thereby achieving directional control of the target intron retention event.

[0069] In some embodiments, the method further comprises S109 verifying whether the level of the target intron retention event in the cell genome reaches the expected directional control.

[0070] In some embodiments, the directional control comprises up-regulation or down-regulation.

[0071] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above-mentioned embodiment methods can be implemented by means of software plus a necessary general hardware platform, and of course by hardware, but in many cases the former is a better implementation method. Based on such an understanding, the technical solution of the present invention, or the part that contributes to the prior art, can be embodied in the form of a software product, which is stored in a storage medium (such as ROM / RAM, magnetic disk, optical disk), and includes a number of instructions for a computer terminal (which can be a mobile phone, computer, server, or network device, etc.) to execute the methods described in each embodiment of the present invention.

[0072] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for controlling intron retention level, characterized in that: The following steps are involved: S101 Obtain transcriptome sequencing data of biological samples; S102 obtaining gene expression matrix data corresponding to the transcriptome sequencing data; S103: processing the transcriptome sequencing data to obtain the levels of all intron retention events in the biological sample and the proportion of intron retention in the introns of all expressed genes, that is, the intron retention level of the whole transcriptome of the biological sample; S104: obtaining a potential RNA binding protein of the biological sample, and obtaining a transcriptome expression level of the potential RNA binding protein in the biological sample based on the gene expression matrix data; S105 calculating the correlation between the potential RNA binding protein and the intron retention level of the whole transcriptome, and taking the potential RNA binding protein correlated with the intron retention level of the whole transcriptome as a candidate regulatory factor; S106 determining that the candidate regulatory factor acts on a cis-regulatory element region of the regulatory target intron; S107: Based on the determined cis-regulatory element region, analyzing the candidate regulatory factors and the regulatory modes of the cis-regulatory element on the target intron retention event, thereby obtaining effective single nucleotide polymorphism sites and / or effective degenerate bases in the sequence of the cis-regulatory element region that can affect the target intron retention event; S108 Using base editing technology to edit the effective single nucleotide polymorphism sites and / or effective degenerate bases in the cell genome, thereby achieving directional control of the target intron retention event.

2. The method according to claim 1, characterized in that The processing method of the step S103 specifically includes: calculating the coverage at the single-base level of each intron region and taking the median as the sequencing depth of the intron region, requiring the overall coverage of the intron region of the intron retention event to be 100%; taking the median of the single-base level coverage of the exon regions upstream and downstream of the intron region as a reference, calculating the level of the intron retention event; and then calculating the proportion of intron retention events occurring in the introns of all expressed genes in the whole transcriptome based on the quantitative results of intron retention, that is, obtaining the intron retention level of the whole transcriptome of the biological sample.

3. The method according to claim 1, characterized in that The biological sample includes one or more of a blood sample, an organ sample and a tumor sample.

4. The method according to claim 1, characterized in that The base editing technology includes PE2.

5. The method according to claim 1, characterized in that The correlation includes a positive correlation or a negative correlation.

6. The method according to claim 1, characterized in that The S106 step specifically includes: using CLIP chromatin immunoprecipitation technology to enrich and sequence the fragments where the candidate regulatory factor is physically bound to the target RNA molecule, and determining the cis-regulatory element region based on the enrichment of the binding signal of the candidate regulatory factor at the location where the target intron retention event occurs.

7. The method according to claim 1, characterized in that Before the S108 step, it also includes: analyzing whether the affinity of the candidate regulatory factor for the target RNA molecule changes after the effective single nucleotide polymorphism site and / or the effective degenerate base change that can affect the target intron retention event in the cis-regulatory element region of the target intron retention event that interacts with the candidate regulatory factor; if so, determining whether the effective single nucleotide polymorphism site and / or the effective degenerate base change can affect the binding of the target RNA molecule with the candidate regulatory factor, thereby achieving directional control of the target intron retention event.

8. The method according to claim 1, characterized in that The method further comprises S109 verifying whether the level of the target intron retention event in the cell genome reaches the expected directional control.

9. The method according to claim 1, characterized in that The method between step S105 and step S106 also includes: screening the RNA binding proteins among the potential RNA binding proteins that have a strong correlation with the intron retention level of the whole transcriptome as the candidate regulatory factors.

10. The method according to claim 1, characterized in that The directional control includes up-regulation or down-regulation.

Images