CRISPR/CasRx based gene editing method and application thereof

文档序号：204248 发布日期：2021-11-05 浏览：13次中文

阅读说明：本技术 基于CRISPR/CasRx的基因编辑方法及其应用 (CRISPR/CasRx based gene editing method and application thereof ) 是由舒易来胡晓湘李耕林郑子文李果崔冲于 2021-08-11 设计创作，主要内容包括：本发明提供了一种靶向Tmc1突变体(c.1235T＞A；p.M412K)的sgRNA、表达载体、CRISPR-CasRx系统、试剂盒及其应用,属于基因工程技术领域。本发明中所述的靶向Tmc1突变体的sgRNA的序列如SEQIDNO.3所示,含有该sgRNA的CasRx在不干扰野生型Tmc1～(+)的情况下,能够使得Tmc1～(Bth)转录物减少82％,将基于AAV-PHP.eB载体的CasRx注入新生贝多芬小鼠内耳,发现Tmc1～(Bth)在2周内减少了70％,整个转录组没有检测到脱靶。本发明提供的sgRNA靶向Tmc1突变体的特异性高,能够提高Tmc1～(Bth)突变小鼠毛细胞存活率,使得纤毛束形态得以恢复,机械转导电流减少,对进行性听力损失有显著改善。(The invention provides sgRNA (small guide ribonucleic acid), an expression vector, a CRISPR-CasRx system, a kit and application of a Tmc1 mutant (c.1235T is more than A; p.M412K) targeting, and belongs to the technical field of genetic engineering. The sequence of sgRNA targeting Tmc1 mutant is shown as SEQ ID NO.3, and CasRx containing the sgRNA does not interfere with wild Tmc1 + In the case of (2), Tmc1 can be made Bth 82% reduction in transcripts, CasRx based on AAV-php. eb vector was injected into the inner ear of neonatal bazedoxifene miceTmc1 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.)

1. A sgRNA targeting a Tmc1 mutant, wherein the sequence of the sgRNA is shown in 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 according to claim 3, wherein the AAV vector is AAV-php.

5. A method of constructing an expression vector targeting the Tmc1 mutant according to any one of claims 2-4, comprising the steps of:

(1) constructing a human-derived 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 CasRx gene to express 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 for targeting a Tmc1 mutant, the kit comprising an expression CasRx protein and the expression vector of claim 3.

10. The use of a sgRNA targeting the Tmc1 mutant according to claim 1, comprising use in the preparation of an immune cell medicament for the treatment of hereditary deafness.

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 approximately 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, almost half of deafness cases are 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 inherited 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 signaling. Mutant TMC1(c.1235t > a; p.m412k) homologous to human TMC1(c.1253t > a; p.m418k), and bedufen (Bth) mice carrying this mutation exhibited DFNA36 hearing loss, and therefore Bth mice were suitable as a model 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 the vesicle glutamate transporter-3 (VGLUT3) 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 CRISPR-Cas9 system is introduced into the inner ear of a Bth model mouse to successfully improve the hearing loss. In addition, the gene function of the barcingto mice with the 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 target and 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 target RNA is transient and reversible, and the sequence and the structure of the gene are not influenced. In recent years, RNA regulation has been used to treat hearing loss in mice. For example, antisense oligonucleotides are used to treat Ush1c (c.216G > A) gene mutations in mouse model hearing that disrupt wild-type splicing, produce frame-shift mutations and translate 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 the Cas13 protein is a CRISPR-Cas system effector protein of type 2 and type VI, has RNA-mediated RNA enzyme digestion activity, is the only protein which is discovered by the second major CRISPR-Cas system at present and can degrade RNA, 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 C2C2), Cas13b, Cas13C, and Cas13 d. PspCas13b and CasRx (RfxCas13d) 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 the CRISPR-Cas13 RNA editing system for the treatment of genetic deafness. Whether the CRISPR-Cas13 RNA editing technology is 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, a 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-stranded sgRNA oligonucleotides, annealing the two single-stranded sgRNAs to form double strands, connecting the sgRNAs to a cloning skeleton by using a BspQI enzyme cutting site, and expressing the sgRNAs 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 fluorescent expression plasmid into an expression vector to be expressed by a CMV promoter.

The invention also provides a CRISPR-CasRx system targeting the 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 (2), Tmc1^BthTranscript 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^BthThe decrease was 70% within 2 weeks and little off-target was detected at the transcriptome level. We found that hair cell survival was increased and ciliary bundle morphology was restored, with a decrease in mechanical transduction current after targeting at 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 an expression vector as described above.

The invention further provides an application of the sgRNA targeting the Tmc1 mutant, and the application of the sgRNA in preparing an immune cell medicament for treating hereditary deafness.

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 born mouse in inner ear hair cells, and successfully reduces Tmc1^BthExpression 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 potential clinical approach to the treatment of genetic deafness.

Drawings

FIG. 1 is a design targeting Tmc1^BthSchematic of sgRNAs of transcripts.

FIG. 2 shows screening targeting Tmc1^BthEfficient specificity of sgRNAs of the transcript; a. constructing sgRNA screening for Cas RNA editing system mediation; constructing a Cas expression vector and mCherry-Tmc1^BthFluorescent reporter gene, mCherry-Tmc1⁺Fluorescent reporter, targeting Tmc1^BthA sgRNA expression vector of the transcript and a non-targeting (NT) sgRNA expression vector; CasRx System mediated targeting of mCherry-Tmc1^BthmRNA and mCherry-Tmc1⁺sgRNA of mRNA and sgRNA of control group (nt) fluorescence intensity ratio, data are expressed as mean ± SD (n ═ 3 bio-independent samples); sgRNA and control sgRNA (NT) system-mediated mCherry-Tmc1 against PspCas13b^BthmRNA and mCherry-Tmc1⁺mRNA fluorescence intensity ratio, data expressed as mean ± SD (n ═ 3 bio-independent samples); mCherry-Tmc1 mediated by CasRx and PspCas13b^BthAnd mCherry-Tmc1⁺The average fluorescence intensity ratio of mRNA interference, of which sgRNAs 3 were the lowest among 30 sgRNAs; integrated fluorescence intensity of cells mediated by the casrx system; and mCherry-Tmc1⁺In comparison to mRNA, sgRNA3 targets mCherry-Tmc1^BthThe fluorescence density of mRNA is significantly reduced; targeting mCherry-Tmc1 with non-targeting sgRNA^BthAnd mCherry-Tmc1⁺Comparison of mRNAThe sgRNA3 targets mCherry-Tmc1⁺A decrease in fluorescence density of the mRNA; data are expressed as mean ± SD (n-2, biologically independent sample), 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 a 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 is Tmc1 from CasRx Selective disruption of Bth mice^Bth(ii) a transcript; a. schematic representation of AAV vector encoding CasRx and sgRNA3 (upper panel) and control NT vector (lower panel); b. summary of in vivo experiments; injecting mice in P1-P2, dissecting and culturing Corti organs at P5 days, analyzing hair cell physiology by P15-P16, sequencing after 2 weeks of injecting mice, performing hearing tests (ABR and DPOAE) after 4, 8 and 12 weeks, immunohistochemistry, and performing scanning electron microscope observation after 10 weeks of injecting; c.Tmcc 1^BthAnd Tmc1⁺Percent deep sequencing; pie chart display Tmc1 in the sample^BthAnd Tmc1⁺Average composition of transcripts, sequence shown as Tmc1^BthAnd Tmc1⁺Single nucleotide differences in transcripts (non-injected, AAV-CasRx + NT injected, and Tmc1 with AAV-CasRx + sgRNA3 injected)^BthTranscripts 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 of non-injected mice (n ═ 3), AAV-CasRx + NT injected mice (n ═ 3) and AAV-CasRx + sgRNA3 injected mice (n ═ 3) Tmc1^BthAnd Tmc1⁺Transcript ratios between, data expressed as mean ± sem, # p<0.01, the p value is determined by one-way analysis of variance using Dunnett multiple comparison test; e. cochlear mRNA expression was tested 2 weeks after RT-qPCR injection (n ═ 6 mice); tmc1 of the ear with AAV-CasRx + sgRNA3 compared to the ear without AAV-CasRx + sgRNA3^BthExpression of transcripts was significantly reduced, data expressed as mean ± sem,. p<0.05, adopting unpaired double-tail t test for p value; f. amplification of Tmc1^BthDetecting 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^+/+Mice (n ═ 16 OHCs), non-injected Tmc1^Bth/BthMice (n ═ 18 OHCs) and Tmc1 injected with AAV-CasRx + sgRNA3^Bth/BthThe 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 are expressed as mean ± SD × p<0.01, p-value was determined unidirectionally using analysis of variance using the Sidak multiple comparison test.

FIG. 5 is a transduction assay of AAV-PHP.eB; cochlear samples were collected 2 weeks after injection and stained with anti-GFP and myostatin 7a antibodies, and images were taken of whole cochlear tissues with a full 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 ears of (a), ABR waveform recorded at 8kHz at 4 weeks, green trace representing threshold; b, c.Tmc1^+/+(Green), Tmc1^Bth/++ AAV-CasRx + sgRNA3 (blue) and Tmc1^Bth/+Pure tone and clickABR thresholds in the non-injected contralateral (red) ear, 4 and 8 weeks later, AAV-CasRx + sgRNA3 vs non-injected 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 thresholds of untreated contralateral (red) ears at 4 and 8 weeks, with a significant decrease in DPOAE thresholds in two frequency AAV-CasRx + sgRNA3 injected ears, 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 ears of wild type mice injected with AAV (AAV-CasRx + sgRNA3 or AAV-CasRx + NT) and non-injected contralateral ears at 4 weeks of age, data are expressed as mean + -SD; c, d. 4 weeks at AAV-CasRx + sgRNA3 injection, non-Bth injected mice and wild-type mice, Click-ABR-wave 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 from Tmc1^+/+、Tmc1^Bth/++ AAV-CasRx + sgRNA3 and Tmc1^Bth/+Non-injected mice (n ═ 5) at positions 8kHz and 16kHz, 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 apical end of the cochlea show the morphology of the fiber hair bundles, and Tmc1 was 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 taken 10 weeks after 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 mismatch with the targeted site is highlighted in color, the 30bp sequence (On-target) targeted by sgRNA3 is shown 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 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.

First, embodiment of the method

(one) in vitro use of CasRx specific knockout Tmc1^BthTranscript

To knock out Tmc1 efficiently and specifically in HEK293T cells^BthmRNA, 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^BthsgRNAs 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 sgRNA1-30 and NT are shown in Table 1, see SEQ ID No.1-31 in the sequence listing), and Tmc1 was targeted^BthSchematic 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 backbone which comprises two direct repetitive sequences cloned by BspQI enzyme; the backbone of the PspCas13b sgRNA clone was constructed containing a direct 3' repeat cloned with the BbsI enzyme. sgRNAs were further synthesized as single-stranded DNA oligonucleotides. The two sgRNA oligonucleotides complementary in the opposite direction 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 the sgRNA expression vector of the PspCas13b system containing the U6 promoter element.

To construct mCherry-Tmc1^BthReporter gene vector, containing the c.1235T > A mutation Tmc1 transcript 90bp sequence, and its cloning to mCherry gene 3' end, the end removed stop codon. To construct mCherry-Tmc1⁺Reporter gene vector, a 90bp sequence of the Tmc1 transcript containing c.1235T was synthesized and cloned into 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 5% CO at 37 ℃₂Culturing under the conditions of (1). Before transfection, cells were seeded on poly D-lysine coated 24-well plates with 60-70% confluency maintained.

Cell transfection EZ Trans reagent (Shanghai Life iLab) was used, following the manufacturer's protocol. At the time of transfection, a CasRx or PspCas13b plasmid (600ng), an sgRNA plasmid (300ng), mCherry-Tmc1 were mixed and expressed in each well^BthOr 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. eb serotype) viral vectors were produced by synechial biotechnology (shanghai) ltd, AAV carrying the double transgene, sgRNA targeting Tmc1 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 had a viral titer of 3.38X 10¹²vg/mL, viral titer of AAV-CasRx + NT 1.73X 10¹²vg/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. Hybrid Beethofen mice (Tmc 1)^Bth/+) Given by Driew Griffith (university of Telaviv, Sackler medical school of human genetics and molecular medicine). Mixing 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 mice injected through round window membrane1.5 μ L of virus. 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 slowly injected into the right cochlea through the round window membrane using glass microtubules. 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/BthOr Tmc1^BthThe mouse cochlea of (1) in DMEM (1X) medium containing 1% fetal bovine serum, at 37 ℃ and 5% CO₂And (5) culturing. Whole cell patch clamp records the expression of inner hair cells at P14-P15, containing in standard artificial perilymph fluid (unit: mM): 137NaCl, 5.8KCl, 1.3CaCl₂、0.9MgCl₂、0.7NaH₂PO₄10HEPES and 5.6D-glucose, adjusting the pH to 7.40 and the osmotic pressure to 300 mmol/kg. Extraction of a recording pipette from a borosilicate glass capillary tube (1B150F-4, world precision instruments, florida, usa), the internal solution contained (unit: mM): 140CsCl, 0.1EGTA, 1MgCl₂10HEPES, 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 hair cell cilia bundle is deflected by spraying a liquid stream through a tube with a tip of-10 μm at a distance of-15 μm. The jet was driven by a piezoelectric disc (27 mm diameter) applying a sinusoidal voltage (40Hz, ± 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 products were 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 anticomplement 5'-GGT GGG ACA GAA CAT CCC CAG GAG GGA CAT-3'), mutant sequences (5'-ATGTCC CTC CTG GGGAAG TTC TGT CCC ACC-3') and its anticomplement (5'-GGT GGGACAGAACTT CCC CAG GAG GGACAT-3').

The calculation formula of the knockout efficiency is as follows:

RT-qPCR of (eleven) cochlea

The cochlea of both wild-type and heterozygous mice was dissected, total mRNA was extracted from the removed cochlea tissue using trizol (invitrogen), and the mRNA was reverse transcribed 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: 5min at 95 ℃, 10s at 95 ℃, 35s at 60 ℃ and 40 cycles.

Primers q-TMC1-F2 and q-TMC1-R2 were designed to detect the total expression level of Tmc 1. In order to detect the expression of mutant transcripts, a forward primer q-TMC1-F4 was designed, the 3' end of which is base A that specifically binds to the mutant sequence, and a reverse primer q-TMC1-R2 (the primer sequences are shown in Table 2).

TABLE 2 primers used for plasmid construction, genotyping, deep sequencing and RT-PCR

(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 using the rnalibry Prep Kit for Illumina (NEB, USA), according to manufacturer's recommendations, and an index code was added to the attribute sequence for 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 for determining differential expression in digital gene expression data using a model based on negative binomial distribution. 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 (10mg/kg) and ketamine (100mg/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 delivered 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 (ground electrode).

The acoustic stimulus was a square pulse of 100 ms duration, with a3 ms duration 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 tones is 1.2(f2/f1), and the f2 level of each pair of f2/f1 is 10dB lower than the f1 level by SPL. 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 level of f2 required to produce DPOAEs above 0 dB.

(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. Perforating the top of the cochlea of the temporal bone, perfusing with 4% paraformaldehyde, incubating overnight at 4 ℃, and decalcifying for 1-3 days at 10% EDTA 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 h, incubated overnight at 4 ℃ with primary anti-rabbit anti-MYO 7A (#25-6790, diluted by Proteus BioSciences, 1: 800), incubated with secondary antibody Cy3 coupled donkey anti-rabbit IgG (AlexJackson ImmunoResearch 711 and 165-152, diluted 1: 500) in the dark for 2 h, and washed three times with PBS at room temperature. 2 weeks old mice were dissected without decalcification, and transduction efficiency of AAV was examined using a chicken-resistant anti-EGFP (Abcam, 1:1000 dilution) by the same procedure as above. The specimens were mounted on an adhesive microscope slide (#188105, Citotest) 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 the 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, 2.5% glutaraldehyde was perfused from the hole, 2.5% glutaraldehyde was fixed at 4 ℃ overnight, 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 3 min. Scanning electron microscope images were obtained with a high vacuum field emission scanning electron microscope (Hitachi 575SU-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.

Second, example of Experimental results

(one) in vitro use of CasRx specific knockout Tmc1^BthTranscript

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). Then the reporter groups, PspCas13b or CasRx expression vectors, were co-transfected, the sgRNA expression vectors were transfected into 293T cells, 48h after transfection, the expression of the fusion RNA was interrupted by the RNA editing system, and the fluorescence intensity of the cells was measured as an indicator of RNA knock-out efficiency. As expected, CasRx and PspCas13b resulted in a significant decrease in mCherry expression in 293T cells (fig. 3). Targeting mCherry-Tmc1 when using the CasRx and PspCas13b systems^BthThe lowest fluorescence intensity ratios of the cells were 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. 2d) and mCherry-Tmc1 was analyzed^BthAnd mCherry-Tmc1⁺mean fluorescence intensity ratio between mRNA interferences. sgRNA3 ratio was lowest in the CasRx system, 0.089113 (fig. 2d), with a significant 82% reduction in mCherry integration density (fig. 2e), indicating Tmc1^BthTranscripts were knocked out efficiently. Taken together, these results indicate that sgRNA3 in CasRx is an effective knock-out of Tmc1^BthTranscripts without interfering with Tmc1⁺Ideal sgRNA of transcript. (II) CasRx mediates specific targeting in vivo

The results of the above studies indicate that sgRNA3 in the CasRx system is directed to Tmc1 in 293T cells^BthThe transcript has the highest targeting efficiency, can be used for judging whether the AAV-CasRx + sgRNA3 is effective in vivo or not, and encodes an AAV vector Tmc1 of sgRNA3 and CasRx^BthDownregulation of Tmc1 in the inner ear of Bth mice^BthThe transcript, non-targeted sgRNA packaged in the same vector as the control: (Fig. 4 a). We used AAV9 variant as a delivery vector, which is 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 organ Corti dissected for immunohistochemistry (fig. 4 b). We observed nearly 100% efficiency of viral transduction in IHCs and OHCs, and found that the efficiency of viral transduction was over 95% with a gradual decrease from top to bottom circles (fig. 5), consistent with our previous study.

To determine the editing ability of CasRx in vivo, whole cochlear tissue was subjected to targeted depth sequencing. Bth mouse cochlea display of AAV-CasRx + sgRNA 3-injected Tmc1^BthTranscripts accounted for 14.88 ± 9.77% of total Tmc1 transcripts, significantly reduced compared to non-injected mice (52.83 ± 5.33%) and mice injected with AAV-CasRx + NT (control AAV, non-targeting sgRNA) (53.39 ± 4.8%) (fig. 4c and table 3). Tmc1 of non-injected, AAV-CasRx + NT, and AAV-CasRx + sgRNA3^BthAnd Tmc1⁺The actual ratios of transcripts were 1.1397. + -. 0.2584, 1.1605. + -. 0.2183 and 0.186. + -. 0.1457, respectively, (FIG. 4d), indicating Tmc1^BthThe gene knockout efficiency was 70%.

In order to further aim at Tmc1^BthAnalysis 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^BthcDNA (FIG. 4f), results show control Tmc1^BthThe mRNA was reduced to 54.69-10.49% (FIG. 4 e).

Further, to detect the specific targeting of CasRx and sgRNA3 to the mutant Tmc1 site, MET current was measured. Tmc1^BthMutations do not affect the sensitivity of hair cell mechanical transduction, but knock-out of Tmc1 results in a decrease in MET current. Both Tmc1 and Tmc2 are essential for MET, Tmc2 is transiently expressed in the first week after birth and then disappears from immunohistochemical static cilia at P10, while Tmc1 is continuously expressed. Therefore, to eliminate the contribution of Tmc2 to the MET current, we used P15-P16Mice intracochlear hair cells, MET current was measured at equal amounts of P15-P16 for corii in vitro cultured organ inner hair cells (fig. 4 g). Wild type Tmc^+/+Mice and no injection of Tmc1^Bth/BthMouse apical inner hair cells showed similar MET current amplitude, whereas Tmc1 injected with AAV-CasRx + sgRNA3^Bth/BthMET current amplitude was significantly reduced in the mouse apical coil inner hair cells (fig. 4h), suggesting that CasRx + sgRNA3 can target mouse inner ear Tmc1^BthmRNA. The above results demonstrate AAV-CasRx + sgRNA 3-mediated Tmc1^BthEfficient 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^BthTranscripts, without affecting Tmc1⁺The transcript, and thus the expected therapeutic effect, is obtained. When Tmc1^BthWhen transcripts were disrupted and levels of deleterious proteins decreased, 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 injected cochlea. We measured pure tone ABRs at frequencies of 4, 8, 16, 24 and 32kHz, and the ABR waveforms recorded at 8kHz showed that the injection of CasRx + sgRNA3 improved hearing function over the uninjected control group (fig. 7 a). Four weeks after injection, Bth mice were injected with AAV-CasRx + sgRNA3 at ABR thresholds (57 + -9 dB, 47 + -11 dB, 65 + -8 dB, 70 + -8 dB, and 75 + -7 dB at 4, 8, 16, 24, 32kHz, respectively) at all frequencies and uninjected contralateral ears (7 at 4, 8, 16, 24, 32kHz, respectively)7 ± 5dB, 67 ± 13dB, 78 ± 4dB and 82 ± 5dB) are all lower (fig. 7 b). ABR thresholds were not reduced in Bth mice 4 weeks after AAV-CasRx + NT injection (fig. 8a), 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, ABR thresholds were raised in both ears, but the thresholds at low frequencies (72 ± 7dB, 65 ± 9dB, 78 ± 7dB, at 4, 8, 16kHz) were still lower in the treated ears compared to the contralateral ears (84 ± 7dB, 82 ± 6dB, 87 ± 5dB, at 4, 8, 16kHz) (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 one-wave amplitude at clickbar peaks of 80dB and 90dB compared to non-injected Bth mice, and the injected Bth mice tended to be normal overall (fig. 8c, d).

We also measured DPOAEs to assess the function of OHCs (fig. 7 d). Ears of mice injected with AAV-CasRx + sgRNA3 had lower DPOAE thresholds at 8kHz and 16kHz (65 + -10 dB, 71 + -9 dB, 8kHz and 16kHz, 4 weeks; 76 + -5 dB and 79 + -3 dB, 8kHz and 16kHz, 8 weeks) at 4 and 8 weeks post-injection, whereas no DPOAE was detected in the non-injected ears, showing loss of function of OHCs. 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 (8kHz zone) of the organ of Corti began to be lost, and the loss of OHCs from the middle coil (16kHz zone) to the bottom coil (32kHz zone) was more severe, with almost no OHCs in the bottom coil (32kHz 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 immunohistochemical number 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. 9c), and the middle loop retained the ciliated bundle (fig. 10). These results are consistent with the ABR data showing protection of hearing 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 over the whole mouse genome, and the 10 genes most likely to be off-target were selected (fig. 11a), and the AAV-CasRx + sgRNA 3-injected group (n-3 mice) was analyzed for expression differences from the non-injected group (n-3 mice). There was no difference in RNA expression of 9 of the 10 genes, one of which (Gm13492) was not detected (fig. 11b and table 4). Nevertheless, there was essentially no difference in RNA expression overall, suggesting that CASRx-mediated RNA knockdown had no off-target effects.

TABLE 4

And (4) conclusion:

the TMC1 mutation accounts 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^BthmRNA transcripts, but not down-regulated Tmc1⁺The transcript (there is only a single base difference between the two transcripts). The hair cells are used as targets, a novel and efficient AAV-PHP.eB delivery system is adopted to deliver CasRx and sgRNA3 to a Bth mouse cochlea, 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 improve dominant-negative effector function by specifically knocking out mutant transcriptsThe force is lost.

The results of the study show that the CasRx RNA editing system shows high knockout efficiency, and the co-transfection of the vector encoding the exogenous Tmc1 sequence, CasRx and sgRNA into 293T cells can enable Tmc1^BthThe 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 AAV have been shown to deliver genes safely and efficiently into the inner ear. Eb vectors have previously demonstrated very high transduction efficiency in cochlear IHCs and OHCs in vivo, delivering CasRx and sgRNA to hair cells with transduction efficiency 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. 8a), 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). It is demonstrated that AAV-CasRx + sgRNA3 specifically inhibits hearing loss, and injection of AAV-CasRx + sgRNA3 is safe and does not affect normal hearing function. Despite the positive results, we have still found that the injection of AAV-CasRx + sgRNA3 still did not fully restore auditory function, particularly in the high frequency region, compared to wild type mice. In future work, there is a need to improve targeting efficiency using 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 study of the present invention also compared the CasRx and PspCas13b systems, and 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 negative effects 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

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<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