Multi-target RNA targeting knockdown system and application

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a multi-target RNA targeting knockdown system and application. The present application constructs a universal multi-target RNA targeting knockdown system based on a CRISPR-CasRx (Cas13d) system, in which two crRNAs are used as the minimum unit, multiple crRNAs are integrated into a single plasmid through a GoldenGate cloning method, and the single plasmid can express multiple crRNAs, so as to realize multi-target coverage of a target sequence. In addition, the crRNA can target the target gene itself or a fluorescent marker sequence such as a GFP sequence. When targeting the fluorescent marker sequence, different crRNAs do not need to be redesigned and screened for different target genes, but the gene can be labeled with GFP, which has good universality, avoids the cumbersome process of designing and screening crRNAs, and has high knockdown efficiency.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a multi-target RNA knockdown system and its applications. Background Technology

[0002] Gene expression regulation typically involves direct manipulation of genes, but this direct manipulation is often irreversible and can lead to cell death. Therefore, intervention at the transcriptional level with RNA provides a new strategy for the study of related lethal genes.

[0003] RNA interference, first implemented in mice by M.V. Lemos et al. in 1978, has become the most commonly used technique. However, RNA interference has strong off-target effects and can even cause gene misexpression. The CRISPR-Cas system, composed of regularly clustered short palindromic repeats and related proteins, was initially discovered in prokaryotes as an immune system to defend against foreign viruses and other exogenous DNA. Its precision has led to its widespread use. The system first recognizes invading DNA fragments and inserts them into CRISPR sequences—regularly clustered short palindromic repeats—forming new spacer sequences. When the host cell recognizes the same invading DNA fragment again, the CRISPR sequence begins transcription to produce a pre-crRNA transcript, which is then cleaved to produce mature CRISPR RNA (crRNA). The mature crRNA contains a fragment that matches the invading DNA fragment, guiding effector complexes such as Cas proteins to cleave the invading DNA fragment, thus protecting itself from invasion.

[0004] With the advent of the CRISPR system, CRISPR-based RNA targeting systems, such as the CRISPR-Cas9 system, began to be applied. Subsequently, researchers discovered the CRISPR-Cas13 system, which is more suitable for targeting RNA; for example, Xinyun Jing et al. used Cas13a for RNA knockdown. These CRISPR-based RNA targeting knockdown systems have improved efficiency and specificity. Their knockdown efficiency and specificity mainly depend on the design of the crRNA, but different genes require screening for suitable crRNAs, a cumbersome process. Therefore, constructing a universal multi-target RNA targeting knockdown or deletion system is of great significance. Summary of the Invention

[0005] In view of this, the present invention provides a multi-target RNA knockdown system and its application. This system has good versatility, is suitable for knocking down most genes at the transcriptional level, avoids the cumbersome process of screening crRNA, and is simple and easy to operate.

[0006] This invention provides the application of crRNA targeting fluorescent genes in constructing a multi-target RNA knockdown system. The crRNA targets a fluorescent tag (such as GFP) sequence on the target gene, ensuring that the target gene transcribed along with it has the same knockdown effect at the RNA level. In some specific embodiments, the fluorescent tag gene is the GFP gene, and the crRNA targets at least one nucleic acid fragment from the GFP gene at 468-487 bp, 502-521 bp, 524-543 bp, 549-568 bp, 572-591 bp, and 602-621 bp. The knockdown effect has been verified in subsequent specific embodiments of this invention.

[0007] This invention provides a crRNA targeting the GFP gene, wherein the crRNA targets at least one nucleic acid fragment selected from the following sequences: 468-487 bp, 502-521 bp, 524-543 bp, 549-568 bp, 572-591 bp, and 602-621 bp of the GFP gene. The full length of the GFP gene is shown in SEQ ID NO: 1. The crRNA targeting the GFP gene contains 1 to 6 crRNA fragments, each crRNA fragment including a target recognition sequence and a CasRx (Cas13d) recognition sequence. The targeted recognition sequences specifically identify fragments of the GFP gene at 468-487 bp (sequence shown in SEQ ID NO: 2), 502-521 bp (sequence shown in SEQ ID NO: 3), 524-543 bp (sequence shown in SEQ ID NO: 4), 549-568 bp (sequence shown in SEQ ID NO: 5), 572-591 bp (sequence shown in SEQ ID NO: 6), and 602-621 bp (sequence shown in SEQ ID NO: 7), and are reverse complementary. This invention does not impose any special restrictions on the specific crRNA sequence; conventional design based on the above fragments and the crRNA design principles in this field is sufficient. In some specific embodiments, six crRNA fragments at 468-487 bp, 502-521 bp, 524-543 bp, 549-568 bp, 572-591 bp, and 602-621 bp are targeted, with their nucleic acid sequences shown in SEQ ID NO: 8-13.

[0008] The full-length sequence of the GFP gene is as follows:

[0009] atgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcctgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataa(SEQ ID NO:1).

[0010] The sequence of the fragment at positions 468 - 487 bp is: acaaaagaatggaatcaaag (SEQ ID NO: 2).

[0011] The sequence of the fragment at positions 502 - 521 bp is: agacacaacattgaagatgg (SEQ ID NO: 3).

[0012] The sequence of the fragment at positions 524 - 543 bp is: gcgttcaactagcagaccat (SEQ ID NO: 4).

[0013] The sequence of the fragment from 549 to 568 bp is: acaaaatactccaattggcg (SEQ ID NO: 5).

[0014] The sequence of the fragment from 572 to 591 bp is: gccctgtccttttaccagac (SEQ ID NO: 6).

[0015] The sequence of the segment from 602 to 621 bp is: tgtccacacaatctgccctt (SEQ ID NO: 7).

[0016] The sequence of crRNA targeting GFP gene 468-487bp is as follows:

[0017] agcacgctgatgagtccgtgaggacgaaacgagtaagctcgtccgtgctcactagtgcgaatttgcactagtct aaaacttattctttgattccattcttttgtggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgcttcg gcatggcgaatgggac(seq id no: 8).

[0018] The sequence of crRNA targeting 502-521 bp of the GFP gene is as follows:

[0019] agcacgctgatgagtccgtgaggacgaaacgagtaagctcgtccgtgctcactagtgcgaatttgcactagtct aaaacttattccatcttcaatgttgtgtctggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgctt cggcatggcgaatgggac(seq id no: 9).

[0020] The sequence of crRNA targeting 524-543 bp of the GFP gene is as follows:

[0021] agcacgctgatgagtccgtgaggacgaaacgagtaagctcgtccgtgctcactagtgcgaatttgcactagtct aaaacttattatggtctgctagttgaacgcggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgct tcggcatggcgaatgggac (SEQ ID NO: 10).

[0022] The sequence of the crRNA targeting the 549 - 568 bp of the GFP gene is:

[0023] agcacgctgatgagtccgtgaggacgaaacgagtaagctcgtccgtgctcactagtgcgaatttgcactagtct aaaacttattcgccaattggagtattttgtggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgctt cggcatggcgaatgggac (SEQ ID NO: 11).

[0024] The sequence of the crRNA targeting the 572 - 591 bp of the GFP gene is:

[0025] agcacgctgatgagtccgtgaggacgaaacgagtaagctcgtccgtgctcactagtgcgaatttgcactagtct aaaacttattgtctggtaaaaggacagggcggccggcatggtcccagcctcctcgctggcgccggctgggcaacatg cttcggcatggcgaatgggac (SEQ ID NO: 12).

[0026] The sequence of the crRNA targeting the 602 - 621 bp of the GFP gene is:

[0027] agcacgctgatgagtccgtgaggacgaaacgagtaagctcgtccgtgctcactagtgcgaatttgcactagtct aaaacttattaagggcagattgtgtggacaggccggcatggtcccagcctcctcgctggcgccggctgggcaacatg cttcggcatggcgaatgggac (SEQ ID NO: 13).

[0028] In the crRNA above, the underlined bases are inversely complementary to the corresponding fragments of the GFP gene, thus targeting and recognizing the GFP gene fragments.

[0029] This invention provides a CRISPR / CasRx(Cas13d) complex comprising the CasRx(Cas13d) protein and the crRNA described in this invention.

[0030] This invention also provides a multi-target RNA knockdown system, comprising:

[0031] Plasmid 1 expressing the CasRx(Cas13d) protein in the CRISPR / CasRx(Cas13d) complex, and plasmid 2 targeting the gfp gene or the crRNA of the target gene itself;

[0032] The crRNA targeting GFP or the target gene includes at least two crRNA fragments targeting the GFP gene or the target gene.

[0033] The crRNA targeting GFP or the target gene is integrated into the backbone vector using the Golden Gate cloning method.

[0034] Specifically, plasmid 1 comprises, in sequence: a promoter, a CasRx (Cas13d) coding sequence, and a terminator;

[0035] The plasmid 2 includes, in sequence: a promoter, a coding sequence for a crRNA targeting the GFP gene or the target gene, and a terminator;

[0036] See Figure 1 The crRNA coding sequence targeting GFP or the target gene comprises n coding units, where n is an integer ≥1, and each coding unit contains coding sequences of two gRNA fragments; the gRNA coding sequence targeting GFP or the target gene is obtained by integrating n coding units using the Golden Gate cloning method, through which a single plasmid can generate countless crRNAs.

[0037] In this invention, there are no special restrictions on the specific types of promoters and terminators in plasmids 1 and 2; any type commonly used in the art is acceptable. The promoter can be any one of Pnmt1, Pase1, or Pase1. In some specific embodiments, in plasmid 2, the promoter controlling the expression of crRNA targeting the target gene or GFP gene is Pnmt1, mainly used for subsequent controlled RNA knockdown experiments; the promoter controlling CasRx (Cas13d) expression is Pcam1. The terminator may include, but is not limited to, nmt1terminate.

[0038] In this invention, each crRNA fragment has an HHR ribozyme and an HDVR ribozyme attached to its 5' and 3' ends, respectively, ensuring self-cleavage to generate multiple clean crRNAs. As the plasmid is transcribed to produce primary transcripts, the HHR and HDVR ribozymes at the 5' and 3' ends of the gRNA precisely cleave the primary transcripts, generating multiple mature crRNAs to achieve multi-target targeting of the target sequence and further improve efficiency.

[0039] This invention also provides a method for multi-target RNA targeted knockdown, wherein plasmid 1 and plasmid 2 from the multi-target RNA targeted knockdown system are co-transformed into a host, wherein the target gene to be knocked out or knocked down in the host is labeled with a GFP sequence. Plasmid 2 contains the coding sequence of crRNA targeting the GFP gene or the target gene, and the crRNA is designed to target the target gene or GFP sequence.

[0040] This invention constructs a universal multi-target gene knockdown or knockout system based on the CRISPR-CasRx system. This system uses two crRNAs as the smallest unit, and through Golden Gate cloning, the crRNAs are integrated into a single plasmid. A single plasmid can express multiple crRNAs, thereby achieving multi-target coverage of the target sequence. Furthermore, when the crRNA targets a GFP sequence rather than the target gene itself, there is no need to redesign and screen different crRNAs for different target genes; instead, the gene can be labeled using GFP. This provides excellent versatility, avoiding the tedious process of designing and screening crRNAs, and also achieving high knockdown efficiency. Attached Figure Description

[0041] Figure 1 This diagram illustrates the structure of the smallest crRNA unit and the process of integrating the crRNA coding sequence into a plasmid using Golden Gate cloning to generate crRNA.

[0042] Figure 2 The plasmid maps showing the expression of CasRx (Cas13d) and crRNA are shown respectively.

[0043] Figure 3 A schematic diagram showing the specific sequence of the GFP gene targeted by crRNA (GFP-UC4);

[0044] Figure 4 The live-cell imaging results of Example 2 are shown;

[0045] Figure 5 The results of the average fluorescence intensity measurement of Noc4-GFP in Example 2 are shown.

[0046] Figure 6 The results of real-time quantitative PCR (qPCR) of Noc4-GFP in Example 2 are shown.

[0047] Figure 7 This shows the Western spectral imaging results of the Noc4-GFP protein in Example 2;

[0048] Figure 8 This shows the quantitative statistical results of Noc4-GFP protein expression in Example 2;

[0049] Figure 9 The live-cell imaging results of Example 3 are shown;

[0050] Figure 10 Western spectroscopy results of Bub1-GFP protein in Example 3 are shown;

[0051] Figure 11 The results of real-time quantitative PCR (qPCR) for Bub1 in Example 3 are shown.

[0052] Figure 12 Example 3 illustrates the drop plate experiment used to detect the sensitivity of each bacterial strain to the drug MBC;

[0053] Figure 13 The results of real-time quantitative PCR (qPCR) for emr1 in Example 4 are shown.

[0054] Figure 14 Western spectroscopy results of Emr1-GFP protein in Example 4 are shown.

[0055] Figure 15 The live-cell imaging results of Example 4 are shown;

[0056] Figure 16 The results of mitochondrial morphology analysis in Example 4 are shown. Detailed Implementation

[0057] This invention provides a multi-target RNA knockdown system and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0058] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0059] The effectiveness and specificity of RNA knockdown based on the CRISPR-Cas system mainly depend on the design of the crRNA, and specific crRNAs usually need to be designed and screened for different genes. In order to avoid the cumbersome process of crRNA screening and to further improve the knockdown efficiency, this invention constructs a multi-target RNA targeted knockdown system based on CRISPR-CasRx (Cas13d) and successfully screens a universal crRNA.

[0060] First, to ensure a high success rate and improve knockdown efficiency, the crRNA in this invention is a multi-target sequence (a unique feature), meaning a single plasmid can produce multiple crRNAs. The smallest unit is a combination of two crRNAs, each containing a 20bp direct target sequence. Simultaneously, an HHR ribozyme is added to the 5' end and an HDVR ribozyme to the 3' end of each crRNA, ensuring precise cleavage of the 5' and 3' ends. This design also allows for multiple promoter options; the adjustable nmt1 promoter is used in this invention, facilitating subsequent regulation of RNA knockdown of the target gene. We synthesize multiple smallest units containing combinations of two crRNAs and integrate them into a single plasmid using the Golden Gate cloning method. This method allows for the generation of numerous crRNAs from a single plasmid, meaning that for any gene, we can achieve multi-target full coverage of the gene by crRNA.

[0061] To avoid the tedious process of screening crRNAs, this invention constructs a universal crRNA that targets the fluorescent tag (such as GFP) sequence of the target gene, ensuring that the target gene transcribed along with it has the same knockdown effect at the RNA level. This effect has been verified in subsequent specific embodiments.

[0062] Fission yeast, as a classic model organism, is widely used to study various highly conserved life processes due to its simple structure and clear genetic background. This invention mainly utilizes fission yeast as a model for verification.

[0063] The present invention will be further illustrated below with reference to the embodiments:

[0064] Example 1

[0065] according to Figure 2 The diagram shown represents the construction of plasmid 1 expressing the CasRx(Cas13d) protein, whose expression is primarily controlled by the cam1 promoter.

[0066] Design multiple crRNAs targeting the target gene or GFP gene, using two crRNAs as the smallest unit (e.g., Figure 1 As shown), the company synthesized multiple crRNA minimal units, and then integrated the synthesized multiple crRNAs into a single plasmid (plasmid 2) using the Golden Gate cloning method. This plasmid is controlled by a regulated nmt1 promoter (see...). Figure 2 The number of crRNAs is determined by the size of the target gene, and theoretically, countless crRNAs can be produced. As the plasmid is transcribed to produce primary transcripts, the HHR ribozymes and HDVR ribozymes at the 5' and 3' ends of the gRNA precisely cleave the primary transcripts to produce multiple mature crRNAs, thereby achieving multi-target targeting of the target sequence and further improving efficiency.

[0067] Example 2

[0068] In this embodiment, crRNAs were designed targeting the GFP gene. The crRNAs are sequences that target a specific fluorescent marker protein, GFP, which successfully marks the target gene. Five crRNAs were designed for GFP: GFP-UC1, GFP-UC2, GFP-UC3, GFP-UC4, and GFP-UC5. Each GFP-UC5 contains six crRNAs, targeting the following sequences respectively: GFP-UC1 targets 1-160 bp, GFP-UC2 targets 161-320 bp, GFP-UC3 targets 321-465 bp, GFP-UC4 targets 468-621 bp, and GFP-UC5 targets 624-710 bp.

[0069] GFP-UC4 contains six crRNA fragments that target GFP sequences from 468 bp to 621 bp. The six gRNAs (sequences shown in SEQ ID NO: 8-13) target GFP gene fragments at 468-487 bp, 502-521 bp, 524-543 bp, 549-568 bp, 572-591 bp, and 602-621 bp, respectively. Figure 3 As shown, although crRNA (UC4) targets the GFP sequence, it has the same knockdown effect on the RNA level of the target gene.

[0070] Plasmid 1, expressing the CasRx (Cas13d) protein, and plasmid 2, expressing crRNA (GFP-UC1, GFP-UC2, GFP-UC3, GFP-UC4, GFP-UC5), were constructed according to the method in Example 1. Plasmid 1 and plasmid 2 were transformed into the same yeast strain using a yeast transformation method. Subsequently, the desired target gene was selected, such as the first noc4 gene, for detection. In the yeast strains transformed with both plasmids, the noc4 gene was endogenously labeled with GFP by homologous recombination PCR to form Noc4-GFP, resulting in five strains that simultaneously expressed CasRx (Cas13d) and GFP-UC (GFP-UC1, GFP-UC2, GFP-UC3, GFP-UC4, GFP-UC5, respectively).

[0071] The knockout effect of the above five strains was verified. Among them, the control WT strain was a yeast strain that had the GFP gene labeled with Noc4 and was transformed with plasmid 1 expressing CasRx(Cas13d) protein but not plasmid 2.

[0072] First, high-resolution imaging results showed that, compared to other crRNAs, the fluorescence of Noc4-GFP was significantly weakened in strains expressing GFP-UC4. Figure 4 Further experiments, including three replicates and measurements of the fluorescence intensity of Noc4-GFP, revealed that the fluorescence of Noc4-GFP was significantly weakened in strains expressing the crRNA shown by GFP-UC4 compared to strains transformed with other crRNAs and WT strains. Figure 5 ).

[0073] Next, we conducted tests at the transcriptional level, using only UC1 and UC4 as controls. qPCR results showed that the expression level of the noc4 gene was significantly decreased at the transcriptional level in strains expressing UC4, while no significant change was observed in strains expressing UC1, consistent with the imaging results (see [link to qPCR results]). Figure 6 ).

[0074] We detected the protein expression level of Noc4-GFP using Western blotting, and the statistical results were consistent with the imaging and qPCR results described above: compared with strains expressing other crRNAs, the expression level of Noc4-GFP was significantly reduced in strains expressing UC4 (see...). Figures 7-8 Therefore, we believe that UC4 is the most suitable crRNA for the GFP sequence.

[0075] In summary, the results above indicate that in strains that simultaneously express CasRx(Cas13d) and UC4, CasRx(Cas13d) targets and cleaves the transcript of the GFP sequence under the action of the crRNA shown by GFP-UC4, and knocks down noc4 at the transcriptional level, resulting in a significant reduction in protein expression and a high knockdown efficiency.

[0076] Example 3

[0077] The knockdown efficiency of the method described in this invention was tested by selecting the bub1 gene, which expresses the spindle checkpoint kinase protein, as the second gene. The bub1 gene was directly endogenously labeled with the GFP sequence using the strain expressing CasRx (Cas13d) and UC4 constructed in Example 1.

[0078] Live-cell imaging results showed that in strains simultaneously expressing CasRx(Cas13d) and UC4, the fluorescence intensity of Bub1-GFP was significantly lower than that of WT strains expressing only CasRx(Cas13d). We also used dCasRx(dCas13d) without RNase activity as a control; the fluorescence intensity of Bub1-GFP did not change significantly in either strains expressing dCasRx(dCas13d) alone or simultaneously expressing dCasRx(dCas13d) and UC4 (see [link to image]). Figure 9 This indicates that Bub1-GFP expression is associated with CasRx(Cas13d) and UC4.

[0079] Next, the expression of Bub1 protein was detected by Western blotting. The results of Western blotting and statistical analysis were consistent with the imaging results: the expression level of Bub1-GFP was significantly decreased only in strains that simultaneously expressed CasRx (Cas13d) and UC4. Figure 10 ).

[0080] Similarly, we examined the transcriptional level of the bub1 gene. qPCR results showed that the transcriptional level of the bub1 gene was significantly decreased only in strains simultaneously expressing CasRx (Cas13d) and UC4. Figure 11 ).

[0081] Finally, we also examined whether the function of the Bub1 protein was affected. Bub1, as a spindle checkpoint protein, is sensitive to the drug Carbendazim (MBC). MBC, a commonly used bactericide, affects spindle formation during mitosis, thus impacting cell division. Therefore, when Bub1 is absent, the spindle checkpoint is impaired, limiting the cell's growth on MBC-added plates. Consequently, we conducted serial dilution growth experiments on plates containing different concentrations of MBC for strains simultaneously expressing CasRx (Cas13d) and UC4. The results showed that with increasing MBC concentration, the growth of strains simultaneously expressing CasRx (Cas13d) and UC4 was essentially consistent with that of the Bub1 knockout strain. Figure 12 ).

[0082] In summary, the results indicate that in strains that simultaneously express CasRx(Cas13d) and UC4, CasRx(Cas13d) targets and cleaves the transcript of the GFP sequence under the action of UC4, and knocks down Bub1 at the transcriptional level, resulting in a decrease in its RNA level, which in turn leads to a reduction in protein expression and affects its function.

[0083] Example 4

[0084] The third gene we selected was the endoplasmic reticulum and mitochondrial contact complex regulatory protein gene eMR1. Since the eMR1 gene does not have a GFP marker sequence, crRNAs were designed to target the eMR1 gene. Six crRNAs were designed to target segments of the eMR1 gene at 6-25 bp, 37-56 bp, 82-101 bp, 111-130 bp, 139-158 bp, and 163-182 bp, respectively. (See...) Figure 13 The construction method is the same as in Example 1, which achieves full coverage of the eMR1 sequence.

[0085] First, the transcriptional level of the emr1 gene was detected, using dCasRx (dCas13d) without RNase activity as a control. qPCR results showed that the RNA level of strains simultaneously expressing CasRx (Cas13d) and UC (emr1) was significantly reduced. Figure 13 This indicates that CasRx(Cas13d) successfully knocked down emr1 at the transcriptional level under the action of UC(emr1).

[0086] Next, we detected the protein expression level of Emr1 using Western blotting. The results were consistent with the qPCR results: the Emr1 protein expression level was significantly reduced in strains simultaneously expressing CasRx (Cas13d) and UC (Emr1). Figure 14 ).

[0087] Finally, we also examined the function of Emr1. Emr1 is a regulatory protein of the endoplasmic reticulum and mitochondrial contact complex; its absence leads to mitochondrial fragmentation. Therefore, we used Mito Tracker to stain and observe intracellular mitochondria. We found that the mitochondrial phenotype of strains simultaneously expressing CasRx (Cas13d) and UC (emr1) was similar to that of the emr1 knockout strains, both exhibiting significant fragmentation. Statistical analysis also showed that in strains simultaneously expressing CasRx (Cas13d) and UC (emr1), the emr1 transcription level was significantly decreased, leading to reduced Emr1 protein expression, successfully achieving the goal of gene knockout. Figures 15-16 ).

[0088] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of crRNA targeting the GFP gene and its encoded nucleic acid in constructing a multi-target RNA knockdown system; The crRNA targeting the GFP gene consists of a GFP gene targeting recognition sequence and a CasRx recognition sequence, wherein the targeting recognition sequence targets six nucleic acid fragments as shown in SEQ ID NO: 2-7; The target gene for targeted knockdown is a GFP-tagged gene, and the GFP gene and the target gene are located in the same transcript.

2. A multi-target RNA knockdown system, characterized in that, include: Plasmid 2, plasmid 1 expressing CasRx protein, and the host; The plasmid 2 contains DNA encoding crRNA; the crRNA is the crRNA targeting the GFP gene as described in claim 1. In plasmid 2, the encoding DNA is integrated into the backbone vector via a Golden Gate; The target gene to be knocked out or knocked down in the host is marked with the GFP gene, and the target gene and the GFP gene are located in the same transcript.

3. The multi-target RNA knockdown system according to claim 2, characterized in that, The plasmid 1 comprises, in sequence, a promoter, a CasRx coding sequence, and a terminator; The plasmid 2 comprises, in sequence: a promoter, a DNA encoding crRNA targeting the GFP gene, and a terminator.

4. The application of the multi-target RNA knockdown system according to claim 2 or 3 in RNA-targeted knockdown of target genes.

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