Patent Publication Number: US-2021189386-A1

Title: Nucleic acid construct, medicinal composition, anticancer agent, antiviral agent and antibacterial agent

Description:
TECHNICAL FIELD 
     The present invention relates to a nucleic acid construct, a pharmaceutical composition, an anticancer agent, an antiviral agent, and an antibacterial agent. 
     BACKGROUND ART 
     For genome editing, many application examples are known as CRISPR/Cas9 systems. Genome editing is performed by simultaneously delivering CRISPR/Cas9 enzymes or an expression construct encoding CRISPR/Cas9 to target cells, together with guide nucleic acids. The application of genome editing in therapy has drawbacks in terms of serious side effects (Non-patent Literature (NPL) 1). 
     In addition to genome editing, RNA editing is also known. NPL 2 and NPL 3 disclose C2C2/Cas13 as an RNA editing enzyme. 
     Regulation of mRNA expression in cells has been attempted in cancer therapy using siRNA and shRNA since the early 2000s (NPL 4 and NPL 5). Although this technology had a significant impact on the regulation of protein gene expression in basic study, no effective use for therapy or diagnosis was found in clinical settings including cancer therapy. This is because both siRNA and shRNA molecules consist only of nucleic acid (RNA), the selectivity for target genes was low, and versatility was insufficient in affecting therapeutic targets. 
     Antiviral agents and antibacterial agents have been developed for infectious diseases; however, the use of these agents causes a problem in terms of resistance. 
     CITATION LIST 
     Non-Patent Literature 
     
         
         NPL 1: Kellie A Schaefer, Wen-Hsuan Wu, Diana F Colgan, Stephen H Tsang, Alexander G Bassuk, VinitB Mahajan. “Unexpected mutations after CRISPR-Cas9 editing in vivo.”  Nature Methods.  2017, 14, 547-548 
         NPL 2: Wright A V, Nunez J K, Doudna J A.  Cell.  2016 Jan. 14; 164 (1-2): 29-44 
         NPL 3: Gootenberg J S, Abudayyeh O O, Kellner M J, Joung J, Collins J J, Zhang F. “Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 .” Science.  2018 Feb. 15. 
         NPL 4: Paddison P J, Caudy A A, Bernstein E, Hannon G J, Conklin D S.  Genes  &amp;  Development  (2002). 16 (8): 948-58. 
         NPL 5: Hamilton A, Baulcombe D (1999), “A species of small antisense RNA in posttranscriptional gene silencing in plants.”  Science.  286 (5441): 950-2. 
       
    
     SUMMARY OF INVENTION 
     Technical Problem 
     An object of the present invention is to provide therapeutic techniques for cancer and infectious diseases caused by viruses or bacteria. 
     Solution to Problem 
     The present invention provides the following nucleic acid construct, pharmaceutical composition, anticancer agent, antiviral agent, and antibacterial agent. 
     Item 1. 
     A nucleic acid construct comprising at least one guide RNA portion that binds to one or more target RNAs and an RNA-cleaving Cas protein expression portion, wherein the cne or more target RNAs are derived from a mutation in a vertebrate cell, a virus, or a bacterium. 
     Item 2. 
     The nucleic acid construct according to Item 1, wherein the at least one guide RNA portion and the RNA-cleaving Cas protein expression portion are present in a single nucleic acid sequence. 
     Item 3. 
     The nucleic acid construct according to Item 1, comprising two or more nucleic acids, 
     wherein the at least one guide RNA portion and the RNA-cleaving Cas protein expression portion are present in separate nucleic acid sequences. 
     Item 4. 
     The nucleic acid construct according to any one of Items 1 to 3, which is an RNA construct or a DNA construct. 
     Item 5. 
     The nucleic acid construct according to any one of Items 1 to 4, wherein an RNA-cleaving Cas protein is a Cas13 family protein. 
     Item 6. 
     The nucleic acid construct according to any one of Items 1 to 5, wherein an RNA-cleaving Cas protein is C2C2/Cas13a. 
     Item 7. 
     The nucleic acid construct according to any one of Items 1 to 6, wherein at least one guide RNA targets RNA that corresponds to a mutation in a vertebrate cell. 
     Item 8. 
     The nucleic acid construct according to any one of Items 1 to 7, wherein 
     the mutation in a vertebrate cell is a translocation, and 
     at least one guide RNA targets RNA that corresponds to a gene of the translocation. 
     Item 9. 
     The nucleic acid construct according to any one of Items 1 to 6, wherein the virus is one member selected from the group consisting of an influenza virus, an HIV virus, a herpesvirus, an Ebola virus, an avian influenza virus, a foot-and-mouth disease virus, a SARS coronavirus, a MERS coronavirus, a papillomavirus, a hepatitis virus (hepatitis virus A, B, and C), a measles virus, a rubella virus, a mumps virus, a rotavirus, an RS virus, a norovirus, a herpes zoster virus, a poliovirus, a dengue virus, and an adult T-cell leukemia virus. 
     Item 10. 
     A pharmaceutical composition, comprising the nucleic acid construct of any one of Items 1 to 8 as an active ingredient. 
     Item 11. 
     An anticancer agent, comprising the nucleic acid construct of any one of Items 1 to 8 as an active ingredient. 
     Item 12. 
     An antiviral agent, comprising the nucleic acid construct of any one of Items 1 to 8 as an active ingredient. 
     Item 13. 
     An antibacterial agent, comprising the nucleic acid construct of any one of Items 1 to 8 as an active ingredient. 
     Advantageous Effects of Invention 
     The present invention uses an RNA gene modification technique and is superior to known art in terms of the degree of freedom in selecting a target gene and specificity to the target gene. 
     The nucleic acid construct according to the present invention indiscriminately reduces the expression of mRNA in cancer cells, virally infected cells, or in bacteria, and does not substantially act on normal cells. Thus, the nucleic acid construct reduces side effects and exhibits a more potent antitumor effect, antiviral effect, or antimicrobial effect than known art. Additionally, the nucleic acid construct can be used in combination with conventional therapeutic methods such as anticancer agents, antimicrobial drugs, or antiviral drugs. 
     The nucleic acid construct according to the present invention is a transitory effect development mechanism and involves no genome invasion, thus exhibiting less invasion to normal cells than known art. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1 : Changes in the survival rate due to RNA cleavage specific to synovial sarcoma-specific synovial sarcoma X chromosome (SSX) fusion gene. For SSX, 5 types of guide RNAs were created using the reported fusion site of synovial sarcoma SYO-1 cells as the target, including a negative control (NC). The bold letters (C, G, C, C, and A, each respectively referred to as “SSX1” to “SSX5”) represent PAM sequences. SYO-1 represents cells at 40% confluency. Guide 1 (SSX-1, terminal: C), guide 2 (SSX-2, negative control (nc), terminal: G), guide 3 (SSX-3, terminal: C), guide 4 (SSX-4, terminal: C), and guide 5 (SSX-5, terminal: A). 
         FIG. 2 : Target gene for therapy 1: synovial sarcoma-specific translocation gene t(X;18) (p11.2;q11.2) as a target for therapy. The increase in the dead cell percentage was notable in Guide_SSX-3, SSX-4, and SSX-5. 
         FIG. 3 : Specific RNA cleavage by C2c2_Lsh: Experiment for confirming cleavage of translocation gene cDNA of brain tumor. 
         FIG. 4 : The results of northern blotting on the cleavage of brain tumor (epithelioma)-specific translocation gene C11orf95-RELA (11q13.1) cDNA. Lane 1 shows the electrophoresis results of a substance with a standard molecular weight. Lanes 2 to 10 show the results of guide RNAs 1 to 9 of  FIG. 3 . Lane NC shows the results of guide RNA 10nc of  FIG. 3 . 
         FIG. 5 : crRNA Design based on RNA structure (ssRNA vs dsRNA). 
         FIG. 6 : Diagram explaining a PA magnet system. 
         FIG. 7 : Diagram explaining a PA magnet system. Quantitation of gDNA binding to XIST. 
         FIG. 8 : Target RNA editing from C to U by a RESCUE (RNA Engineering by Substitution of Cytidine to Uridine Edits) system. 
         FIG. 9 : Reduction in AP production by inhibiting 3-secretase cleavage by the RESCUE system. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     The nucleic acid construct according to the present invention may be either DNA or RNA, and may contain both DNA and RNA. 
     The nucleic acid construct according to the present invention contains (1) at least one guide RNA portion that binds to one or more target RNAs and (2) an RNA-cleaving Cas protein expression portion. The guide RNA (gRNA) portion as used here refers to guide RNA itself when the nucleic acid is RNA. The guide RNA portion refers to DNA capable of expressing guide RNA in a cell into which the nucleic acid construct is introduced when the nucleic acid is DNA. The “guide RNA portion” includes both guide RNA itself and DNA capable of expressing guide RNA in meaning, and may be either one or both. The RNA-cleaving Cas protein expression portion refers to RNA capable of expressing an RNA-cleaving Cas protein (e.g., a portion that corresponds to mRNA containing the post-splicing coding region of an RNA-cleaving Cas protein) when the nucleic acid is RNA. The RNA-cleaving Cas protein expression portion refers to DNA capable of expressing an RNA-cleaving Cas protein (e.g., DNA that contains a promoter and a coding region of the RNA-cleaving Cas protein (introns may be contained)) when the nucleic acid is DNA. The RNA-cleaving Cas protein expression portion may be composed of one portion of DNA or RNA encoding an RNA-cleaving Cas protein; 
     the DNA or RNA encoding an RNA-cleaving Cas protein may be divided into two or more portions such that their expression products collaborate intracellularly to exhibit RNA-cleaving activity (e.g., the system illustrated in  FIG. 7 ). 
     In the present specification, “a mutation in a vertebrate cell” refers to, for example, translocation, inversion, or deletion or insertion of multiple bases, and is a mutation associated with cancerization; RNA derived from a mutation is produced in cancer cells of a vertebrate, and not produced in normal cells. This mutation is present in post-splicing RNA, and does not include a mutation of an intron. The mutation in the present invention does not also include single-nucleotide polymorphisms (SNPs). In an embodiment of the present invention, RNA derived from a mutation is a target with which a guide RNA hybridizes. In another embodiment of the present invention, a target RNA is produced in a vertebrate cell infected with a target virus, and not produced in a cell uninfected with the target virus. Further, in another embodiment of the present invention, a target RNA is produced in a target bacterium, and not produced in a vertebrate cell including a human cell. 
     The guide RNA contains a sequence complementary to a target RNA and a PAM sequence. The guide RNA for use can be those used in genome editing. The number of bases of the sequence complementary to a target RNA is 20 to 30, preferably 22 to 30, more preferably 24 to 29, still more preferably 26 to 29, and most preferably 28. The PAM sequence depends on the origin and type of the organism from which the RNA-cleaving Cas protein is derived. The PAM sequence is, for example, more preferably A, but may be C or U. In the present invention using RNA editing, the PAM sequence is different from that of genome editing using Cas9, and the PAM sequence for use in RNA editing is short. 
     When the nucleic acid construct is RNA, the loop portion of the guide RNA may be formed beforehand. The guide RNA may be sgRNA in which crRNA (CRISPR RNA) and tract RNA (trans-activating RNA) are linked to each other, or may be guide RNA prepared by synthesizing crRNA and tract RNA as separate RNAs and hybridizing these RNAs to form a complex. 
     Examples of RNA-cleaving Cas proteins include Cas13 family proteins. The RNA-cleaving Cas protein is preferably, for example, Cas13a/C2C2, Cas13b, or Cas13c, and more preferably Cas13a/C2C2. “Cas13a” and “C2C2” both refer to the same RNA-cleaving Cas protein. 
     When the nucleic acid construct according to the present invention is DNA, the nucleic acid construct can be incorporated into a plasmid or a virus vector. When a nucleic acid construct in the form of DNA is used, intracellular transcription occurs to form a nucleic acid construct in the form of RNA, thereby forming an RNA-cleaving Cas protein and guide RNA in cytoplasm. When a single nucleic acid construct contains a nucleic acid (DNA or RNA) capable of expressing an RNA-cleaving Cas protein and guide RNA, the RNA-cleaving Cas protein and the guide RNA are preferably linked via a hammerhead ribozyme (HHR) sequence. When the nucleic acid construct according to the present invention contains multiple guide RNAs, adjacent guide RNAs are preferably linked via a hammerhead ribozyme sequence. The hammerhead ribozyme sequence is intracellularly cleaved by the self-cleavage function, and each guide RNA as well as RNA capable of expressing the RNA-cleaving Cas protein are produced in cells. 
     The hammerhead ribozyme (HHR) can cleave the RNA phosphodiester bond in a specific site, and a minimal hammerhead ribozyme that cleaves a trans-cleaving ribozyme is prepared by modifying natural HHR. The hammerhead ribozyme is used in reducing the expression of a target gene in vivo by RNA-mediated gene regulation. 
     The expression product of the nucleic acid construct according to the present invention contains one or more guide RNAs and an RNA-cleaving Cas protein. These guide RNAs and RNA-cleaving Cas protein act in cytoplasm of target vertebrate cells (cancer cells or virally infected cells) and/or of a bacterium. Specifically, when a target RNA that hybridizes with a guide RNA is present in cytoplasm, the guide RNA forms a hybrid with the target RNA, which leads the RNA-cleaving Cas protein to cleave and decompose not only the hybrid RNA but also RNAs present around in the cytoplasm, thereby killing the cells. For example, cells that have become cancerous by chromosome translocation contain RNA that corresponds to the translocation in the cells. Thus, introducing the nucleic acid construct of the present invention into the cancer cells kills the cancer cells, but does not affect normal cells because normal cells involve no translocation. Thus, introducing the nucleic acid construct of the present invention into the cells of a whole body of a vertebrate kills only cancer cells, while the nucleic acid construct decomposes in the cytoplasm, thus causing almost no side effects or toxicity to the normal cells. The case above has been explained with translocation as an example of mutations. However, the nucleic acid construct of the present invention can also selectively kill cancer cells caused by other mutations such as inversion, insertion, or deletion, as long as no target RNA is present in normal cells, and the target RNA is present only in cancer cells. RNAs to which Cas13 binds as a target are preferably composed of a short-chain sequence. Thus, in extracting and designing a crRNA sequence, optimal sequence extraction based on the prediction of a target RNA sequence and conformation is required in the present invention. 
     In a preferable embodiment according to the present invention, the RNA-cleaving Cas protein can be divided into two components. The functional activity of the RNA-cleaving Cas protein can be regulated externally by fusing each divided fragment with a PA magnet system for fusing protein fragments that form a dimer depending on specific wavelength stimulation (research paper for reference: Yuta Nihongaki et al., “Photoactivatable CRISPR-Cas9 for optogenetic genome editing,” Nature Biotechnology, Published online 15 Jun. 2015). The use of this mechanism enables the regulation of RNA-cleaving Cas protein functions in a site affected after the uptake of the divided fragments in vivo by using an adeno-associated virus (AAV) vector (the site irradiated with light from an optical source embedded in vivo). For example, β-secretase is known to cleave amyloid precursor protein (APP) to produce amyloid β-protein (Aβ). With the system in which mRNA of β-secretase is the target RNA, and the production of β-secretase is inhibited in the hippocampus only during irradiation with light from the light source embedded in the hippocampus, Alzheimer&#39;s disease can be treated, reducing side effects. This is because the inhibition of β-secretase can be regulated by light irradiation. 
     In a preferable embodiment according to the present invention, an RNA-cleaving Cas protein, which is the expression product of the nucleic acid construct of the present invention, can edit RNA by using an RNA-cleaving active mutant ( FIG. 8 ). Specifically, RNA-non-cleaving Cas13 (dCas13) is fused with the active site of an RNA-editing enzyme (APOBEC1), and the RNA-condensation-activating domain of A1CF protein, which is a coenzyme of APOBEC protein, is further addition-fused thereto. The target RNA hauled in by Cas13 and crRNA is presented by the A1CF domain to the APOBEC1 domain, and the RNA sequence in a specific region is edited (editing C→U in  FIG. 8 ). 
     In the present specification, vertebrates include humans, chimpanzees, monkeys, cows, horses, swine, sheep, rabbits, mice, rats, dogs, cats, chickens, wild ducks, and domesticated ducks, with humans, domestic animals (e.g., cows, swine, and chickens), and pets (e.g., dogs and cats) being preferable. 
     When the guide RNA according to the present invention forms a hybrid with RNA derived from a virus, but not with RNA of vertebrate cells, only vertebrate cells infected with the virus are killed, and non-virally infected cells are not affected. Thus, the nucleic acid construct according to the present invention can treat virus infection without causing serious side effects. 
     When the guide RNA according to the present invention forms a hybrid with RNA derived from a bacterium, but not with RNA of vertebrate cells, only the bacterium is killed, thereby causing almost no toxicity to the vertebrate. Thus, the nucleic acid construct according to the present invention can treat bacterial infection. Additionally, the nucleic acid construct according to the present invention is useful as an antimicrobial cleaning agent or disinfectant. The antimicrobial action of the nucleic acid construct according to the present invention is also useful for antibiotic drug-resistant bacteria such as multidrug-resistant bacteria because such bacteria do not develop resistance to the antimicrobial action of the nucleic acid construct. 
     The nucleic acid construct according to the present invention may contain in a single nucleic acid sequence (1) at least one guide RNA that binds to one or more target RNAs or DNA encoding the at least one guide RNA and (2) RNA encoding an RNA-cleaving Cas protein or DNA encoding the RNA (the RNA or DNA may be composed of a single sequence or of divided two or more sequences). The nucleic acid construct according to the present invention may also contain in separate nucleic acid sequences (1) the guide RNA or DNA encoding the guide RNA and (2) RNA encoding an RNA-cleaving Cas protein or DNA encoding the RNA. In this case, the nucleic acid construct according to the present invention is a composition containing multiple nucleic acid sequences. 
     Multiple types of guide RNAs or multiple guide RNAs of an identical type may be present. Additionally, several guide RNAs that target the same RNA but that are composed of different nucleic acid sequences may be present. For example, when there are multiple translocation sequences involved in cancerization, introducing multiple guide RNAs into vertebrate cells kills multiple types of cancer cells at the same time. When multiple target RNAs are produced by a single translocation site, multiple guide RNAs can kill cancer cells more reliably. 
     By containing multiple types of guide RNAs that correspond to the sequences unique to many different types of influenza viruses (e.g., influenza A virus and B virus), the nucleic acid construct according to the present invention can provide an antiviral agent effective against all types of influenza viruses that became epidemic in the past. Additionally, by containing multiple types of guide RNAs that correspond to the sequences unique to many types of bacteria (in particular, pathogenic bacteria), the nucleic acid construct according to the present invention can provide, for example, an antibacterial agent or disinfectant effective against many bacterial infectious diseases. A viral or bacterial infectious disease can be caused by only one type of virus or bacterium. However, a viral or bacterial infectious disease can also be caused by simultaneous infection with multiple types of viruses and/or bacteria in some cases. The use of guide RNAs that address multiple types of viruses and/or bacteria enables the treatment of viral or bacterial infectious diseases without strictly specifying the kind and type of viruses or bacteria. 
     Cancers treatable with the nucleic acid construct according to the present invention include cancers caused by gene mutation, such as synovial sarcoma, brain tumor, leukemia, malignant lymphoma, lung cancer, prostate cancer, and renal cell cancer. The nucleic acid construct according to the present invention can kill only cancer cells in which RNA involved in translocation is present. Cancerization involves gene mutation. Cancer cells in which RNA unique to gene mutation is produced can be killed all together with cells that have become cancerous by mutations other than translocation by the nucleic acid construct according to the present invention that contains at least one guide RNA that corresponds to the unique RNA. The anticancer agent according to the present invention can be used in both the primary focus and the metastatic focus, and can also be used in the prevention of recurrence after surgery. The anticancer agent according to the present invention can also be used in combination with at least one other anticancer agent. 
     The nucleic acid construct according to the present invention is also useful as a therapeutic agent for Alzheimer&#39;s disease by regulating the production of amyloid β-protein. 
     In a preferable embodiment according to the present invention, due to lack of the nuclear localization signal, the nucleic acid construct containing RNA according to the present invention does not move into a nucleus, making no direct action on the chromosomes, DNA, and genes. This is one reason why the nucleic acid construct has a low degree of side effects. 
     In a preferable embodiment according to the present invention, the nucleic acid construct of the present invention is introduced into vertebrate cells or bacteria, in particular, the cytoplasm. In the case of a vertebrate, the introducing agent for introducing a nucleic acid construct such as RNA or DNA into cells is not particularly limited; any known introducing agent is usable. For example, the introducing agent is a liposome, exosome, liposome-exosome hybrid, Sendai virus, or virus vector (e.g., an adenovirus vector), preferably an exosome, Sendai virus, or virus vector, and particularly preferably an exosome. An exosome is suitable for use in introducing a nucleic acid construct into cells (e.g., cancer cells and virally infected cells) because the nucleic acid construct can be easily introduced into cells by mixing an exome with the nucleic acid construct. The nucleic acid construct according to the present invention includes pharmaceutical compositions that contain the introducing agent described above and the nucleic acid construct. When a bacterium is targeted, the nucleic acid construct can be dissolved, dispersed, or suspended in a calcium-ion-containing medium in order to introduce the nucleic acid construct into the cells of the bacterium. Examples of such a medium include water, buffers, and water-miscible organic solvents, such as ethanol. 
     Examples of viruses targeted by the antiviral agent include influenza viruses (including influenza A virus and B virus), HIV virus, herpesvirus, Ebola virus, avian influenza virus, foot-and-mouth disease virus, SARS coronavirus, MERS coronavirus, papillomavirus, hepatitis viruses (hepatitis A virus, B virus, and C virus), measles virus, rubella virus, mumps virus, rotavirus, RS virus, norovirus, herpes zoster virus, poliovirus, dengue virus, Zika virus, and adult T-cell leukemia virus. 
     Examples of bacteria targeted by the antibacterial agent include  Shigella, Mycobacterium tuberculosis, cholera bacillus, serratia, vulnificus, aeromonad, pertussis, Brucella, Bartonella, Legionella pneumophila, Coxiella, gonococcal, campylobacter, Helicobacter pylori, Staphylococcus aureus, Streptococcus pyogenes , anthrax, gas gangrene,  Clostridium botulinum, Listeria monocytogenes, Corynebacterium diphtheriae, mycoplasma, Chlamydia pneumonia, pneumococcus, Clostridium tetani, Yersinia pestis , enterohemorrhagic  Escherichia coli  (e.g., 0157),  Vibrio parahaemolyticus, Salmonella, Clostridium welchii , hemolytic  Streptococcus, meningococcus , Proteobacteria,  Pseudomonas aeruginosa, Citrobacter, Acinetobacter, Enterobacter, Klebsiella, Clostridium , and  Trichophyton  fungi. 
     The nucleic acid construct according to the present invention used as a medical drug (e.g., anticancer agents, antiviral agents, and antibacterial agents) can be administered at a dose of about 1 ng to 1000 mg per day for an adult, once daily or in 2 to 4 divided doses daily. Examples of dosage forms of the medical drug include injectable drugs, tablets, capsules, inhalants, fluid medicines, drinkable preparations, suppositories, spray agents, plasters, ointments, and ophthalmic solutions. 
     EXAMPLES 
     The present invention will be described in more detail below based on Examples. 
     Example 1 
     (1) Plasmid Construction and Target Guide 
     The DNA sequence of C2C2 Lsh ( Leptotrichia shahii ) was amplified and fused with the pX458 plasmid using Hifi DNA Assembly (NEB). An Lsh-specific scaffold RNA sequence was inserted using Hifi DNA Assembly (NEB). Phosphorylated oligonucleotides encoding an sgRNA sequence were ligated to Bbs1-digested scaffold constructs to prepare sgRNA-targeting XistRNAs, C11orf95-RELA fusion RNAs, and SS18-SSX fusion RNAs. The platform thereof was named “pLMT” (pLMTXist plasmids). The 15 different pLMTXist plasmids each contained any of the 15 types of guide RNAs shown in Table 1. 
     Table 1 below shows the sequences of 5 different guide RNAs introduced into synovial sarcoma cells (SYO-1 containing a SS18-SSX fusion gene) (SSX-1, SSX-2, SSX-3, SSX-4, and SSX-5), and 10 different guide RNAs introduced into epithelioma cells (HEK293T into which a translocation gene C11orf95-RELA (11q13.1) associated with brain tumor was introduced).  FIG. 1  shows 5 different guide RNAs introduced into the synovial sarcoma cells, and the experimental conditions.  FIG. 3  shows 10 different guide RNAs introduced into the HEK293T. The sequences in Tables 1 to 4 show the genetic information of the DNA templates. 
     
       
         
           
               
               
               
             
               
                 TABLE  1   
               
               
                   
               
             
            
               
                   
                   
                 h.C11orf95RELA fusion 
               
               
                   
                 Epithelioma 
                  Guide RNA list 
               
               
                   
               
               
                   
                 Guide1 
                 GCCCTTGGGCGGGCAAGCTGGGACACCG 
               
               
                   
               
               
                   
                 Guide2nc 
                 TGGGCCCTTGGGCGGGCAAGCTGGGACA 
               
               
                   
               
               
                   
                 Guide3 
                 TTCTGGGCCCTTGGGCGGGCAAGCTGGG 
               
               
                   
               
               
                   
                 Guide4nc 
                 AGTTCTGGGCCCTTGGGCGGGCAAGCTG 
               
               
                   
               
               
                   
                 Guide5 
                 GGGAACAGTTCTGGGCCCTTGGGCGGGC 
               
               
                   
               
               
                   
                 Guide6 
                 AGGGGGAACAGTTCTGGGCCCTTGGGCG 
               
               
                   
               
               
                   
                 Guide7 
                 ATGAGGGGGAACAGTTCTGGGCCCTTGG 
               
               
                   
               
               
                   
                 Guide8 
                 AAGATGAGGGGGAACAGTTCTGGGCCCT 
               
               
                   
               
               
                   
                 Guide9 
                 GGGAAGATGAGGGGGAACAGTTCTGGGC 
               
               
                   
               
               
                   
                 Guide 10n.C. 
                 AACAGTTCTGGGCCCTTGGGCGGGCAAG 
               
               
                   
               
               
                   
                 Synovial 
                 SS18.SSX fusion Guide  
               
               
                   
                 sarcoma 
                 Guide RNA List 
               
               
                   
               
               
                   
                 Guide_1 
                 GCATGATCTGGTCATATCCATAAGGCCT 
               
               
                   
               
               
                   
                 Guide_2 (nC) 
                 TTGGGCATGATCTGGTCATATCCATAAG 
               
               
                   
               
               
                   
                 Guide_3 
                 GCTTCTTGGGCATGATCTGGTCATATCC 
               
               
                   
               
               
                   
                 Guide_4 
                 CTGGCTTCTTGGGCATGATCTGGTCATA 
               
               
                   
               
               
                   
                 Guide_5 
                 CCTCTGCTGGCTTCTTGGGCATGATCTG 
               
               
                   
               
            
           
         
       
     
     (2) Cell Culture 
     The HEK293T and SYO-1 synovial sarcoma cell lines were maintained in D-MEM (low-glucose) medium supplemented with 1% penicillin, streptomycin, and 10% fetal bovine serum (FBS). The obtained cells were cultured in a humidified atmosphere at 37° C. in 5% CO 2 . 
     The HEK293T was obtained by introducing a translocation gene C11orf95-RELA (11q13.1) associated with brain tumor. The SYO-1 contained a SS18-SSX fusion gene. 
     (3) Northern Blotting 
     The HEK293T cells at 30% confluency were transfected with the pLMT_Xist plasmids using ScreenFect A (Wako). The 10 different pLMT_Xist plasmids each contained any guide RNA selected from the “Epithelioma h.C11orf95RELA fusion Guide RNA list” in Table 1. After being cultured for 48 hours, the cells were harvested. The RNAs were precipitated using ISOGEN. Northern blotting was performed using a DIG Northern Starter kit (Roche). The membrane was hybridized with a DIG-labeled probe targeting XistRNA and C11-orf95-RELA in a hybridization buffer (7% SDS, 0.5 M Na-phosphate buffer (pH 7.2), 10 mM EDTA), and washed with a washing buffer (1% SDS, Na-phosphate buffer (pH 7.2), 10 mM EDTA).  FIG. 4  shows the results of northern blotting. 
     (4) Trypan Blue Assay 
     The SYO-1 cells at 30% confluency were transfected with the pLMT_Xist plasmids by using ScreenFect A (Wako). After being cultured for 48 hours, the cells were harvested. The cell suspension and a trypan blue solution, 0.4%, were mixed at 1:1. The live cells and dead cells were counted with a hemocytometer to determine the proportion of dead cells.  FIG. 2  shows the results. 
     Example 2 
     (1) Plasmid Construction and Target Guide 
     The DNA sequence of C2C2 Lsh ( Leptotrichia shahii ) was amplified and fused with the pX458 plasmid using Hifi DNA Assembly (NEB). An Lsh-specific scaffold RNA sequence was inserted using Hifi DNA Assembly (NEB). A phosphorylated oligonucleotide encoding an sgRNA sequence was ligated to Bbs1-digested scaffold constructs to prepare sgRNA-targeting XistRNAs and fusion RNAs. Tables 2 to 4 below show 71 different guide RNA sequences designed based on RNA conformation prediction and introduced into the HEK293T.  FIG. 5  shows the 71 introduced guide RNAs, the experimental conditions, and the results. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Guides targeting the ss Regions in loops in the XIST trascript 
               
               
                 (Total crRNAs = 37) 
               
            
           
           
               
               
               
               
            
               
                 SENSE 
                 ANTISENSE 
                 EXON 
                 TARGET SITE 
               
               
                   
               
               
                 AGACTAGGGGTTTGCTGGGAGCAGGGCT 
                 AGCCCTGCTCCCACAAACCCCTAGTCT 
                 1 
                 2816-2823 
               
               
                   
               
               
                 GGGGCTAGACTAGGGGTTTGCTGGGAGC 
                 GCTCCCAGCAAACCCCTAGTCTAGCCCC 
                 1 
                 2822-2830 
               
               
                   
               
               
                 GGGGGGTTAGGGGACTGGGGCTGGGGCA 
                 TGCCCCAGCCCCAGTCCCCTAACCCCCC 
                 1 
                 2877-2904 
               
               
                   
               
               
                 GGACTGGGGCTAGGGCTGGGGGGTTAG 
                 CTAACCCCCCAGCCCTAGCCCCAGTCCC 
                 1 
                 2895-2922 
               
               
                   
               
               
                 GCTGGGATTACAGGTGTGAGCCACCACA 
                 TGTGGTGGCTCACACCTGTAATCCCAGC 
                 1 
                 5242-5269 
               
               
                   
               
               
                 CCCAAAGTGCTGGGATTACAGGTGTGAG 
                 CTCACACCTGTAATCCCAGCACTTTGGG 
                 1 
                 5250-5277 
               
               
                   
               
               
                 AAGGGATCTTCCCACCTCAGCCTCCCAA 
                 TTGGGAGGCTGAGGTGGGAAGATCCCTT 
                 1 
                 5273-5300 
               
               
                   
               
               
                 CCTGGCAGTAAGGGATCTTCCCACCTCA 
                 TGAGGTGGGAAGATCCCTTACTGCCAGG 
                 1 
                 5282-5309 
               
               
                   
               
               
                 CTCAAACTCCTGGCAGTAAGGGATCTTC 
                 GAAGATCCCTTACTGCCAGGAGTTTGAG 
                 1 
                 5290-5317 
               
               
                   
               
               
                 TAATGTTGGCAAGGCTGGTCTCAAACTC 
                 GAGTTTGAGACCAGCCTGGCCAACATTA 
                 1 
                 5503-5556 
               
               
                   
               
               
                 TAAAGGATTATAAAATTTAGGTAGTTTT 
                 AAAACTACCTAAATTTTATAATCCTTTA 
                 1 
                 10818 10845 
               
               
                   
               
               
                 CCAGATGAAGAAATTAAAGGATTATAAA 
                 TTTATAATCCTTTAATTTCTTCATCTGG 
                 1 
                 10832-10859 
               
               
                   
               
               
                 CAGGTGCTCCAGATGAAGAAATTAAAGG 
                 CCTTTAATTTCTTCATCTGGAGCACCTG 
                 1 
                 10840-10867 
               
               
                   
               
               
                 AGGGGCAGGTGCTCCAGATGAAGAAATT 
                 AATTTCTTCATCGGAGCACCTGCCCCT 
                 1 
                 10845-10872 
               
               
                   
               
               
                 GAATAAGTAGGGGCAGGTGCTCCAGAT 
                 ATCTGGAGCACCTGCCCCTACTTATTTC 
                 1 
                 10854-10881 
               
               
                   
               
               
                 TTACTGCAATCTTCTTGAAATAAGTAGGG 
                 CCCTACTTATTTCAAAGAAGATTGCAGTAA 
                 1 
                 10869-10897 
               
               
                   
               
               
                 TTTACTGCAATCTTCTTGAAATAAAGTAGG 
                 CCTACTTATTTCAAGAAGATTGCAGTAAA 
                 1 
                 10870-10898 
               
               
                   
               
               
                 ATGTTCCCTCATTTAATCGTTTTACTGC 
                 GCAGTAAAACGATTAAATGAGGGAACAT 
                 1 
                 10891-10918 
               
               
                   
               
               
                 CTGCATATGTTCCCTCATTTAATCGTTT 
                 AAACGATTAAATGAGGGAACATATGCAG 
                 1 
                 10897-10924 
               
               
                   
               
               
                 TCCCTCATTTAATCGTTTTACTGCAATC 
                 GATTGCAGTAAAACGATTAAATGAGGGA 
                 1 
                 10887-10914 
               
               
                   
               
               
                 AAGGAGACATGACTACTAAGGACACATG 
                 CATGTGTCCTTAGTAGTCATGTCTCCTT 
                 2 
                 11397-11414 
               
               
                   
               
               
                 GACTACTAAGGACACATGCAGCGTGGTA 
                 TACCACGCTGCATGTGTCCTTAGTAGTC 
                 2 
                 11387-11414 
               
               
                   
               
               
                 ACTAAGGACACATGCAGCGTGGTATCTT 
                 AAGATACCACGCTGCATGTGTCCTTAGT 
                 6 
                 11383-11410 
               
               
                   
               
               
                 CCAATTGGCTCAAAAACTAAGAATGATT 
                 AAATCATTCTTAGTTTTTGAGCCAATTGG 
                 6 
                 14306-14333 
               
               
                   
               
               
                 CAAAAAACTAAGAATGATTTTGACCTTAT 
                 ATAAGGTCAAAAAATCATTCTTAGTTTTTG 
                 6 
                 14296-14323 
               
               
                   
               
               
                 AAGAATGATTTTGACCTTATAAAAACGT 
                 ACGTTTTTATAAGGTCAAAAAATCATTCTT 
                 6 
                 14288-14315 
               
               
                   
               
               
                 TTGACCTTATAAAAACGTTGTTTAAAAA 
                 TTTTTAAACAACGTTTTTATAAGGTCAA 
                 6 
                 14278-14305 
               
               
                   
               
               
                 GTTTAAAAAACAAATATGTAACAGAAAC 
                 GTTTCTGTTACATATTTGTTTTTTAAAC 
                 6 
                 14259-14286 
               
               
                   
               
               
                 ATGTAACAGAAACCATATGGCCCACAGT 
                 ACTGTGGGCCATATGGTTTCTGTTACAT 
                 6 
                 14244-14271 
               
               
                   
               
               
                 CTAAAGTATTTATGATTTGACCCCTTAC 
                 GTAAGGGGTCAAATCATAAATACTTTAG 
                 6 
                 14216-14243 
               
               
                   
               
               
                 TTGACCCCTTACAGAAAAACTGTGGACC 
                 GGTCCACAGTTTTTCTGTAAGGGGTCAAA 
                 6 
                 14200-14227 
               
               
                   
               
               
                 GAACAGCAGGCCAAATCCAATTGGCTCA 
                 TGAGCCAATTGGATTTGGCCTGCTGTTC 
                 6 
                 14248-14275 
               
               
                   
               
               
                 GGTAAGCTATGAACAGCAGGCCAAATCC 
                 GGATTTGGCTGCTGTTCATAGCTTACC 
                 6 
                 14332-14349 
               
               
                   
               
               
                 ACCTATTGGCACCCGAATATATTTGTAG 
                 CTACAAATATATTCGGGTGCCAATAGGT 
                 6 
                 17428-17455 
               
               
                   
               
               
                 GGCACCCGAATATATTTGTAGAATGAAT 
                 ATTCATTCTACAAATATATTCGGGTGCC 
                 6 
                 17421-17448 
               
               
                   
               
               
                 TATACCAAGTACCTATTGGCACCCGAAT 
                 ATTCGGGTGCCAATAGGTACTTGGTATA 
                 6 
                 17438-17465 
               
               
                   
               
               
                 GGGGCCAAAAAACCTTATACCAAGTACCT 
                 AGGTACTTGGTATAAGGTTTTTGGCCCC 
                 6 
                 17452-17479 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3  
               
             
            
               
                   
               
               
                 Guides targeting the ds Regions in stem 
               
               
                 in the XIST trascript (total crRNAs = 12) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 TARGET 
               
               
                   
                 SENSE 
                 ANTISENSE 
                 EXON 
                 SITE 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 AGCGGTAGGTACA 
                 TTGGTGGTGTGTGA 
                 1 
                 840- 
               
               
                   
                 CTCACACACCACC 
                 GTGTACCTACCGCT 
                   
                 867 
               
               
                   
                 AA 
                   
                   
                   
               
               
                   
               
               
                   
                 ATCCGCCATTTTGG 
                 CTTTGTTAGGTTGT 
                 1 
                 1240- 
               
               
                   
                 ACAACCTAACAAAG 
                 CCAAAATGGCGGAT 
                   
                 1267 
               
               
                   
               
               
                   
                 TGAATTCTACAAAT 
                 TATTAAGAGGCTTT 
                 1 
                 2357- 
               
               
                   
                 AAAGCCTCTTAATA 
                 TTTTGTAGAATTCA 
                   
                 2384 
               
               
                   
               
               
                   
                 GTGGCCAACACAGT 
                 ATCTTTTCTTGTGT 
                 1 
                 3300- 
               
               
                   
                 ACACAAGAAAAGAT 
                 ACTGTGTTGGCCAC 
                   
                 3327 
               
               
                   
               
               
                   
                 ACAAATACAATCAC 
                 GCCTCCCAATATGT 
                 1 
                 6010- 
               
               
                   
                 ACATATTGGGAGGC 
                 GTGATTGTATTTGT 
                   
                 6037 
               
               
                   
               
               
                   
                 CCAGACGATTATAA 
                 CATGTTGTGTGTGA 
                 1 
                 6290- 
               
               
                   
                 TCACACACAACATG 
                 TTATAATCGTCTGG 
                   
                 6317 
               
               
                   
               
               
                   
                 ACTGATGGCTGAA 
                 TGATTGTCCCATTT 
                 1 
                 10134- 
               
               
                   
                 AAATGGGACAATC 
                 TTTCAGCCCATCAG 
                   
                 10161 
               
               
                   
                 A 
                 T 
                   
                   
               
               
                   
               
               
                   
                 TTCTATCCACAGAC 
                 ACTGAGGGTGGTGG 
                 6 
                 10695- 
               
               
                   
                 CCACCACCCTCAGT 
                 GTCTGTGGATAGAA 
                   
                 10722 
               
               
                   
               
               
                   
                 CACTAGAAATCCCA 
                 AGGATTCTGGGGTT 
                 6 
                 13823- 
               
               
                   
                 AACCCCAGAATCCT 
                 TGGGATTTCTAGTG 
                   
                 12850 
               
               
                   
               
               
                   
                 CAAAATTACCAGAG 
                 ATTTGTGTTTGCTG 
                 6 
                 14659- 
               
               
                   
                 CAGCAAACACAAAT 
                 CTCTGGTAATTTTG 
                   
                 14686 
               
               
                   
               
               
                   
                 CGGAAAAGGTCAAA 
                 AGGCCTGGCTGGGC 
                 6 
                 18192- 
               
               
                   
                 GCCCAGCCAGGCCT 
                 TTTGACCTTTTCCG 
                   
                 18210 
               
               
                   
               
               
                   
                 TGACATTTATCTAT 
                 AAGTGGAGAAGGAA 
                 6 
                 18648- 
               
               
                   
                 TTCCTTCTCCACTT 
                 ATAGATAAATGTCA 
                   
                 18675 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Guides targeting the ss and ds regions 
               
               
                 in stem-loopjunction in the XIST  
               
               
                 transcript (Total crRNAs = 22) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 TARGET 
               
               
                   
                 SENSE 
                 ANTISENSE 
                 EXON 
                 SITE 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 GAGAGAAGCTGGGC 
                 AGTTCCTCAGTCCC 
                 1 
                 2929- 
               
               
                   
                 GGGACTGAGGAACT 
                 GCCCAGCTTCTCTC 
                   
                 2956 
               
               
                   
               
               
                   
                 TTTCGAGAGAAGCT 
                 CCTCAGTCCCGCCC 
                 1 
                 2933- 
               
               
                   
                 GGGCGGGACTGAGG 
                 AGCTTCTCTCGAAA 
                   
                 2960 
               
               
                   
               
               
                   
                 AGTGACTTTCGAGA 
                 TCCCGCCCAGCTTC 
                 1 
                 2969- 
               
               
                   
                 GAAGCTGGGCGGGA 
                 TCTCGAAAGTCACT 
                   
                 2999 
               
               
                   
               
               
                   
                 CAGGGCAATTGTCT 
                 AAAAAAAAAAAGTA 
                 1 
                 5339- 
               
               
                   
                 TACTTTTTTTTTTT 
                 AGACAATTGCCCTG 
                   
                 5366 
               
               
                   
               
               
                   
                 AAATTGTCTTACTT 
                 ATTAAAAAAAAAAA 
                 1 
                 5333- 
               
               
                   
                 TTTTTTTTTTTTAA 
                 AAAGTAAGACAATT 
                   
                 5360 
               
               
                   
                 T+0 
                   
                   
                   
               
               
                   
               
               
                   
                 TTACTTTTTTTTTT 
                 GGCCAACATTAAAA 
                 1 
                 5326- 
               
               
                   
                 TTTTAATGTTGGCC 
                 AAAAAAAAAAGTAA 
                   
                 5355 
               
               
                   
               
               
                   
                 ATATGCTTTTTAAA 
                 TATGCAGAGGTGCT 
                 1 
                 10918- 
               
               
                   
                 GCACCTCTGCATA 
                 TTTAAAAAGCATAT 
                   
                 10945 
               
               
                   
               
               
                   
                 AAAGGTGGCATATG 
                 GTGCTTTTAAAAAG 
                 1 
                 10927- 
               
               
                   
                 CTTTTTAAAAGCAC 
                 CATATGCCACCTTT 
                   
                 10954 
               
               
                   
               
               
                   
                 GTGGCATATGCTTT 
                 AGAGGTGCTTTTAA 
                 1 
                 10923- 
               
               
                   
                 TTAAAAAAGCACCT 
                 AAAAGCATATGCCA 
                   
                 10950 
               
               
                   
                 CT 
                 C 
                   
                   
               
               
                   
               
               
                   
                 GGCATATGCTTTTT 
                 GCAGAGGTGCTTTT 
                 1 
                 1092- 
               
               
                   
                 AAAAGCACCTCTGC 
                 AAAAAGCATATGCC 
                   
                 10948 
               
               
                   
               
               
                   
                 CTCACATGCTCAGA 
                 CTCCTCTTGGACAT 
                 2 
                 11428- 
               
               
                   
                 TGTCCAAGAGGAG 
                 TCTGAGCATGTGAG 
                   
                 11455 
               
               
                   
               
               
                   
                 GCTCAGAATGTCCA 
                 CCTTAGGCTCCTCT 
                 2 
                 11421- 
               
               
                   
                 AGAGGAGCCTAAGG 
                 TGGACATTCTGAGC 
                   
                 11448 
               
               
                   
               
               
                   
                 CAGAATGTCCAAGA 
                 TCTCCTTAGGCTCC 
                 2 
                 11418- 
               
               
                   
                 GGAGCCTAAGGAGA 
                 TCTTGGACATTCTG 
                   
                 11445 
               
               
                   
               
               
                   
                 GGTCTCACATGCTC 
                 CTCTTGGACATTCT 
                 2 
                 11431- 
               
               
                   
                 AGAATGTCCAAGAG 
                 GAGCATGTGAGACC 
                   
                 11456 
               
               
                   
               
               
                   
                 GAATAACAAATAAT 
                 CCACCCCCTGATGT 
                 6 
                 14356- 
               
               
                   
                 ACATCAGGGGGTGG 
                 ATTATTTTTTATTC 
                   
                 14385 
               
               
                   
               
               
                   
                 AATACATCAGGGGG 
                 TTCATAGCTTACCA 
                 6 
                 14347- 
               
               
                   
                 TGGTAAGCTATGAA 
                 CCCCCTGATGTATT 
                   
                 14374 
               
               
                   
               
               
                   
                 AAATAATACATCAG 
                 TAGCTTACCACCCC 
                 6 
                 14351- 
               
               
                   
                 GGGGTGGTAAGCTA 
                 CTGATGTATTATTT 
                   
                 14378 
               
               
                   
               
               
                   
                 AATAACAAATAATA 
                 ACCACCCCCTGATG 
                 6 
                 14357- 
               
               
                   
                 CATCAGGGGGTGGT 
                 TATTATTTGTTATT 
                   
                 14384 
               
               
                   
               
               
                   
                 GGAAGGCATGCATT 
                 AGACATGGGAAAAA 
                 6 
                 17482- 
               
               
                   
                 TTTTTCCCATGTCT 
                 AATGCATGCCTTCC 
                   
                 17509 
               
               
                   
               
               
                   
                 CTCTGGGAAGGCAT 
                 TGGAAAAAAATGCA 
                 6 
                 17487- 
               
               
                   
                 GCATTTTTTTCCCA 
                 TGCCTTCCCAGAG 
                   
                 17514 
               
               
                   
               
               
                   
                 GCATGCATTTTTTT 
                 CCCAGAGACATGGG 
                 6 
                 17477- 
               
               
                   
                 CCCATGTCTCTGGG 
                 AAAAAAATGCATGC 
                   
                 17507 
               
               
                   
               
               
                   
                 CATGCATTTTTTTC 
                 CCCCAGAGACATGG 
                 6 
                 17476- 
               
               
                   
                 CCATGTCTCTGGGG 
                 GAAAAAAATGCATG 
                   
                 17503 
               
               
                   
               
            
           
         
       
     
     Example 3 
     (1) Plasmid Construction and Target Guide 
     To create a PA magnet system, the DNA sequence of dead Lwa (also known as “dCas13a”; from  Leptotrichia wadei ) was amplified in two parts and fused with pcDNA3.1-PA by using Hifi DNA Assembly (NEB) (which is referred to as “pPA-dCas13-EGFP”). A Lwa-specific scaffold RNA sequence was inserted using Hifi DNA Assembly (NEB). A phosphorylated oligonucleotide encoding an sgRNA sequence was ligated to Bbs1-digested scaffold constructs to prepare sgRNA-targeting XistRNAs and fusion RNAs. 
     (2) Confirmation of Target RNA Localization 
     A short-chain-sequence-targeting sgRNA designed based on RNA conformation prediction and pPA-dCas13-EGFP were introduced into the HEK293T cells. The binding to the target XIST RNA was observed under a fluorescence microscope ( FIG. 6 ). 
     (3) Evaluation of Target RNA Function: ChIP-qPCR 
     The binding of the target XIST RNA to an arbitrary sequence on chromatin was confirmed. A short-chain-sequence-targeting sgRNA designed based on RNA conformation prediction and pPA-dCas13-EGFP were introduced into the HEK293T cells. Thereafter, the XIST-sgRNA-Cas13-EGFP complex on chromatin was crosslinked with 1% paraformaldehyde, and the Cas13-EGFP fusion proteins in the cell extract were immunoprecipitated with anti-GFP antibodies. Thereafter, the co-immunoprecipitated XIST and XIST-binding genomic sequences were extracted. The extracted genomic sequences were detected by using the qPCR method to confirm the polymerization of XIST-chromatin. Histone was used as a positive control, while non-specific IgG was used as a negative control ( FIG. 7 ). 
     Example 4 
     (1) Plasmid Construction and Target Guide 
     To create an RNA-editing system, the EGFP domain of pPA-dCas13-EGFP was re-written into a domain in which APOBEC1 domain and A1CF domain were fused using Hifi DNA Assembly (NEB) (referred to as “RESCUE system”; pPA-dCas13-ABC1A1,  FIG. 8 ). A phosphorylated oligonucleotide encoding an sgRNA sequence was ligated to Bbs1-digested scaffold constructs to prepare an RNA fused with sgRNA-targeting RNA of APP protein with Aβ cleavage sequence recognition. 
     (2) Testing of Target RNA Gene Editing Effect 
     The function of the target APP protein, a short-chain-sequence-targeting sgRNA designed based on RNA conformation prediction, and pPA-dCas13-ABC1A1 were introduced into the HEK293T cells. The effect of inhibiting cleavage by β-secretase on the target APP protein was confirmed by western blot analysis ( FIG. 9 ).