CN113604469B - CRISPR/CasRx based gene editing method and application thereof - Google Patents

CRISPR/CasRx based gene editing method and application thereof Download PDF

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CN113604469B
CN113604469B CN202110919746.2A CN202110919746A CN113604469B CN 113604469 B CN113604469 B CN 113604469B CN 202110919746 A CN202110919746 A CN 202110919746A CN 113604469 B CN113604469 B CN 113604469B
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tmc1
casrx
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bth
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舒易来
胡晓湘
李耕林
郑子文
李果
崔冲
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Eye and ENT Hospital of Fudan University
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Abstract

The invention provides sgRNA (small guide ribonucleic acid), an expression vector, a CRISPR-CasRx system, a kit and application of a targeting Tmc1 mutant (c.1235T is more than A; p.M412K), and belongs to the technical field of genetic engineering. The sequence of sgRNA of the targeting Tmc1 mutant is shown in SEQ ID NO.3, and CasRx containing the sgRNA does not interfere with wild Tmc1 + In the case of (2), tmc1 can be set to Bth Casrx based on AAV-php. Eb vector was injected into the inner ear of neonatal bazedoxifene mice with 82% reduction in transcript, and Tmc1 was found Bth The reduction was 70% in 2 weeks and no off-target was detected throughout the transcriptome. The sgRNA-targeted Tmc1 mutant provided by the invention has high specificity, and can improve Tmc1 Bth The survival rate of the mutant mouse hair cells leads the form of the cilia bundle to be recovered, the mechanical transduction current is reduced, and the progressive hearing loss is obviously improved.

Description

CRISPR/CasRx based gene editing method and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a CRISPR-CasRx-based gene editing method and application thereof.
Background
According to the World Health Organization (WHO) data, hearing loss is one of the most common sensory defects, with about 5% of the world population suffering from hearing loss, of which 3400 ten thousand are children. In children, hearing loss affects cognitive, linguistic and psychosocial development, with almost half of deafness cases being caused by genetic factors, and of the different types of hereditary hearing loss, 20-25% of non-syndromic hearing loss (NSHL) cases are autosomal dominant. To date, over 100 genes have been shown to be associated with NSHL, and most of the autosomal dominant inheritance is progressive hearing loss with a sufficient time window for gene therapy.
TMC1 (transmembrane channel class 1) is the sixth most common hereditary deafness gene, with TMC1 mutations leading to dominant or recessive NSHL. There is evidence that TMC1, as a component of the channel complex, contains 10 transmembrane domains and is involved in sensory nerve signal transduction. Mutant Tmc1 (c.1235T > A; p.M412K) homologous to human TMC1 (c.1253T > A; p.M418K), and Beethoven (Bth) mice carrying this mutation exhibited DFNA36 hearing loss, and therefore Bth mice were suitable as models for the study of non-syndromic deafness in this study.
Currently, there are few clinical treatments that can slow or reverse hereditary deafness. With the increasing understanding of the relationship between inheritance and hearing loss, people are interested in the gene therapy of hearing loss. The gene is expressed in the inner ear of a mouse with a vesicle glutamate transporter-3 (VGLUT 3) gene deletion by using a gene replacement method, and the hearing of the mouse is successfully restored for the first time. Subsequent studies have also demonstrated the effectiveness of gene replacement therapy for hereditary hearing loss, but gene replacement does not allow precise regulation of gene expression according to the needs of the cell, and therapeutic efficacy is reduced when the gene mutation is dominant negative.
The gene editing technology is used as a novel gene therapy method for treating hereditary hearing loss, and the research on introducing a CRISPR-Cas9 system into the inner ear of a Bth model mouse successfully improves the hearing loss. In addition, the gene function of the barcingto mice with recessive point mutation of the Tmc1 gene was restored using the double AAVs-packaged cytosine base editor, which indicates that the base editing can partially and temporarily restore the auditory function in vivo, and the gene therapy of the inner ear is expected to be an ideal treatment method for hereditary deafness. Potential off-target risks exist in the CRISPR-Cas9 system, and the Cas9 protein may be targeted to cut a DNA sequence close to the sgRNA sequence. While single base editing systems have demonstrated a large number of off-target edits, these pose significant safety risks for disease treatment and clinical applications, which limit the utility of this technology, particularly in therapeutic and clinical applications. The operation of the targeted RNA is transient reversible and does not affect the sequence and structural changes of the gene. In recent years, RNA regulation has been used to treat hearing loss in mice. For example, antisense oligonucleotides are used to treat Ush c (c.216G > A) gene mutation in mouse model hearing, which disrupts wild-type splicing, produces frame-shift mutations and translates into truncated proteins; in addition, RNA interference (RNAi) and artificial micrornas are also used to prevent hearing loss. However, off-target effects of these traditional RNA regulatory tools, which are still present at the level of gene transcription, are a concern.
The CRISPR-Cas13 is a novel RNA editing system, wherein Cas13 protein is a CRISPR-Cas system effector protein of type 2 type VI, has RNA-mediated RNA enzyme digestion activity, is the only protein which can degrade RNA and is discovered by the CRISPR-Cas system in the second main class at present, is originally used for relieving virus infection in bacteria, and has higher specificity than a traditional RNA intervention tool. The Cas13 protein family has identified 4 members, including Cas13a (formerly C2), cas13b, cas13C, and Cas13d. PspCas13b and CasRx (RfxCas 13 d) have been reported to have higher activity and specificity than other Cas13 s. As the current smallest Cas13 enzyme, casRx can be easily packaged into AAVs vectors, which makes the CRISPR-CasRx system convenient for delivery in vivo. CasRx has been applied as a therapeutic tool in mouse models of liver and eye diseases, and RNA editing systems can provide safer gene silencing methods without changing the genome compared to other gene editing systems. Furthermore, PAM sequences are essential in genome editing, which limits the selection of sgRNA sequences, particularly in specific disease point mutations. However, there are no restrictions in this system like this PAM sequence, so gRNAs can be designed and screened more flexibly.
There is still a lack of research on the use of CRISPR-Cas13 RNA editing systems for the treatment of genetic deafness. Whether the CRISPR-Cas13 RNA editing technology can be utilized to provide a method for treating hereditary hearing loss and improve the specificity and efficiency of the existing gene editing system becomes a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to solve the technical problems, and researches related to treatment of hereditary hearing loss based on a CRISPR-Cas13 RNA editing system are carried out. In the invention, sgRNA targeting Tmc1 mutant, an expression vector thereof, a CRISPR-CasRx system, a related kit and application thereof are provided.
The invention firstly provides sgRNA targeting a Tmc1 mutant, and the sequence of the sgRNA is shown in SEQ ID NO. 3.
The invention also provides an expression vector targeting the Tmc1 mutant, which comprises the sgRNA sequence as described above.
The expression vector targeting the Tmc1 mutant comprises the sgRNA sequence and the AAV vector, wherein the AAV vector is AAV-PHP.eB.
Further, the construction method of the expression vector of the invention is as follows:
(1) Constructing a human codon-optimized CasRx gene expression plasmid, firstly synthesizing a CasRx gene, connecting two nuclear localization signal peptides, constructing into a mammalian expression vector and guiding expression by a CAG promoter.
(2) Constructing an sgRNA expression plasmid, firstly synthesizing two complementary single-chain sgRNA oligonucleotides, annealing the two single-chain sgRNA to form a double chain, connecting the sgRNA to a cloning skeleton by using a BspQI enzyme cutting site, and expressing the sgRNA by using a U6 promoter on the skeleton.
(3) Constructing a fluorescent expression plasmid of mCherry-Tmc1 containing a mutant gene, firstly synthesizing a 90bp sequence containing c.1234T > A mutant site, connecting the mCherry fluorescent gene at the 3' end of the sequence, and constructing the expression plasmid to be expressed by a CMV promoter.
The invention also provides a CRISPR-CasRx system targeting a Tmc1 mutant comprising a sgRNA sequence as described above and a gene sequence encoding a CasRx protein.
The invention adopts CasRx technology to improve the hearing of a human DFNA36 animal model Beethoven mouse, firstly, 30 sgRNAs are screened in 293T cells, and CasRx containing sgRNA3 is found not to interfere with a wild type (Tmc 1) + ) In the case of (1), tmc1 is set Bth Transcript reduction was 82%. Then, casRx based on AAV vector (AAV-php. Eb) was injected into the inner ear of neonatal bazedoxifene mice, and Tmc1 was found Bth The decrease was 70% within 2 weeks and little off-target was detected at the transcriptome level. We found that hair cell survival was increased, ciliary bundle morphology was restored, and mechanical transduction current was reduced after targeting to Tmc 1. Importantly, hearing measured as Auditory Brainstem Response (ABR) and distortion product otoacoustic emission (DPOAE) improved significantly at all ages within 8 weeks. Therefore, we validated the CRISPR-CasRx-based RNA editing strategy for its effectiveness in treating autosomal dominant hearing loss, paving the way for its further application in hearing and many other genetic diseases.
The invention also provides a kit for targeting the Tmc1 mutant, which comprises an expression CasRx protein and the expression vector.
The invention further provides an application of sgRNA targeting the Tmc1 mutant, and the application of the sgRNA in preparation of an immune cell medicament for treating hereditary hearing loss.
The invention has the beneficial effects that:
the invention screens 30 sgRNAs which are matched with single point mutation at all possible positions, takes Tmc1 pathogenic allele as a target point, and compares the editing specificity and efficiency of PspCas13b and CasRx systems to select the optimal sgRNA. The invention uses AAV vector (AAV-PHP. EB), has higher transduction efficiency in transferring CasRx and sgRNA to cochlea of a birth mouse in inner ear hair cells, and successfully reduces Tmc1 Bth Expression of transcripts altering Tmc1 Bth /Tmc1 + mRNA ratio, preventing progressive hearing loss, improved hair cell and ciliated bundle morphology, and no off-target effects. These results indicate that the CasRx RNA editing system is a treatment for hereditary deafnessPotential clinical methods.
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FIG. 1 is a design targeting Tmc1 Bth Schematic of sgRNAs of transcripts.
FIG. 2 screening for targeting Tmc1 Bth Efficient specificity of sgRNAs of the transcript; a. constructing sgRNA screening for Cas RNA editing system mediation; constructing a Cas expression vector and mCherry-Tmc1 Bth Fluorescent reporter gene, mCherry-Tmc1 + Fluorescent reporter gene, targeting Tmc1 Bth A sgRNA expression vector of the transcript and a non-targeting (NT) sgRNA expression vector; casRx System mediated targeting of mCherry-Tmc1 Bth mRNA and mCherry-Tmc1 + sgRNA to control sgRNA (NT) fluorescence intensity ratio of mRNA, data expressed as mean ± SD (n =3 bio-independent samples); mCherry-Tmc1 mediated by sgRNA and control sgRNA (NT) against PspCas13b system Bth mRNA and mCherry-Tmc1 + Fluorescence intensity ratio of mRNA, data expressed as mean ± SD (n =3 bio-independent samples); casRx and PspCas13b mediated mCherry-Tmc1 Bth And mCherry-Tmc1 + The average fluorescence intensity ratio of mRNA interference is that of the sgRNAs 3 in 30 sgRNAs, the average fluorescence intensity ratio is lowest; integrated fluorescence intensity of cells mediated by the casrx system; with mCherry-Tmc1 + Compared with mRNA, sgRNA3 targets mCherry-Tmc1 Bth The fluorescence density of mRNA is significantly reduced; non-targeting sgRNA targeting mCherry-Tmc1 Bth And mCherry-Tmc1 + Compared with mRNA, sgRNA3 targets mCherry-Tmc1 + A decrease in fluorescence density of the mRNA; data are expressed as mean ± SD (n =2, biologically independent samples), p<0.05，**p<0.01，***p<0.001，****p<0.0001; assumed values were determined by one-way analysis of variance and Sidak multiple comparison test.
FIG. 3 is a representative fluorescence image of 293T cells; a. transfection of 30 sgRNAs and non-target sgRNAs with the CasRx System to target Tmc1 Bth (ii) a transcript; b. cells were transfected with the PspCas13b system containing 30 sgRNAs and non-target sgRNAs to target Tmc1 Bth (ii) a transcript; mCherry intensity indicates RNA knock-out efficiency.
FIG. 4 shows Tmc1 in CasRx Selective disruption of Bth mice Bth Transcript(ii) a a. Schematic representation of AAV vectors encoding CasRx and sgRNA3 (top panel) and control NT vectors (bottom panel); b. summary of in vivo experiments; injecting a mouse from P1 to P2, dissecting and culturing a Corti organ on P5 days, analyzing hair cell physiology by P15 to P16, sequencing the injected mouse after 2 weeks, performing hearing tests (ABR and DPOAE) after 4, 8 and 12 weeks, immunohistochemistry, and performing scanning electron microscope observation after 10 weeks; c.Tmcc 1 Bth And Tmc1 + Percent deep sequencing; pie chart shows Tmc1 in the sample Bth And Tmc1 + Average composition of transcripts, sequence shows Tmc1 Bth And Tmc1 + Single nucleotide differences in transcripts (non-injected, AAV-CasRx + NT injected, and Tmc1 with AAV-CasRx + sgRNA3 injected) Bth Transcripts 52.83 ± 5.33%, 53.39 ± 4.8% and 14.88 ± 9.77%, respectively, n =3 mice, data expressed as mean ± SD); d. deep sequencing analysis Tmc1 mice were not injected (n = 3), AAV-CasRx + NT injected (n = 3) and AAV-CasRx + sgRNA3 injected (n = 3) Bth And Tmc1 + Transcript ratios between, data expressed as mean ± sem, # p<The 0.01,p value was determined using a one-way analysis of variance with Dunnett's multiple comparison test; e. cochlear mRNA expression was tested 2 weeks after RT-qPCR injection (n =6 mice); tmc1 in the AAV-CasRx + sgRNA 3-injected ear compared to the AAV-CasRx + sgRNA 3-uninjected ear Bth Expression of transcripts was significantly reduced, data expressed as mean ± sem,. P<0.05, p value is tested by unpaired two-tailed t test; f. amplification of Tmc1 Bth Detecting the sequence by using a primer specifically combined with a mutant template, wherein the primer cannot be combined with a wild template for amplification; g. mechanical Electrical Transduction (MET) current recordings and maximum MET current amplitudes of top-ring IHCs at P15-P16; h.Tmc +/+ Mouse (n =16 OHCs), non-injected Tmc1 Bth/Bth Mice (n =18 OHCs) and Tmc1 injected with AAV-CasRx + sgRNA3 Bth/Bth The MET current amplitudes of mice (n =16 OHCs) were 461.134 ± 74.978pA, 442.458 ± 82.805pA, 344.409 ± 114.591pA, respectively, and the data were expressed as mean ± SD,. Times.p<The 0.01 p value was determined in one-way format using the analysis of variance using the Sidak multiple comparison test.
FIG. 5 is a transduction assay of AAV-PHP.eB; collecting cochlear samples 2 weeks after injection, staining with anti-GFP and myostatin 7a antibodies, and imaging whole cochlear tissues with a total length of 300 μm; scale bar: 100 μm.
Figure 6 is the improvement of hearing by mRNA down-regulation; at Tmc1 Bth/+ In mice, casRx-mediated gene deletion was used and not used for experiments to prevent progressive hearing loss.
FIG. 7 is an improvement in hearing function of Bth mice by CasRx; a. injection of Tmc1 Bth/+ Right, left and wild-type ear of (a), ABR waveform recorded at 8kHz at 4 weeks, green trace represents threshold; b, c.Tmc1 +/+ (Green), tmc1 Bth/+ + AAV-CasRx + sgRNA3 (blue) and Tmc1 Bth/+ Pure tone and clickABR thresholds of non-injected contralateral (red) ear, 4 and 8 weeks later, AAV-CasRx + sgRNA3 with no injection of Tmc1 Bth/+ Ear comparison, wherein the average ABR threshold is obviously reduced, and the statistical analysis adopts Tukey post-test of bidirectional variance analysis and multiple comparison; tmcc 1 Bth/+ + AAV-CasRx + sgRNA3 (blue) and Tmc1 Bth/+ DPOAE threshold in untreated contralateral ears (red) at 4 and 8 weeks, with a significant decrease in DPOAE threshold in ears injected with AAV-CasRx + sgRNA3 at two frequencies, statistically analyzed using a two-way Bonferroni multiple comparison test<0.05,**p<0.01,***p<0.001，****p<0.0001, and the error value is represented by + -SD.
FIG. 8 is ABR values from AAV-injected mice; a.4 weeks of age, injecting control AAV and heterozygous mice without injected contralateral ear hearing threshold; threshold values for wild type mouse ears injected with AAV (AAV-CasRx + sgRNA3 or AAV-CasRx + NT) and uninjected contralateral ears at 4 weeks of age, data are presented as mean + -SD; c, d. 4 weeks after AAV-CasRx + sgRNA3 injection, non-Bth injected mice and wild type mice, click-ABR amplitudes and latencies of 80dB and 90dB, × P <0.05, × P <0.01, × P <0.0001; mean ± SD, statistical analysis using Tukey.
FIG. 9 protection of hair cells and cilia bundles without AAV-CasRx + sgRNA3 injection; a. confocal images of 100 μm cochlear sections taken 10 weeks after injection, specimens stained with myosin7a (red), these images being taken at Tmc1 +/+ 、Tmc1 Bth/+ + AAV-CasRx + sgRNA3 and Tmc1 Bth/+ Non-injected mice (n)= 5), positions 8kHz and 169hz respectively, ihcs and OHCs are indicated in the figure, scale bar: 20 μm; b. number of OHCs (upper) and IHCs (lower) per 100 μm cochlea, data expressed as mean ± SD, p<0.05，**p<0.01，***p<0.001 and<0.0001, performing statistical analysis by adopting a bidirectional Sidak multiple comparison test; c. scanning electron microscope images of the sensory epithelium at the top of the cochlea show the morphology of the fiber hair bundles, and Tmc1 is collected 10 weeks after injection +/+ 、Tmc1 Bth/+ + AAV-CasRx + sgRNA3 and Tmc1 Bth/+ Non-injected sample, scale bar: 20 μm (top); 3 μm (bottom).
FIG. 10 is a scanning electron microscope image of the morphology of the cochlear medial sensory epithelial fibrohair bundles; tmc1 at 10 weeks post-injection +/+ 、Tmc1 Bth/+ + AAV-CasRx + sgRNA3 and Tmc1 Bth/+ Non-injected samples, arrows indicate loss of cilia bundles, scale bar: 20 μm (upper) and 3 μm (lower).
FIG. 11 is an off-target analysis of RNA sequences editing RNA in vivo; off-target-1 to Off-target-10 are 10 Off-target sites detected by RNA sequences, the unmatched target sites are highlighted by color, and the sgRNA3 targeted 30bp sequence (On-target) is displayed in the first row; b. comparing the expression levels of 10 decoy sites in the ears injected with AAV-CasRx + sgRNA3 or AAV-CasRx + NT, and in the ears injected with AAV-CasRx + sgRNA3 or not, the data are shown as mean. + -. SEM, ns indicates no significant difference, and statistical analysis is performed by using multiple unpaired t-test.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.
1. Examples of the methods
(one) in vitro use of a CasRx specific knockout Tmc1 Bth Transcript
To knock Tmc1 out efficiently and specifically in HEK293T cells Bth mRNA, first comparing two RNA editing systems, pspCas13b and CasRx, both of which have been shown to be effective in knocking out endogenous transcripts. Then 30 targeting Tmc1 were designed Bth sgRNAs of the transcripts, in both PspCas13b and CasRx systems, were 30bp in length, and a30bp GFP-targeting sgRNA (denoted NT) was used as a control (the sequences of sgRNAs 1-30 and NT are shown in Table 1, see SEQ ID No.1-31 of the sequence Listing), targeting Tmc1 Bth Schematic design of sgRNAs of transcripts is shown in fig. 1, where bases a are inserted sequentially at positions 1-30.
TABLE 1
sgRNA1 5’-CAA ATA AGT CAA ACA GGG TGG GAC AGA ACT-3’
sgRNA2 5’-AAA TAA GTC AAA CAG GGT GGG ACA GAA CTT-3’
sgRNA3 5’-AAT AAG TCA AAC AGG GTG GGA CAG AAC TTC-3’
sgRNA4 5’-ATA AGT CAA ACA GGG TGG GAC AGA ACT TCC-3’
sgRNA5 5’-TAA GTC AAA CAG GGT GGG ACA GAA CTT CCC-3’
sgRNA6 5’-AAG TCA AAC AGG GTG GGA CAG AAC TTC CCC-3’
sgRNA7 5’-AGT CAAACA GGG TGG GAC AGA ACT TCC CCA-3’
sgRNA8 5’-GTC AAA CAG GGT GGG ACA GAA CTT CCC CAG-3’
sgRNA9 5’-TCA AAC AGG GTG GGA CAG AAC TTC CCC AGG-3’
sgRNA10 5’-CAA ACA GGG TGG GAC AGA ACT TCC CCA GGA-3’
sgRNA11 5’-AAA CAG GGT GGG ACA GAA CTT CCC CAG GAG-3’
sgRNA12 5’-AAC AGG GTG GGA CAGAAC TTC CCC AGG AGG-3’
sgRNA13 5’-ACA GGG TGG GAC AGA ACT TCC CCA GGA GGG-3’
sgRNA14 5’-CAG GGT GGG ACA GAA CTT CCC CAG GAG GGA-3’
sgRNA15 5’-AGG GTG GGA CAG AAC TTC CCC AGG AGG GAC-3’
sgRNA16 5’-GGG TGG GAC AGA ACT TCC CCA GGA GGG ACA-3’
sgRNA17 5’-GGT GGG ACA GAA CTT CCC CAG GAG GGA CAT-3’
sgRNA18 5’-GTG GGA CAG AAC TTC CCC AGG AGG GAC ATT-3’
sgRNA19 5’-TGG GAC AGA ACT TCC CCA GGA GGG ACA TTA-3’
sgRNA20 5’-GGG ACA GAA CTT CCC CAG GAGGGA CAT TAC-3’
sgRNA21 5’-GGA CAG AAC TTC CCC AGG AGG GAC ATT ACC-3’
sgRNA22 5’-GAC AGA ACT TCC CCA GGA GGG ACA TTA CCA-3’
sgRNA23 5’-ACA GAA CTT CCC CAG GAG GGA CAT TAC CAT-3’
sgRNA24 5’-CAG AAC TTC CCC AGG AGG GAC ATT ACC ATG-3’
sgRNA25 5’-AGA ACT TCC CCA GGA GGG ACA TTA CCA TGT-3’
sgRNA26 5’-GAA CTT CCC CAG GAG GGA CAT TAC CAT GTT-3’
sgRNA27 5’-AAC TTC CCC AGG AGG GAC ATT ACC ATG TTC-3’
sgRNA28 5’-ACT TCC CCA GGA GGG ACA TTA CCA TGT TCA-3’
sgRNA29 5’-CTT CCC CAG GAG GGA CAT TAC CAT GTT CAT-3’
sgRNA30 5’-TTC CCC AGG AGG GAC ATT ACC ATG TTC ATT-3’
NT 5’-ACC AGG ATG GGC ACC ACC CCG GTG AAC AGC-3’
(II) construction of plasmid
The human codon-optimized CasRx gene was synthesized under the control of the CAG promoter and cloned into a mammalian expression vector with two NLS (nuclear localization sequences). The human codon optimized PspCas13b gene was synthesized and cloned into NES (nuclear export sequence) mammalian expression vector under the control of CAG promoter.
Then constructing a CasRx sgRNA cloning main chain, wherein the CasRx sgRNA cloning main chain comprises two direct repetitive sequences cloned by BspQI enzyme; construction of a PspCas13b sgRNA cloning backbone comprising a direct 3' repeat cloned with BbsI enzyme. sgRNAs were further synthesized as single-stranded DNA oligonucleotides. The two sgRNA oligonucleotides complementary in the opposite directions anneal to form a double strand, are ligated to the sgRNA expression vector of the CasRx system containing the U6 promoter via a BspQI cleavage site, and are cloned under the U6 promoter. The double-stranded sgRNA was also cloned using the BbsI cleavage site into a sgRNA expression vector of the PspCas13b system containing the U6 promoter element.
To construct mCherry-Tmc1 Bth Reporter gene vector, synthesizing 90bp sequence of Tmc1 transcript containing c.1235T > A mutation, cloning it to the 3' end of mCherry gene, and removing the stop codon from the end. To construct mCherry-Tmc1 + The reporter gene vector was synthesized as a 90bp sequence of the Tmc1 transcript containing c.1235T, and cloned at the 3' -end of the mCherry gene with the stop codon removed.
(III) cell culture and transfection
293T cells were cultured in DMEM medium (Gibco) supplemented with 10% Fetal Bovine Serum (FBS) (v/v) and the% CO was 5% at 37 ℃% 2 Culturing under the conditions of (1). Cells were seeded on poly D-lysine coated 24-well plates and maintained at 60-70% confluency prior to transfection.
Cell transfection EZ Trans reagent (Shanghai Life iLab) was used, following the manufacturer's protocol. At the time of transfection, a plasmid expressing CasRx or PspCas13b (600 ng), a plasmid expressing sgRNA (300 ng), mCherry-Tmc1 were mixed in each well Bth Or mCherry-Tmc1 + A reporter gene. Mu.g DNA and 3. Mu.L EZ transfection reagents were diluted with 40. Mu.L DMEM, respectively, the diluted EZ transfection reagent was added to the diluted DNA solution, gently mixed, incubated at room temperature for 15 minutes to form DNA-EZ complexes, then the DNA-EZ complexes were added directly to each well, gently mixed by shaking the plate back and forth, after transfection for 6h, the complexes were removed, and 0.5mL of complete growth medium was added to the cells.
(IV) cellular imaging
48h after transfection, 3 images of each well were observed and captured using a Nikon TI-E microscope. ImageJ was used to quantify the fluorescence intensity of the mCherry signal.
(V) flow cytometry (FACS)
293T cells were harvested and detected by flow cytometry 48 hours after transfection. The mCherry signal was detected immediately on a BD LSRFortessa flow cytometer (BD Biosciences) by FCS Express 5 software (DeNovo software). A total of 10,000 cell samples were collected and each sample was analyzed using FlowJo software.
(VI) measurement of Virus production
AAV(PHP.eBSerotype) viral vectors were produced by and biotechnology (shanghai) ltd, AAV carrying the double transgene, a tgc 1-targeted sgRNA driven by the U6 promoter and RfxCas13 driven by the CMV promoter as therapeutic vectors; the control AAV was constructed in accordance with the therapeutic vector except that the sgRNA sequence was replaced with the NT sequence. Transduction efficiency was tested using the same serotype AAV encoding the EGFP protein. AAV-CasRx + sgRNA3 has a viral titer of 3.38X 10 12 vg/mL, viral titer of AAV-CasRx + NT 1.73X 10 12 vg/mL. Virus isolates were isolated in small volumes and stored at-80 ℃ to avoid repeated freeze-thaw cycles.
(VII) test mice
All test mice were housed in the same facility and housed for a 12 hour light-dark cycle. Heterozygous Beethofen mice (Tmc 1) Bth/+ ) Given by Driew Griffith (university of Telaviv, sackler medical school of human genetics and molecular medicine). Adding Tmc1 Bth/+ Mouse and Tmc1 +/+ Or Tmc1 Bth/+ C3HeB/FeJ (C3H) background mice (Jackson Laboratories) were inbred to breed young mice. Adopting mixed protease-K lysate, incubating for 8h at 55 ℃, then incubating for 1h at 85 ℃, extracting DNA from tail clamp biopsy specimen, carrying out PCR by using 20 mu L system, and carrying out genotype identification by product sequencing.
(VIII) inner ear injection
Tmc1 Bth/+ Or wild type P1 and P2 pups were injected with 1.5 μ L of virus through the round window membrane. The young mice were anesthetized on ice for 2-3 minutes until unconsciousness was lost. After anesthesia, the cochlea viewing round window membrane was exposed at the retroauricular incision. The virus was injected slowly through the round window membrane into the right cochlea using glass microtubes. After injection, the skin incision was closed, and the young mouse was placed on a 42 ℃ heating pad for recovery, and after the young mouse was completely recovered, the mouse was returned to the mother mouse for feeding.
(nine) Hair cell electrophysiology
Collection of injections Tmc1 at P4-P5 +/+ 、Tmc1 Bth/Bth Or Tmc1 Bth The mouse cochlea of (4) was treated at 37 ℃ with 5% CO in DMEM (1X) medium containing 1% fetal bovine serum 2 And (4) culturing. Recording the expression of inner hair cells in P14-P15 by using whole-cell patch clamp, and performing standard artificial stranguriaBar liquid contains (unit: mM): 137NaCl, 5.8KCl, 1.3CaCl 2 、0.9MgCl 2 、0.7NaH 2 PO 4 10HEPES and 5.6D-glucose, adjusting the pH to 7.40, and the osmotic pressure to 300mmol/kg. Extraction of recording pipettes from borosilicate glass capillaries (1B 150F-4, world precision instruments, florida, usa) the internal solutions contained (units: mM): 140CsCl, 0.1EGTA, 1MgCl 2 10HEPES, 2Mg-ATP and 0.3Na-ATP (pH 7.20, osmolality-295 mmol/kg). MET current was recorded using a voltage clamp with a potential of-80 mV maintained by an EPC10/2 amplifier (HEKA, lambrrecht/Pfalz, germany) driven by a PC-terminal Patchmaster (HEKA). The current signal was filtered at 2kHz and digitized at 200 kHz. The liquid flow is sprayed at a distance of 15 mu m through a tube with the tip of 10 mu m, so that the hair cell cilia bundle is deflected. The jet was driven by a piezoelectric disc (27 mm diameter) applying a sinusoidal voltage (40 Hz, ± 120V).
(Ten) analysis of target deep sequencing data
To analyze the sequence of the CasRx knockout mutant at the RNA level, total RNA and cDNA were obtained from the cochlea, the site sequence of interest was amplified with primers TMC1-lib-F and TMC1-lib-R (primer sequences as in Table 2), the PCR product was visualized on a 2% agarose gel, purified with a purification kit (Qiagen), and generated 150 base pairs on the Illumina MiSeq platform. Reads from heterozygous samples were isolated based on the presence of wild type sequences (5'-ATG CCT CCT GGG GAT GTT CTG TCC CAC C-3' and anti-complement 5'-GGT GGG ACA GAA CAT CCC CAG GAG GGA CAT-3'), mutant (5'-ATGTCC CTC CTG GGGAAG TTC TGT CCC ACC-3') and its anti-complement (5'-GGT GGGACAGAACTT CCC CAG GAG GGACAT-3').
The calculation formula of the knockout efficiency is as follows:
Figure BDA0003207028370000061
RT-qPCR of (eleven) cochlea
The cochlea of both wild-type and heterozygous mice was dissected, total mRNA was extracted from the extracted cochlear tissue using TRIzol (Invitrogen) and reverse transcribed for mRNA using cDNA synthesis super mix (YEASEN) according to the manufacturer's protocol, and 1 μ L of the RT product was added to an RT-qPCR SYBR kit (YEASEN) for subsequent RT-qPCR detection with the following steps: 95 ℃ for 5min,95 ℃ for 10s,60 ℃ for 35s, and 40 cycles.
Primers q-TMC1-F2 and q-TMC1-R2 are designed to detect the total expression level of Tmc 1. In order to detect the expression of the mutant transcript, a forward primer q-TMC1-F4 is designed, the 3' end of the forward primer is a base A which is specifically combined with the mutant sequence, and a reverse primer q-TMC1-R2 is designed (the primer sequence is shown in Table 2).
TABLE 2 primers used for plasmid construction, genotyping, deep sequencing and RT-PCR
Figure BDA0003207028370000062
(twelve) off-target assay
To analyze off-target RNA editing sites across transcriptomes, total RNA from different treatment samples was collected using the RNeasy Plus Miniprep Kit (Qiagen), 1g of RNA was taken for each sample as input material for RNA sample preparation, sequencing libraries were used with the rnalry Prep Kit for Illumina (NEB, USA), according to the manufacturer's recommendations, and an index code was added to the attribute sequence of each sample, with at least 500 million reads per sample. Differential expression analysis was performed on both groups using the DESeq 2R package. DESeq2 provides a statistical routine that uses a model based on negative binomial distribution to determine differential expression in digital gene expression data. The p value result is adjusted by Benjamini and Hochberg methods to control the error discovery rate. Genes found to have a p value > 0.05 by DESeq2 were considered differentially expressed.
(thirteen) auditory test
ABRs and DPOAEs were recorded in a sound-proof booth using the BioSigRZ system (Take-Davis technologies, alachua, FL, USA). Mice were anesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg) intraperitoneally. The stimuli were generated by a digital input/output card (national instruments PXI-4461) on a PXI-1042Q chassis, amplified by an SA-1 speaker driver (tuner Davis Technologies, inc.) and transmitted to the ear under study by two electrostatic drivers (CUI Miniature Dynamics), the ear canal sound pressure was recorded using an electret microphone (electret condenser), and the ABR signal was collected using a hypodermic needle electrode implanted in the pinna (movable electrode), apex (reference electrode), and hip (grounded electrode).
The acoustic stimulus was a square pulse of 100 msec duration, with a3 msec tone burst, and frequencies of 4, 8, 16, 24, and 32khz, respectively. The sound level is raised from 20db below the threshold to 90db, every 5db, and the electrical signal is repeated 512 times on average. The ABR threshold is visually defined as the lowest sound pressure level (dB SPL) where any wave can be detected. And taking the ABR threshold average value of each experimental group and carrying out statistical analysis. The first wave amplitude is defined as the difference between the first wave peak value and the baseline average value 1ms before stimulation. DPOAE data were collected and recorded under the same conditions as ABR. When DPOAEs are generated at 2f1-f2, the frequency ratio of the primary tone is 1.2 (f 2/f 1), and the f2 level of each pair of f2/f1 is 10dB SPL lower than the f1 level. The f2 stage sweeps from 20dB to 80dB in 5dB steps. The waveform and average spectrum are used at each level to improve the signal-to-noise ratio of the recorded ear canal sound pressure. At each level, the amplitude of DPOAE at 2f1-f2 was extracted from the average spectrum and noise. And interpolating an equal response curve according to the relation between the DPOAE amplitude and the sound level. This threshold is defined as the f2 level required to produce DPOAEs above 0dB.
(fourteen) immunohistochemistry of cochlea
After 10 weeks old adult mice die at cervical dislocation, immunohistochemistry with cochlear injection and without cochlear injection on the opposite side was performed. Perforation of the apical part of the cochlea of the temporal bone, perfusion with 4% paraformaldehyde, overnight incubation at 4 ℃ and 10% EDTA decalcification for 1-3 days at 4 ℃. PBS full-patch immunofluorescence staining of decalcified cochlear sections. Tissue was infiltrated with 1% Triton X-100, blocked with 10% donkey serum at 4 ℃ for 12-16 hours, then incubated overnight at 4 ℃ with primary anti-rabbit anti-MYO 7A (# 25-6790, dilution of protein BioSciences, 1, 800) and incubated with secondary antibody Cy3 coupled donkey anti-rabbit IgG (AlexJackson ImmunoResearch 711-165-152, 500 dilution) for 2 hours in the dark, washed three times with PBS at room temperature. Cochlea was removed from 2-week-old mice, no decalcification was performed during dissection, and AAV transduction efficiency was examined using a chicken-resistant anti-EGFP (Abcam, 1, 1000 dilution) by the same procedure as above. The specimens were mounted on an adherent microscope slide (# 188105, cittest) and confocal images were obtained with a come TCS SP8 microscope, 40 × glycerol immersion lens.
(fifteen) Hair cell count
The z-stacks are merged using ImageJ's maximum intensity projection of each segmented z-stack. MYO7A positive IHCs and IHCs were counted on 100 μm cochlear slices in 8kHz and 16kHz regions, respectively, with the approximate frequency perceived by each region determined on command. The outer three rows of cells were arranged as OHCs, and the inner one row was arranged as IHCs.
(sixteen) scanning Electron microscope Observation
After cervical dislocation of 10-week-old adult mice was sacrificed, the cochlea was punched out, 2.5% glutaraldehyde was perfused from the hole, 2.5% glutaraldehyde was fixed overnight at 4 ℃, 10% EDTA decalcification was performed at 4 ℃ for 3-5 days, the decalcified cochlea was divided into small pieces in 0.1M PB medium, the dissected tissue was put into 2.5% glutaraldehyde, washed 3 times with 0.1M PB, and fixed in 1% osmium acid at 4 ℃ for 2 hours. The tissue was dehydrated in an ethanol gradient, then dried in a HCP-2 desiccator for 2 hours, and the dried tissue was attached to a sample stand and sprayed with an IB-3 ion sputter for 3min. Scanning electron microscope images were obtained with a high vacuum field emission scanning electron microscope (Hitachi 575 SU-8010) at 2.5kV (low magnification) and 10.0kV (high magnification).
(seventeen) statistics of data
All data are expressed as standard deviation means or SEM means, results were analyzed statistically using GraphPad Prism (GraphPad Prism, version 8.0), significant differences between means were determined using student's t-test, and multiple comparisons were performed using one-way analysis of variance (ANOVA) or two-way analysis of variance, with a significance level P < 0.05.
2. Examples of the results of the experiments
(one) in vitro use of CasRx specific knockout Tmc1 Bth Transcript
To screen for the best sgrnas, two mCherry fluorescent reporter genes containing wild-type or mutant Tmc1 sequences were constructed, fused at the 5' end of the sequence (fig. 2 a). These reporter groups, either the PspCas13b or the CasRx expression vectors, were then co-transfected,transfecting an sgRNA expression vector into 293T cells, and after transfecting for 48h, interrupting the expression of fusion RNA by an RNA editing system, and measuring the fluorescence intensity of the cells to be used as an index of RNA knockout efficiency. As expected, casRx and PspCas13b resulted in a significant decrease in mCherry expression in 293T cells (figure 3). Targeting mCherry-Tmc1 when using the CasRx and PspCas13b System Bth The lowest fluorescence intensity ratio of the cells was 9.2. + -. 0.13% and 17.44. + -. 0.48%, respectively (FIG. 2b, c). Further, to compare the specificity of the two systems, we used mCherry-Tmc1 + The mean fluorescence intensity was measured for the target (FIG. 2 d) and mCherry-Tmc1 was analyzed Bth And mCherry-Tmc1 + mean fluorescence intensity ratio between mRNA interferences. The sgRNA3 ratio in the CasRx system was the lowest, 0.089113 (FIG. 2 d), with a significant 82% reduction in mCherry integration density (FIG. 2 e), indicating Tmc1 Bth Transcripts were knocked out efficiently. Taken together, these results indicate that sgRNA3 in CasRx is an effective knock-out of Tmc1 Bth Transcripts without interfering with Tmc1 + Ideal sgRNA of transcript. (II) CasRx mediates specific targeting in vivo
The results of the above studies show that sgRNA3 in the CasRx system is directed to Tmc1 in 293T cells Bth The transcript has the highest targeting efficiency, and can be used for judging whether AAV-CasRx + sgRNA3 is effective in vivo or not, and AAV vectors Tmc1 for coding sgRNA3 and CasRx Bth Down-regulation of Tmc1 in the inner ear of Bth mice Bth The transcript, non-targeting sgRNA, was packaged in the same vector as the control (fig. 4 a). We used AAV9 variants as delivery vectors, a more efficient and further developed AAV vector of the php. To verify the ability of AAV-php.eb vectors to introduce genes into Inner Hair Cells (IHCs) and Outer Hair Cells (OHCs), we injected AAV-php.eb encoded EGFP through the round window membrane into the right inner ear of postnatal P1-P2 mice. Cochlea were harvested 2 weeks after injection and Corti organs were dissected for immunohistochemistry (fig. 4 b). We observed near 100% efficiency of viral transduction in IHCs and OHCs, and found that the efficiency of viral transduction was over 95% with a gradual decline in efficiency from top to bottom circles (fig. 5), consistent with our previous study.
To determine the editing capacity of CasRx in vivo, whole cochlear tissue was targetedAnd (4) deep sequencing. Bth mouse cochlea display of AAV-CasRx + sgRNA 3-injected Bth mouse Tmc1 Bth Transcripts accounted for 14.88 ± 9.77% of total Tmc1 transcripts, significantly reduced compared to non-injected mice (52.83 ± 5.33%) and AAV-CasRx + NT (control AAV, non-targeting sgRNA) injected mice (53.39 ± 4.8%) (fig. 4c and table 3). Tmc1 of non-injected, AAV-CasRx + NT and AAV-CasRx + sgRNA3 Bth And Tmc1 + The actual ratios of transcripts were 1.1397. + -. 0.2584, 1.1605. + -. 0.2183 and 0.186. + -. 0.1457, respectively, (FIG. 4 d), indicating Tmc1 Bth The gene knockout efficiency was 70%.
To further aim at Tmc1 Bth Analysis of expression gene expression was measured at the RNA level using RT-qPCR. Increased CasRx expression and decreased total Tmc1 expression was observed compared to non-injected cochlea (fig. 4 e). RT-qPCR specifically targets Tmc1 with a pair of primers Bth cDNA (FIG. 4 f), results show control Tmc1 Bth mRNA was reduced to 54.69-10.49% (FIG. 4 e).
Further, in order to detect the specific targeting of CasRx and sgRNA3 to the mutant Tmc1 site, MET current was measured. Tmc1 Bth Mutations do not affect the sensitivity of hair cell mechanical transduction, but the knockout of Tmc1 results in a decrease in MET current. Both Tmc1 and Tmc2 are essential for MET, with Tmc2 being transiently expressed in the first week after birth and then eliminated from immunohistochemical static cilia at P10, while Tmc1 is continuously expressed. Therefore, to eliminate the contribution of Tmc2 to MET current, we measured MET current at equal amounts of P15-P16 in vitro organ cultured organ inner hair cells of Corti using P15-P16 mouse intracochlear hair cells (fig. 4 g). Wild type Tmc +/+ Mice and non-injected Tmc1 Bth/Bth Mouse inner hair cells in the apical loop showed similar MET current amplitude, whereas Tmc1 injected with AAV-CasRx + sgRNA3 Bth/Bth MET current amplitude was significantly reduced in the mouse apical loop inner hair cells (fig. 4 h), suggesting that CasRx + sgRNA3 can target mouse inner ear Tmc1 Bth mRNA. The above results confirm AAV-CasRx + sgRNA 3-mediated Tmc1 Bth Efficient and selective knock-out of transcripts in vivo.
TABLE 3
Samples Total reads Wt reads Mut reads Mut ratio Background
HE-T-1 628,899 457,113 161,862 0.261500061 0.015780
HE-T-2 693,555 623,612 59,743 0.087426008 0.014707
HE-T-4 646,412 573,649 61,993 0.097528168 0.016661
Het-CT-1 740,710 307,819 421,008 0.577651487 0.016043
Het-CT-2 979,293 425,342 502,410 0.541534807 0.052631
Het-CT-3 1,128,677 571,231 532,729 0.482561868 0.021899
HE-ut-1 1,943,326 781,915 1,123,868 0.589714569 0.019319
HE-ut-2 571,456 283,888 276,354 0.49327612 0.019624
HE-ut-3 801,693 391,520 394,741 0.502048302 0.019249
(III) in vivo RNA knock-out to prevent progressive hearing loss
CasRx disrupts Tmc1 guided by sgRNA3 Bth Transcript without affecting Tmc1 + The transcript, and thus the expected therapeutic effect, is obtained. When Tmc1 Bth When transcripts were disrupted and levels of harmful proteins were reduced, hearing was protected in Bth mice, while progressive hearing loss occurred in control mice following injection of AAV encoding non-targeting (NT) RNA (fig. 6). Since the Tmc1 mutation may lead to progressive hearing loss, ABR tests were performed every 4 weeks to measure the hearing function of the cochlea. We measured pure tone ABRs at frequencies of 4, 8, 16, 24 and 32kHz, and recording ABR waveforms at 8kHz showed that injection of CasRx + sgRNA3 improved hearing function over the uninjected control group (fig. 7 a). Four weeks after injection, bth mice injected AAV-CasRx + sgRNA3 had lower ABR thresholds at all frequencies (57 + -9 dB, 47 + -11 dB,65 + -8 dB, 70 + -8 dB, and 75 + -7 dB at 4, 8, 16, 24, and 32kHz, respectively) compared to the uninjected contralateral ear (77 + -5 dB, 67 + -13 dB,78 + -4 dB, and 82 + -5 dB at 4, 8, 16, 24, and 32kHz, respectively) (FIG. 7 b). ABR thresholds were not reduced in Bth mice 4 weeks after AAV-CasRx + NT injection (fig. 8 a), and both AAV-CasRx + sgRNA3 and AAV-Cas + NT had an effect on hearing in wild type mice (fig. 8 b). At 8 weeks post-injection, the ABR thresholds were raised in both ears, but the thresholds for treatment of the ears at low frequencies (72 ± 7dB,65 ± 9dB,78 ± 7dB, at 4, 8, 16 kHz) were still lower compared to the contralateral ear (84 ± 7dB,82 ± 6dB,87 ± 5dB, at 4, 8, 16 kHz) (fig. 7 b).
The clickABR hearing test was further performed and it was found that the in-ear threshold significantly decreased in the injected mice 4 and 8 weeks after injection, which is consistent with the pure tone ABR results, i.e., injection of AAV-CasRx + sgRNA3 slowed the progressive hearing loss in Bth mice (FIG. 7 c). At 4 weeks post-injection, bth mice injected with AAV-CasRx + sgRNA3 had increased amplitude at the clickABR peaks at 80dB and 90dB compared to Bth mice not injected, and the injected Bth mice were overall normalized (fig. 8c, d).
We also measured DPOAEs to assess the function of OHCs (fig. 7 d). At 4 and 8 weeks post-injection, ears of mice injected with AAV-CasRx + sgRNA3 had lower DPOAE thresholds (65 + -10 dB, 71 + -9 dB,8kHz and 169z, 4 weeks; 76 + -5 dB and 79 + -3 dB,8kHz and 16kHz,8 weeks) at 8kHz and 16kHz, whereas DPOAE was not detected in the non-injected ears, showing loss of OHCs function. These results indicate that AAV-CasRx + sgRNA 3-mediated mRNA knockdown can improve hearing function for over 8 weeks.
(IV) CasRx mediated protection of hair cell and statical fiber bundle morphology
To determine whether CasRx and sgRNA3 were able to protect hair cell and cilia bundle morphology, we sacrificed mice at 10 weeks of age for confocal and Scanning Electron Microscopy (SEM) analysis. It was found that OHCs in the apical coil (8 kHz zone) of the Corti organ began to be lost, and that the loss of OHCs from the middle coil (16 kHz zone) to the bottom coil (32 kHz zone) was more severe, with almost no OHCs in the bottom coil (32 kHz zone) (FIG. 9 a). The top coil IHCs remained intact, the middle coil IHCs were lost and the bottom coil IHCs were completely absent (fig. 9 a). These results are consistent with previous findings. After cochlear injection of AAV-CasRx + sgRNA3, both IHCs and OHCs had improved survival, with an increase in the number of OHCs per 100 μm per cochlea in both the 8kHz and 16kHz regions (37.0 + -1.6, 39.8 + -1.6 vs. 26.2 + -10.1, 22.6 + -9.1), and an increase in the number of immunohistochemistry in the 16kHz region (11.4 + -2.5 vs. 1.2 + -1.3) (FIG. 9 b).
Then, the fiber bundle morphology was observed by scanning electron microscope. Wild type Tmc1 +/+ OHCs and IHCs were aligned at 10 weeks of age in mice, whereas uninjected mice developed severe ciliary disturbance. Both apical loop IHCs and OHCs morphology remained normal following AAV-CasRx + sgRNA3 injection (fig. 9 c), and the middle loop retained the ciliated bundle (fig. 10). These results are consistent with the ABR data showing hearing protection in the lower frequency region (4-8 kHz).
(V) off-target analysis of CASRx-mediated RNA knockout in vivo
We performed RNA sequencing of cochlea collected 2 weeks after AAV injection. The sgRNA30bp sequences were aligned through the whole mouse genome, 10 genes most likely to be off-target were selected (fig. 11 a), and the expression difference between the AAV-CasRx + sgRNA 3-injected group (n =3 mice) and the non-injected group (n =3 mice) was analyzed. There was no difference in RNA expression of 9 of the 10 genes, one of which (Gm 13492) was not detected (fig. 11b and table 4). Nevertheless, there was essentially no difference in RNA expression overall, suggesting that CASRx-mediated RNA knock-out had no off-target effects.
TABLE 4
Figure BDA0003207028370000091
And (4) conclusion:
TMC1 mutations account for 4% -8% of the cases of inherited hearing loss worldwide. In the present invention, we down-regulated Tmc1 in a Bth mouse model of human genetic deafness using the CRISPR-CasRx System Bth mRNA transcripts, but not down-regulated Tmc1 + The transcript (there is only a single base difference between the two transcripts). A novel and efficient AAV-PHP.eB delivery system is adopted to deliver CasRx and sgRNA3 to a Bth mouse cochlea by taking hair cells as targets, and the auditory function is protected by improving the survival of the hair cells and improving ciliary bundle disorder. These results indicate that the CasRx system can successfully ameliorate dominant-negative effect hearing loss by specifically knocking out mutant transcripts.
The research result shows that the CasRx RNA editing system shows high knockout efficiency, and the Tmc1 can be caused by co-transfecting a vector for encoding exogenous Tmc1 sequence, casRx and sgRNA into 293T cells Bth The mRNA transcript was knocked out by more than 80%. CasRx RNA also showed in vivo RNA knockdown rates of over 70%, two major factors determining the efficient knockdown outcome: first, casRx is the smallest protein in the Cas13 family, which makes it easy to package into one AAV; second, different AAV serotypes have different transfection efficiencies, and several improved AAVs have been demonstrated to safely and efficiently deliver genes into the inner ear. eB vectors have previously been demonstrated to have extreme activity in cochlear IHCs and OHCs in vivoHigh transduction efficiency, it can deliver CasRx and sgRNA to hair cells, the transduction efficiency is over 95% (fig. 5).
CASRx-mediated RNA knockout can improve the auditory function of Bth mice. In our results, we detected that Bth mice had about 10-20dB improvement in hearing at 8 weeks and still prevented hearing loss at 12 weeks (data not shown), indicating that their hearing protection effect is comparable to Cas 9-based DNA editing techniques. The number of IHCs and OHCs in the treated group was greater than in the untreated group, which was essentially consistent with improved hearing. At the basement membrane bottom circle, bth mice disorganize the ciliary bundles, which prevents the transmission of sound, resulting in a higher ABR threshold at low frequencies. This explains why the hearing threshold at low frequencies is high, despite the presence of hair cells. The results show that the ciliated bundle morphology of IHCs and OHCs in the injected ear is significantly better than in the non-injected ear at the subcoil site, and hearing remains good at low frequencies. Furthermore, injection of control AAV (AAV-CasRx + NT) did not improve hearing in heterozygous mice (fig. 8 a), and we then injected AAV-CasRx + sgRNA3 and control AAV on wild type mice, and found no change in ABR threshold was observed compared to non-injected mice (fig. 8 b). The AAV-CasRx + sgRNA3 specifically inhibits hearing loss, and the AAV-CasRx + sgRNA3 injection is safe and does not affect normal hearing function. Despite the positive results, we have still found that AAV-CasRx + sgRNA3 injection still does not fully restore hearing function, especially in the high frequency region, compared to wild type mice. In future work, there is a need to improve targeting efficiency with more efficient AAV vectors to restore auditory function to a greater extent.
RNA editing techniques based on the CasRx system have certain advantages in the treatment of disease. At the level of RNA expression, casRx-mediated knockdown can significantly reduce off-target effects compared to RNA interference knockdown. Our RNA-seq data also showed little off-target effects. At the gene editing level, targeting RNA using CRISPR systems can avoid the risks associated with permanent DNA changes. The above studies of the present invention also compared the CasRx and PspCas13b systems, and it was found that CasRx has higher efficiency (see FIG. 2b, c) and specificity (see FIG. 2 d). A recent study showed that CasRx had no toxic effects, while PguCas13b and PspCas13b had a negative impact on embryonic development. These findings suggest that the CasRx system may be a safe and efficient RNA knock-out tool for future clinical applications.
In conclusion, the CasRx RNA is well applied to knock-out treatment of dominant-negative effect hearing loss, which shows that CasRx has great potential in treating dominant-negative effect hearing loss of human beings in the future.
Sequence listing
<110> eye, ear, nose and throat department hospital affiliated to the university of Compound Dan
<120> CRISPR/CasRx-based gene editing method and application thereof
<141> 2021-08-11
<160> 37
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 1
caaataagtc aaacagggtg ggacagaact 30
<210> 2
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 2
aaataagtca aacagggtgg gacagaactt 30
<210> 3
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 3
aataagtcaa acagggtggg acagaacttc 30
<210> 4
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 4
ataagtcaaa cagggtggga cagaacttcc 30
<210> 5
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 5
taagtcaaac agggtgggac agaacttccc 30
<210> 6
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 6
aagtcaaaca gggtgggaca gaacttcccc 30
<210> 7
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 7
agtcaaacag ggtgggacag aacttcccca 30
<210> 8
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 8
gtcaaacagg gtgggacaga acttccccag 30
<210> 9
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 9
tcaaacaggg tgggacagaa cttccccagg 30
<210> 10
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 10
caaacagggt gggacagaac ttccccagga 30
<210> 11
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 11
aaacagggtg ggacagaact tccccaggag 30
<210> 12
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 12
aacagggtgg gacagaactt ccccaggagg 30
<210> 13
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 13
acagggtggg acagaacttc cccaggaggg 30
<210> 14
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 14
cagggtggga cagaacttcc ccaggaggga 30
<210> 15
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 15
agggtgggac agaacttccc caggagggac 30
<210> 16
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 16
gggtgggaca gaacttcccc aggagggaca 30
<210> 17
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 17
ggtgggacag aacttcccca ggagggacat 30
<210> 18
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 18
gtgggacaga acttccccag gagggacatt 30
<210> 19
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 19
tgggacagaa cttccccagg agggacatta 30
<210> 20
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 20
gggacagaac ttccccagga gggacattac 30
<210> 21
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 21
ggacagaact tccccaggag ggacattacc 30
<210> 22
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 22
gacagaactt ccccaggagg gacattacca 30
<210> 23
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 23
acagaacttc cccaggaggg acattaccat 30
<210> 24
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 24
cagaacttcc ccaggaggga cattaccatg 30
<210> 25
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 25
agaacttccc caggagggac attaccatgt 30
<210> 26
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 26
gaacttcccc aggagggaca ttaccatgtt 30
<210> 27
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 27
aacttcccca ggagggacat taccatgttc 30
<210> 28
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 28
acttccccag gagggacatt accatgttca 30
<210> 29
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 29
cttccccagg agggacatta ccatgttcat 30
<210> 30
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 30
ttccccagga gggacattac catgttcatt 30
<210> 31
<211> 30
<212> DNA/RNA
<213> Artificial Sequence
<400> 31
accaggatgg gcaccacccc ggtgaacagc 30
<210> 32
<211> 6367
<212> DNA/RNA
<213> Artificial Sequence
<400> 32
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaacgg gccctctaga ctcgagatgg tgagcaaggg cgaggaggat aacatggcca 960
tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt 1020
tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga 1080
aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt 1140
acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct 1200
tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg 1260
tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca 1320
ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct 1380
ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc 1440
tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg 1500
tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg 1560
actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg 1620
acgagctgta caagagtgga tacctcatct tttgggctgt gaagcgatcc caggagttcg 1680
cccagcaaga tcctgacacc cttgggtggt gggaaaaaaa tgaaatgaac atggtaatgt 1740
ccctcctggg gaagttctgt cccaccctgt ttgacttatt tgctgaactg gaagattacc 1800
atcctctcat tgctctgaag tggctcctgg ggcgcatttt tgctcttctt ctaggcaact 1860
tgtatgtatt cattctcgcc ttgatggatg agattaacaa caagattgaa gaggagaagc 1920
ttgtgaaggc caataagctt aagtttaaac cgctgatcag cctcgactgt gccttctagt 1980
tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 2040
cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 2100
tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 2160
aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc 2220
tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 2280
acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 2340
ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 2400
ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 2460
ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 2520
acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 2580
tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 2640
atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa 2700
agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 2760
ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 2820
attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 2880
gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 2940
ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 3000
tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat 3060
gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg 3120
tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg 3180
tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 3240
ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc 3300
cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg 3360
aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca 3420
tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc 3480
aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg 3540
atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg 3600
cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 3660
tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg 3720
accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 3780
gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 3840
tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca 3900
agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt 3960
gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 4020
gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag 4080
caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 4140
gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt 4200
ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 4260
caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 4320
cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 4380
gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 4440
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 4500
ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4560
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4620
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4680
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4740
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4800
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4860
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4920
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4980
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 5040
cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 5100
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtttttttgt 5160
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 5220
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 5280
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 5340
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 5400
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 5460
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 5520
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 5580
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 5640
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 5700
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 5760
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 5820
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 5880
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 5940
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 6000
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 6060
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 6120
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 6180
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 6240
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 6300
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 6360
tgacgtc 6367
<210> 33
<211> 6367
<212> DNA/RNA
<213> Artificial Sequence
<400> 33
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaacgg gccctctaga ctcgagatgg tgagcaaggg cgaggaggat aacatggcca 960
tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt 1020
tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga 1080
aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt 1140
acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct 1200
tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg 1260
tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca 1320
ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct 1380
ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc 1440
tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg 1500
tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg 1560
actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg 1620
acgagctgta caagagtgga tacctcatct tttgggctgt gaagcgatcc caggagttcg 1680
cccagcaaga tcctgacacc cttgggtggt gggaaaaaaa tgaaatgaac atggtaatgt 1740
ccctcctggg gatgttctgt cccaccctgt ttgacttatt tgctgaactg gaagattacc 1800
atcctctcat tgctctgaag tggctcctgg ggcgcatttt tgctcttctt ctaggcaact 1860
tgtatgtatt cattctcgcc ttgatggatg agattaacaa caagattgaa gaggagaagc 1920
ttgtgaaggc caataagctt aagtttaaac cgctgatcag cctcgactgt gccttctagt 1980
tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 2040
cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 2100
tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 2160
aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc 2220
tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 2280
acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 2340
ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 2400
ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 2460
ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 2520
acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 2580
tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 2640
atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa 2700
agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 2760
ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 2820
attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 2880
gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 2940
ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 3000
tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat 3060
gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg 3120
tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg 3180
tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 3240
ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc 3300
cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg 3360
aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca 3420
tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc 3480
aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg 3540
atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg 3600
cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 3660
tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg 3720
accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 3780
gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 3840
tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca 3900
agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt 3960
gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 4020
gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag 4080
caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 4140
gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt 4200
ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 4260
caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 4320
cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 4380
gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 4440
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 4500
ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4560
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4620
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4680
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4740
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4800
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4860
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4920
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4980
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 5040
cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 5100
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtttttttgt 5160
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 5220
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 5280
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 5340
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 5400
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 5460
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 5520
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 5580
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 5640
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 5700
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 5760
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 5820
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 5880
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 5940
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 6000
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 6060
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 6120
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 6180
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 6240
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 6300
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 6360
tgacgtc 6367
<210> 34
<211> 8830
<212> DNA/RNA
<213> Artificial Sequence
<400> 34
gaacgcgcgg cgcaccggga agccctcgcc ctcgaaaccg ctgggcgcgg tggtcacggt 60
gagcacggga cgtgcgacgg cgtcggcggg tgcggatacg cggggcagcg tcagcgggtt 120
ctcgacggtc acggcgggca tgcggccgca taacttacgg taaatggccc gcctggctga 180
ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 240
atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 300
gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 360
cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 420
tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc 480
cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt 540
gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag 600
gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga 660
aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg 720
cgggcgggga gtcgctgcga cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc 780
gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc 840
cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc 900
tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg 960
gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct 1020
gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg 1080
cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc 1140
ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc 1200
ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta 1260
cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg 1320
gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggcccccgga 1380
gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg 1440
agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc 1500
gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg 1560
gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg 1620
gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct 1680
ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc 1740
tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattgg 1800
aattcgccgc caccatgcct aaaaagaaaa gaaaggtggg ttctggtatg gagaagaaga 1860
agagcttcgc caagggcatg ggagtgaaga gcaccctggt gtccggctct aaggtgtaca 1920
tgaccacatt tgctgaggga agcgacgcca ggctggagaa gatcgtggag ggcgatagca 1980
tcagatccgt gaacgaggga gaggctttca gcgccgagat ggctgacaag aacgctggct 2040
acaagatcgg aaacgccaag ttttcccacc caaagggcta cgccgtggtg gctaacaacc 2100
cactgtacac cggaccagtg cagcaggaca tgctgggact gaaggagaca ctggagaaga 2160
ggtacttcgg cgagtccgcc gacggaaacg ataacatctg catccaggtc atccacaaca 2220
tcctggatat cgagaagatc ctggctgagt acatcacaaa cgccgcttac gccgtgaaca 2280
acatctccgg cctggacaag gatatcatcg gcttcggaaa gttttctacc gtgtacacat 2340
acgacgagtt caaggatcca gagcaccacc gggccgcttt taacaacaac gacaagctga 2400
tcaacgccat caaggctcag tacgacgagt tcgataactt tctggataac cccaggctgg 2460
gctacttcgg acaggctttc ttttctaagg agggcagaaa ctacatcatc aactacggaa 2520
acgagtgtta cgacatcctg gccctgctga gcggactgag gcactgggtg gtgcacaaca 2580
acgaggagga gtctcggatc agccgcacct ggctgtacaa cctggacaag aacctggata 2640
acgagtacat ctccacactg aactacctgt acgacaggat caccaacgag ctgacaaaca 2700
gcttctccaa gaactctgcc gctaacgtga actacatcgc tgagaccctg ggcatcaacc 2760
cagctgagtt cgctgagcag tacttcagat tttccatcat gaaggagcag aagaacctgg 2820
gcttcaacat cacaaagctg agagaagtga tgctggacag aaaggatatg tccgagatca 2880
ggaagaacca caaggtgttc gattctatca gaaccaaggt gtacacaatg atggactttg 2940
tgatctacag gtactacatc gaggaggatg ccaaggtggc cgctgccaac aagagcctgc 3000
ccgacaacga gaagtctctg agcgagaagg atatcttcgt gatcaacctg agaggctcct 3060
ttaacgacga tcagaaggac gctctgtact acgatgaggc caacaggatc tggagaaagc 3120
tggagaacat catgcacaac atcaaggagt tccggggaaa caagacccgc gagtacaaga 3180
agaaggacgc tccaaggctg cctaggatcc tgcctgctgg aagggacgtg agcgccttca 3240
gcaagctgat gtacgccctg acaatgtttc tggacggaaa ggagatcaac gatctgctga 3300
ccacactgat caacaagttc gacaacatcc agtcttttct gaaagtgatg cctctgatcg 3360
gcgtgaacgc taagttcgtg gaggagtacg ccttctttaa ggacagcgcc aagatcgctg 3420
atgagctgcg gctgatcaag tcctttgcca ggatgggaga gccaatcgct gacgctagga 3480
gagctatgta catcgatgcc atccggatcc tgggaaccaa cctgtcttac gacgagctga 3540
aggctctggc cgacaccttc agcctggatg agaacggcaa caagctgaag aagggcaagc 3600
acggaatgcg caacttcatc atcaacaacg tgatcagcaa caagcggttt cactacctga 3660
tcagatacgg cgacccagct cacctgcacg agatcgctaa gaacgaggcc gtggtgaagt 3720
tcgtgctggg acggatcgcc gatatccaga agaagcaggg ccagaacgga aagaaccaga 3780
tcgaccgcta ctacgagacc tgcatcggca aggataaggg aaagtccgtg tctgagaagg 3840
tggacgctct gaccaagatc atcacaggca tgaactacga ccagttcgat aagaagagat 3900
ctgtgatcga ggacaccgga agggagaacg ccgagagaga gaagtttaag aagatcatca 3960
gcctgtacct gacagtgatc taccacatcc tgaagaacat cgtgaacatc aacgctagat 4020
acgtgatcgg cttccactgc gtggagcgcg atgcccagct gtacaaggag aagggatacg 4080
acatcaacct gaagaagctg gaggagaagg gctttagctc cgtgaccaag ctgtgcgctg 4140
gaatcgacga gacagccccc gacaagagga aggatgtgga gaaggagatg gccgagagag 4200
ctaaggagag catcgactcc ctggagtctg ctaaccctaa gctgtacgcc aactacatca 4260
agtactccga tgagaagaag gccgaggagt tcaccaggca gatcaacaga gagaaggcca 4320
agaccgctct gaacgcctac ctgaggaaca caaagtggaa cgtgatcatc cgggaggacc 4380
tgctgcgcat cgataacaag acctgtacac tgttccggaa caaggctgtg cacctggagg 4440
tggctcgcta cgtgcacgcc tacatcaacg acatcgccga ggtgaactcc tactttcagc 4500
tgtaccacta catcatgcag aggatcatca tgaacgagag atacgagaag tctagcggca 4560
aggtgtctga gtacttcgac gccgtgaacg atgagaagaa gtacaacgat agactgctga 4620
agctgctgtg cgtgcctttc ggatactgta tcccacggtt taagaacctg agcatcgagg 4680
ccctgttcga ccgcaacgag gctgccaagt ttgataagga gaagaagaag gtgagcggca 4740
actccggttc tggtctcgag cccaagaaga agaggaaagt cctcgaggct actaacttca 4800
gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctatgcat atggtgagca 4860
agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 4920
acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 4980
ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 5040
ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 5100
tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 5160
acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 5220
tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 5280
acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 5340
tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 5400
agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 5460
cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 5520
tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa acgctagcta 5580
gaatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 5640
tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 5700
tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 5760
gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 5820
tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 5880
tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 5940
gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 6000
cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 6060
caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 6120
tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg 6180
tcgacctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 6240
tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 6300
tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 6360
ggggaggatt gggaagacaa tagcaggcat gctggggata actttaaata attggcatta 6420
tttaaagtta acgcgtacaa gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat 6480
aaatatcaat atattaaatt agattttgca taaaaaacag actacataat actgtaaaac 6540
acaacatatc cagtcactat gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 6600
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 6660
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 6720
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 6780
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 6840
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 6900
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 6960
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 7020
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 7080
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 7140
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 7200
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 7260
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 7320
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 7380
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 7440
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 7500
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 7560
caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 7620
gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 7680
gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 7740
ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 7800
attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 7860
gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 7920
tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 7980
agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 8040
gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 8100
actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 8160
tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 8220
attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 8280
tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 8340
tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 8400
aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 8460
tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 8520
cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 8580
acctataaaa atcaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa 8640
attgatgagc aatgcttttt tataatgcca actttgtaca aaaaagcagg ctgtcgacga 8700
tgtaggtcac ggtctcgaag ccgcggtgcg ggtgccaggg cgtgcccttg ggctccccgg 8760
gcgcgtactc cacctcaccc atctggtcca tcatgatgaa cgggtcgagg tggcggtagt 8820
tgatcccggc 8830
<210> 35
<211> 8478
<212> DNA/RNA
<213> Artificial Sequence
<400> 35
gaacgcgcgg cgcaccggga agccctcgcc ctcgaaaccg ctgggcgcgg tggtcacggt 60
gagcacggga cgtgcgacgg cgtcggcggg tgcggatacg cggggcagcg tcagcgggtt 120
ctcgacggtc acggcgggca tgcggccgca taacttacgg taaatggccc gcctggctga 180
ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 240
atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 300
gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 360
cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 420
tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc 480
cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt 540
gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag 600
gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga 660
aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg 720
cgggcgggga gtcgctgcga cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc 780
gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc 840
cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc 900
tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg 960
gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct 1020
gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg 1080
cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc 1140
ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc 1200
ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta 1260
cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg 1320
gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggcccccgga 1380
gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg 1440
agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc 1500
gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg 1560
gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg 1620
gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct 1680
ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc 1740
tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattgg 1800
aattcgccgc caccatgaac atccccgctc tggtggaaaa ccagaagaag tactttggca 1860
cctacagcgt gatggccatg ctgaacgctc agaccgtgct ggaccacatc cagaaggtgg 1920
ccgatattga gggcgagcag aacgagaaca acgagaatct gtggtttcac cccgtgatga 1980
gccacctgta caacgccaag aacggctacg acaagcagcc cgagaaaacc atgttcatca 2040
tcgagcggct gcagagctac ttcccattcc tgaagatcat ggccgagaac cagagagagt 2100
acagcaacgg caagtacaag cagaaccgcg tggaagtgaa cagcaacgac atcttcgagg 2160
tgctgaagcg cgccttcggc gtgctgaaga tgtacaggga cctgaccaac cactacaaga 2220
cctacgagga aaagctgaac gacggctgcg agttcctgac cagcacagag caacctctga 2280
gcggcatgat caacaactac tacacagtgg ccctgcggaa catgaacgag agatacggct 2340
acaagacaga ggacctggcc ttcatccagg acaagcggtt caagttcgtg aaggacgcct 2400
acggcaagaa aaagtcccaa gtgaataccg gattcttcct gagcctgcag gactacaacg 2460
gcgacacaca gaagaagctg cacctgagcg gagtgggaat cgccctgctg atctgcctgt 2520
tcctggacaa gcagtacatc aacatctttc tgagcaggct gcccatcttc tccagctaca 2580
atgcccagag cgaggaacgg cggatcatca tcagatcctt cggcatcaac agcatcaagc 2640
tgcccaagga ccgcatccac agcgagaagt ccaacaagag cgtggccatg gatatgctca 2700
acgaagtgaa gcggtgcccc gacgagctgt tcacaacact gtctgccgag aagcagtccc 2760
ggttcagaat catcagcgac gaccacaatg aagtgctgat gaagcggagc agcgacagat 2820
tcgtgcctct gctgctgcag tatatcgatt acggcaagct gttcgaccac atcaggttcc 2880
acgtgaacat gggcaagctg agatacctgc tgaaggccga caagacctgc atcgacggcc 2940
agaccagagt cagagtgatc gagcagcccc tgaacggctt cggcagactg gaagaggccg 3000
agacaatgcg gaagcaagag aacggcacct tcggcaacag cggcatccgg atcagagact 3060
tcgagaacat gaagcgggac gacgccaatc ctgccaacta tccctacatc gtggacacct 3120
acacacacta catcctggaa aacaacaagg tcgagatgtt tatcaacgac aaagaggaca 3180
gcgccccact gctgcccgtg atcgaggatg atagatacgt ggtcaagaca atccccagct 3240
gccggatgag caccctggaa attccagcca tggccttcca catgtttctg ttcggcagca 3300
agaaaaccga gaagctgatc gtggacgtgc acaaccggta caagagactg ttccaggcca 3360
tgcagaaaga agaagtgacc gccgagaata tcgccagctt cggaatcgcc gagagcgacc 3420
tgcctcagaa gatcctggat ctgatcagcg gcaatgccca cggcaaggat gtggacgcct 3480
tcatcagact gaccgtggac gacatgctga ccgacaccga gcggagaatc aagagattca 3540
aggacgaccg gaagtccatt cggagcgccg acaacaagat gggaaagaga ggcttcaagc 3600
agatctccac aggcaagctg gccgacttcc tggccaagga catcgtgctg tttcagccca 3660
gcgtgaacga tggcgagaac aagatcaccg gcctgaacta ccggatcatg cagagcgcca 3720
ttgccgtgta cgatagcggc gacgattacg aggccaagca gcagttcaag ctgatgttcg 3780
agaaggcccg gctgatcggc aagggcacaa cagagcctca tccatttctg tacaaggtgt 3840
tcgcccgcag catccccgcc aatgccgtcg agttctacga gcgctacctg atcgagcgga 3900
agttctacct gaccggcctg tccaacgaga tcaagaaagg caacagagtg gatgtgccct 3960
tcatccggcg ggaccagaac aagtggaaaa cacccgccat gaagaccctg ggcagaatct 4020
acagcgagga tctgcccgtg gaactgccca gacagatgtt cgacaatgag atcaagtccc 4080
acctgaagtc cctgccacag atggaaggca tcgacttcaa caatgccaac gtgacctatc 4140
tgatcgccga gtacatgaag agagtgctgg acgacgactt ccagaccttc taccagtgga 4200
accgcaacta ccggtacatg gacatgctta agggcgagta cgacagaaag ggctccctgc 4260
agcactgctt caccagcgtg gaagagagag aaggcctctg gaaagagcgg gcctccagaa 4320
cagagcggta cagaaagcag gccagcaaca agatccgcag caaccggcag atgagaaacg 4380
ccagcagcga agagatcgag acaatcctgg ataagcggct gagcaacagc cggaacgagt 4440
accagaaaag cgagaaagtg atccggcgct acagagtgca ggatgccctg ctgtttctgc 4500
tggccaaaaa gaccctgacc gaactggccg atttcgacgg cgagaggttc aaactgaaag 4560
aaatcatgcc cgacgccgag aagggaatcc tgagcgagat catgcccatg agcttcacct 4620
tcgagaaagg cggcaagaag tacaccatca ccagcgaggg catgaagctg aagaactacg 4680
gcgacttctt tgtgctggct agcgacaaga ggatcggcaa cctgctggaa ctcgtgggca 4740
gcgacatcgt gtccaaagag gatatcatgg aagagttcaa caaatacgac cagtgcaggc 4800
ccgagatcag ctccatcgtg ttcaacctgg aaaagtgggc cttcgacaca taccccgagc 4860
tgtctgccag agtggaccgg gaagagaagg tggacttcaa gagcatcctg aaaatcctgc 4920
tgaacaacaa gaacatcaac aaagagcaga gcgacatcct gcggaagatc cggaacgcct 4980
tcgatcacaa caattacccc gacaaaggcg tggtggaaat caaggccctg cctgagatcg 5040
ccatgagcat caagaaggcc tttggggagt acgccatcat gaaggccggt agtgggagca 5100
acggcagcag cggatccctg cctccacttg aaagactgac actggggtcc ggtagatccc 5160
tcgaggctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac 5220
cttagctaga atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac 5280
tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt 5340
gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat 5400
gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca 5460
acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc 5520
cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg 5580
gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct 5640
tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct 5700
tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt 5760
ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat 5820
cgataccgtc gacctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 5880
ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 5940
aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 6000
acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggataac tttaaataat 6060
tggcattatt taaagttaac gcgtacaagt ttgtacaaaa aagctgaacg agaaacgtaa 6120
aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac 6180
tgtaaaacac aacatatcca gtcactatgc tgcattaatg aatcggccaa cgcgcgggga 6240
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 6300
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 6360
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 6420
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 6480
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 6540
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 6600
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 6660
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 6720
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 6780
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 6840
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 6900
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 6960
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 7020
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 7080
aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 7140
ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 7200
acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 7260
ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 7320
gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 7380
taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 7440
tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 7500
gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 7560
cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 7620
aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 7680
cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 7740
tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 7800
gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 7860
tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 7920
gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 7980
ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 8040
cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 8100
agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 8160
gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 8220
tgacattaac ctataaaaat caaataatga ttttattttg actgatagtg acctgttcgt 8280
tgcaacaaat tgatgagcaa tgctttttta taatgccaac tttgtacaaa aaagcaggct 8340
gtcgacgatg taggtcacgg tctcgaagcc gcggtgcggg tgccagggcg tgcccttggg 8400
ctccccgggc gcgtactcca cctcacccat ctggtccatc atgatgaacg ggtcgaggtg 8460
gcggtagttg atcccggc 8478
<210> 36
<211> 4995
<212> DNA/RNA
<213> Artificial Sequence
<400> 36
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 60
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 120
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 180
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 240
gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 300
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 360
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 420
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 480
tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 540
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 600
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 660
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 720
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 780
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 840
cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 900
ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagat 960
ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1020
agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 1080
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 1140
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 1200
cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 1260
gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 1320
tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 1380
tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 1440
aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 1500
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 1560
cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 1620
aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 1680
ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 1740
tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 1800
ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 1860
gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 1920
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 1980
cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 2040
agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 2100
aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 2160
gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 2220
aaatttaacg cgaattttaa caaaatatta acgcttacaa tttgccattc gccattcagg 2280
ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagcccaag 2340
ctaccatgat aagtaagtaa tattaaggta cgggaggtac ttggagcggc cgcaataaaa 2400
tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat 2460
acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg 2520
caagtgcagg tgccagaaca tttctctatc gataggtacc gattagtgaa cggatctcga 2580
cggtatcgat cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc 2640
atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat 2700
ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt 2760
gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact 2820
tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgcaagt 2880
aaacccctac caactggtcg gggtttgaaa cagaagagcc tcgaggctct tctcaagtaa 2940
acccctacca actggtcggg gtttgaaacg aagacttttt ttttcgcttc ctcgctcact 3000
gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 3060
atacggtcct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt 3120
ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa 3180
gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga 3240
gatccacttt ggccgcggct cgagggggtt ggggttgcgc cttttccaag gcagccctgg 3300
gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg gcgccgaccc 3360
tgggactcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct 3420
tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt 3480
cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc actagtaccc tcgcagacgg 3540
acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca atagcggctg 3600
ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg 3660
ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca agcctccgga 3720
gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc cccaggggga 3780
tccaccggag cttaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat 3840
cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga 3900
gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc 3960
cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta 4020
ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca 4080
ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt 4140
cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg 4200
caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc 4260
cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg 4320
cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct 4380
gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa 4440
gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga 4500
cgagctgtac aagtaacgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg 4560
accccatgca tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 4620
ctttctagag tcggggcggc cggccgcttc gagcagacat gataagatac attgatgagt 4680
ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg 4740
ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca 4800
ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc 4860
tctacaaatg tggtcgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 4920
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4980
gcaggaaaga acatg 4995
<210> 37
<211> 4814
<212> DNA/RNA
<213> Artificial Sequence
<400> 37
ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 60
cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 120
ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 180
cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 240
tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 300
tgtggaaagg acgaaacacc gaagtcttcg atatcgaaga cttgttgtgg aaggtccagt 360
tttgaggggc tattacaact ttttttaaag aattctcgac ctcgagacaa atggcagtat 420
tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag 480
tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc 540
aaaattttcg ggtttattac agggacagca gagatccact ttggccgcgg ctcgaggggg 600
ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg 660
cgtggttccg ggaaacgcag cggcgccgac cctgggactc gcacattctt cacgtccgtt 720
cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct 780
ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa 840
gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc 900
cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc 960
cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc 1020
gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt 1080
gaccgaatca ccgacctctc tccccagggg gatccaccat ggtgagcaag ggcgaggagc 1140
tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt 1200
tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca 1260
tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg 1320
gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg 1380
ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca 1440
agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg 1500
gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca 1560
gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga 1620
tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc 1680
ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc 1740
tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg 1800
ccgggatcac tctcggcatg gacgagctgt acaagtaagt acctttaaga ccaatgactt 1860
acaaggcagc tgtagatctt agccactttc tagagtcggg gcggccggcc gcttcgagca 1920
gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 1980
tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat 2040
aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg 2100
gaggtttttt aaagcaagta aaacctctac aaatgtggtc gcttcctcgc tcactgactc 2160
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 2220
gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 2280
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 2340
cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 2400
ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 2460
taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg 2520
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 2580
ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 2640
aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 2700
tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac 2760
agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 2820
ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 2880
tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 2940
tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 3000
cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 3060
aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 3120
atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 3180
cttaccatct ggccccagtg ctgcaatgat accgcgggac ccacgctcac cggctccaga 3240
tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 3300
atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 3360
taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 3420
tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 3480
gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 3540
cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 3600
cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 3660
gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 3720
aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 3780
accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 3840
ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 3900
gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 3960
aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 4020
taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag 4080
cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 4140
cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 4200
tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 4260
cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 4320
gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 4380
aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc 4440
gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 4500
caaaatatta acgtttacaa tttcccattc gccattcagg ctgcgcaact gttgggaagg 4560
gcgatcggtg cgggcctctt cgctattacg ccagcccaag ctaccatgat aagtaagtaa 4620
tattaaggta cgggaggtac ttggagcggc cgcaataaaa tatctttatt ttcattacat 4680
ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca tcaaaacaaa 4740
acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg tgccagaaca 4800
tttctctatc gata 4814

Claims (10)

1. A sgRNA targeting a Tmc1 mutant is characterized in that the sequence of the sgRNA is shown as SEQ ID NO. 3.
2. A fluorescent reporter vector targeting a Tmc1 mutant comprising the sgRNA sequence of claim 1.
3. A fluorescent reporter vector targeting a Tmc1 mutant comprising the sgRNA sequence of claim 1 and an AAV vector.
4. The expression vector targeting the Tmc1 mutant of claim 3, wherein the AAV vector is AAV-PHP.eB.
5. A method of constructing an expression vector targeting a Tmc1 mutant as claimed in any one of claims 2 to 4, comprising the steps of:
(1) Constructing a human source codon optimized CasRx gene expression plasmid;
(2) Constructing an sgRNA expression plasmid;
(3) Constructing a fluorescent expression plasmid of mCherry-Tmc1 containing a mutant gene.
6. The construction method according to claim 5, characterized by comprising the following specific steps:
(1) Constructing a human codon-optimized CasRx gene expression plasmid: firstly, synthesizing a CasRx gene, connecting two nuclear localization signal peptides, constructing the CasRx gene into a mammal expression vector, and leading the expression by a CAG promoter;
(2) Constructing an sgRNA expression plasmid: firstly, synthesizing two complementary single-stranded sgRNA oligonucleotides, annealing the two single-stranded sgRNAs to form a double strand, connecting the double-stranded sgRNAs to a cloning skeleton by using a BspQI enzyme cleavage site, and expressing the double-stranded sgRNAs by using a U6 promoter on the skeleton;
(3) Constructing a fluorescent expression plasmid of mCherry-Tmc1 containing a mutant gene: firstly, a 90bp sequence containing c.1234T > A mutation sites is synthesized, and the 3' end of the sequence is connected with an mCherry fluorescent gene to construct an expression vector, and the mCherry fluorescent gene is expressed by a CMV promoter.
7. The method of claim 6, wherein the annealing temperature is 55 ℃.
8. A CRISPR-CasRx system targeting a Tmc1 mutant comprising the sgRNA sequence of claim 1 and a gene sequence encoding a CasRx protein.
9. A kit targeting a Tmc1 mutant, comprising an expression CasRx protein and the expression vector of claim 3.
10. The use of a Tmc1 mutant-targeted sgRNA of claim 1, comprising use in the preparation of an immune cell medicament for the treatment of hereditary deafness.
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