Compositions for use in treating haploinsufficiency diseases

Abstract

Provided herein are compositions and methods to treat a disease or condition associated with haplo-insufficient genes comprising administering to a subject in need thereof a dCas protein and one or more gRNA, wherein the one or more gRNA is complementary to a miRNA response element (MRE) on target mRNA expressed from the functioning allele of the haplo-insufficient gene so as to cause dCas / gRNA binding to the MRE thereby blocking miRNA repression of the functioning allele.

Description

[0001] COMPOSITIONS FOR USE IN TREATING HAPLOINSUFFICIENCY DISEASES

[0002] PRIORITY

[0003] This application claims the benefit of the filing date of U.S. provisional application No. 63 / 650,751, filed on May 22, 2024, the disclosures of which is incorporated by reference herein in its entirety.

[0004] GOVERNMENT GRANT SUPPORT

[0005] This invention was made with government support under R01NS121223 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0006] Appropriate protein levels are a fundamental feature of gene expression. In fact, haploinsufficiency (loss-of-function of one allele leading to reduced protein levels) can result in devastating diseases, such as epilepsy and autism spectrum disorder.

[0007] SUMMARY

[0008] Identifying strategies to restore protein levels to a physiological range is paramount for developing treatment to disease with low or insufficient expression. MicroRNAs (miRNAs) can tightly regulate protein levels by repressing translation of their mRNA targets. Intriguingly, of the -700 human haplo-insufficient genes known to cause disease, a majority is strongly targeted by miRNAs, suggesting that miRNAs can exacerbate these diseases. Thus, a method to restore pathologically low levels of haplo-insufficient genes by removing miRNA repression from the remaining healthy allele is provided herein. However, this method has not yet been explored as the current tools for mapping and manipulating miRNA-target interactions (MTIs) lack the necessary spatial resolution. This bottleneck was tackled herein through the use of cutting-edge methods to map MTIs in, for example, specific brain cell types, the first step in identifying target sequences for rescuing levels of haplo-insufficient genes.

[0009] The state-of-the-art tool for manipulating MTIs is target site blockers (TSBs), which are antisense oligonucleotides (ASOs) designed to compete with specific MTIs. Since ASOs that modulate splicing are already FDA-approved drugs, TSBs, which have not yet been tested in humans, have enormous clinical potential. Despite these advantages, ASOs need to be administered repeatedly to maintain proper dosage, a problem exacerbated for neurological disorders where ASOs must be delivered intrathecally. For this reason, a novel RNA-protein complex was engineered that prevents miRNAs from repressing their target mRNAs, thereby permanently restoring protein levels with a single treatment. As proof-of-concept, the approach was tested on in vitro models of haplo- insufficiency, focusing on the gene Sptbnl. It found that Sptbnl is strongly repressed by miRNAs, and its haploinsufficiency is known to cause intellectual disability and autistic features. The results show that the novel RNA-protein complex can restore Sptbnl protein levels and rescue the typical axonal defects observed in mouse neurons that are heterozygous for Sptbnl in vitro. This approach can be applied to any haplo-insufficient gene that is repressed by miRNAs within and outside the brain.

[0010] Provided herein are compositions and methods for mapping and manipulating miRNA-mRNA target interactions (MTIs) to de-repress the target, for unraveling miRNA- mediated mechanisms underlying, for example, neural development, and developing therapeutics for treating diseases caused by reduced protein levels.

[0011] RNA-targeting catalytically inactive nuclease “dead” Casl3 proteins (dCasl3) was repurposed to bind to specific miRNA binding sites on mRNAs to outcompete the miRNA machinery and remove miRNA regulation from that site. dCasl3s can be programmed to bind specific sequences by designing their guide RNAs (gRNAs) complementary to that sequence. dCasl3s have been used to outcompete the splicing machinery and to track RNA for visualization but this the first to show that it can disrupt MTIs, providing novel uses.

[0012] Also provided herein is the use of a modified Ago2 CLIPseq (crosslinking immunoprecipitation and sequencing of Argonaute 2 (Ago2), the main effector protein of miRNAs) method. The method relies on a conditional knock in mouse line in which endogenous Ago2 is tagged with SpyTag3 only in Cre-expressing cells. UV light is used on the sample cells or tissue to crosslink and stabilize the miRNA-Ago2-mRNA target complex. The mRNA is fragmented with RNase so that Ago2-protected target sequences are spared. Beads couple to SpyCatcher3, which forms a covalent bond with SpyTag3, are then used to pull down the complex and the miRNA and mRNA fragments are sequenced to identify naturally occurring MTIs. The method was used to map MTIs different neuronal subtypes of the mouse brain to identify bona fide miRNA binding sites (specific sequences within the footprint of Ago2, mostly in the 3’UTR) that are potentially amenable to derepression. Intriguingly, in cortical excitatory pyramidal neurons, cortical GABAergic interneurons and cerebellar Purkinje cells, 148 targets were identified that are haploinsufficient genes (where loss of one allele results in reduced protein levels and abnormal phenotypes) known to cause ASD or epilepsy. These targets include Abcdl, Actb, Adnp, Ahdcl, Akt3, Ank2, Ankrdl l, Apls2, Arid la, Arid2, Arx, Ash 11, Asxll, Atplal, Atp8a2, Atrx, Auts2, Brwd3, Camtal, Cavl, Cdkl5, Chll, Cic, Cnksr2, Crebbp, Ctcf, Ctnnbl, Ctnnd2, Dex, Ddx3x, Dffb, Dmd, Dnmt3a, Dpp6, Dyrkla, Erf, Fgfrl, Flna, Foxp2, Fus, Gabral, Gatad2b, Gria2, Gria3, Grinl, Grin2a, Grin2b, Gtf2i, Hdac4, Hicl, Hivep2, Hmga2, Hnmpk, Hnmpu, Huwel, Ids, Iqsec2, Kenai, Kcnab2, Kcnbl, Kcnq2, Kdm6a, Kifla, Kiflb, Kmt2a, Kmt2b, Kmt2c, Kmt2d, Llcam, Magel2, Magtl, Map2kl, Mapt, Mbd5, Mecp2, Medl31, Mef2c, Meis2, Mllt3, Mytll, Nckapl, Nexmif, Nfl, Nf2, Nfia, Nfix, Nrxnl, Nsdl, Ocrl, Pafahlbl, Pak2, Pak3, Pax6, Pcdhl9, Pdhal, Pgapl, Phf6, Phip, Plpl, Pnpla6, Prrt2, Pten, Puml, Pura, Qrichl, Rbfoxl, Rein, Rps6ka3, Rtn4r, Satb2, Senia, Scn2a, Scn8a, Setbpl, Setd5, Shank3, Six3, Slc2al, Slc5a3, Slc6al, Slc6a8, Slc9a6, Smarca2, Smarccl, Smcla, Snca, Son, Soxl l, Sox8, Sptanl, Sptbnl, Sptbn2, Stxbpl, Suzl2, Syngapl, Tbkl, Tbrl, Tcf4, Trio, Tscl, Tspan7, Vampl, Wdfy3, Wdr26, Ywhae, Zdhhc9, Zeb2 or Zic2. The miRNA binding sites that were identified can be targeted to outcompete miRNA repression at the site to restore protein levels and rescue disease phenotypes. This can be achieved either via dCasl3 or other forms of MTI manipulation like antisense oligonucleotides (ASOs) called target site blockers (TSBs).

[0013] Further provided is the identification of miRNA binding sites in the 3’UTR (ACAAAGTAATGTTACTCTAATGGTTACTTGCTTGTGCGTGGCCACACTGTTA TAATTTGCTTCATTACCCTGCTATTTGATACATAGTGTGCATTTCTCTGTCACT GTAACTATTGTAATAACAAATTTTCATCTTACTGCACAATCAAAATGGCATCT ATAGTAATGACCTCCAGAGTCTGAGCTTGGACAGAGAGTGGGCGCTCAGGCC TGGTGCTCCATCATACGACCTGTACCTCTCAACTTTTGCCCTGTTAAATATAT GCTATGTCATTAAATGCTTTTAAATCTA), specifically the stretch of sequence AATGGTTACTTGCTTGTGCGTGGCCACACT of a disease-associated gene, Sptbnl and the demonstration that dCasl3 (such as the ortholog dCasRx) can restore Sptbnl protein levels and rescue the typical axonal defects observed in mouse neurons heterozygous for Sptbnl in vitro.

[0014] One embodiment provides a method treat a disease or condition associated with haplo-insufficient genes comprising administering to a subject in need thereof a dCas protein and one or more gRNA, wherein the one or more gRNA is complementary to a miRNA response element (MRE) on target mRNA expressed from the functioning allele of the haplo-insufficient gene so as to cause dCas / gRNA binding to the MRE thereby blocking miRNA repression of the functioning allele.

[0015] Another embodiment provides a method to increase haplo-insufficient gene protein levels comprising removing miRNA repression from the functioning allele of the haplo- insufficient gene, wherein the miRNA repression is removed by introducing a dCas protein and one or more gRNA into a cell, wherein the one or more gRNA is complementary to the miRNA response element (MRE) on a target mRNA expressed from the functioning allele of the haplo-insufficient gene so as to block miRNA repression from the remaining functioning allele and increase said protein levels.

[0016] One embodiment provides a method to prevent miRNA repression of their target mRNAs comprising introducing dCas protein and one or more gRNA into a cell, wherein the one or more gRNA are complementary to the miRNA response element (MRE) on the target mRNA expressed by the functioning allele of the haplo-insufficient gene so as to block miRNA repression of the target mRNA.

[0017] One embodiment provides a method to de-repress at least one target RNA comprising introducing a dCas protein and one or more gRNA into a cell, wherein the one or more gRNA are complementary to at least one miRNA response element (MRE) on the at least one target RNA so as to cause the dCas / gRNA to bind to the at least one MRE thereby de-repressing miRNA repression of the functioning allele of the at least one target RNA.

[0018] In one embodiment, the dCas protein is a dCasl3d protein, dCasl3b protein or ortholog or variant thereof, such as dRfxCasl3d (dCasRx), k87Casl3d (dCask87), 212Casl3d (dCas212) and / or PspCasl3b (dCasl3b). In one embodiment, the dCas protein is fused to at least one localization signal, such as a nuclear export signal (NES).

[0019] In one embodiment, the dCas protein and the one or more gRNAs are provided by one or more polynucleotide molecules coding for the dCas protein and / or the one or more gRNA, and wherein the one or more polynucleotide molecules are operably configured to express the dCas protein and / or the one or more gRNA. In one embodiment, the one or more polynucleotide molecules comprise one or more promoters, such as inducible promoters or one or more tissue specific promoters. In one embodiment, the one or more polynucleotide molecules are contained within one or more vectors. In another embodiment, the one or more polynucleotide molecules are contained within a vector, such as a viral vector.

[0020] In one embodiment, the viral vector comprises one or more retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, or herpes simplex viral vectors. In one embodiment, the adeno-associated viral vector is a recombinant AAV (rAAV), such as AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-anc80, AAV-PHP.eB, AAV- PHP.eB, AAV-PHP.S, AAV.CAP-B10, AAV.CAP-B22, AAV.CAP-Mac, AAV- MaCPNSl, AAV-MaCPNS2, AAVrh32.33 or AAV rh.74. In one embodiment, the rAAV is rAAV-9.

[0021] In one embodiment, the target mRNA is coded by one or more genes comprising Atrx, Cacnala, Cdkl5 Grin2b, Iqsec2, Kcnq2, Kmt2a, Mecp2, Nfl, Pcdhl9, Pten, Scn2a, Sptbnl, Stxpbl, Syngapl, and / or Tscl.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGS.1A-1F. Manipulating MicroRNA-Target Interactions to Restore Gene Expression. A) A subset of genes cannot tolerate loss of one allele. For example, 262 of 660 haplo-insufficient genes are known to cause various neurological disorders (ASD, epilepsy, schizophrenia etc). B) MicroRNAs are post-transcriptional repressors. miRNAs bind and repress their mRNA targets via the Ago protein. Ago then recruits Tnrc6, which coordinates downstream repression. Haplo-insufficient genes generally have more conserved MREs than other genes. C) De-repression from miRNAs to restore protein levels. Restore haploinsufficient gene expression levels by de-repressing the healthy allele from miRNAs. D) Depicting Spy3-Ago2 pull-down and sequencing (SAP-seq) technology. Mapping cell type-specific miRNA-target interactions (MTIs). MTIs were mapped in mouse cortical pyramidal neurons, with over 100 target genes that are haploinsufficient identified. Mapping can identify bona fide MTIs that cannot be determined through computational prediction tools. Predicted versus mapped MTIs in Syngapl and Slc6al 3’ UTRs. E) Competing with MTIs to de-repress miRNA targets. Target Site Blockers (TSBs; e.g., antisense oligonucleotides) and RNA binding CRISPR system (delivery via, for example, viral vectors (such as AAV); cell type specific; no permanent change to DNA). F) dCasl3 can de-repress miRNA sensor in cells, such as neurons. As an example, dCasRx, a type of dCasl3, can efficiently de-repress a miRNA sensor by -50% in cultured neurons.

[0024] FIGS. 2A-2C. A) Screening for efficient dCasl3 effector. Different dCasl3s and gRNA lengths have varying efficiencies. B) dCasRx can de-repress the haplo-insufficient gene Sptbnl and increase axonal length; mapped MREs for Sptbnl; dCasRx efficiently increases with a gRNA that targets the miR-132 MRE compared to a control non-targeting gRNA (NT). dCasRx can rescue the axonal defect caused by Sptbnl haploinsufficiency. C) Mapping and manipulating miRNA-target interactions in patient-derived iPSCs.

[0025] FIGS. 3A-3G. Screening dCasl3 effectors for disrupting MTIs in cultured cortical neurons. (A) Top: Schematic for target site blocker (TSB) function. TSBs are designed to bind to specific MREs and prevent miRNAs from accessing the site. Bottom: dCasl3 directed by a gRNA complementary to an MRE will outcompete the miRNA-Ago complex. (B) The dCasl3 effector and gRNA lengths tested. (C) Schematic for lentiviral dCasl3 expression and AAV gRNA expression constructs. LTR, long terminal repeat. NES, nuclear export signal. ITR, inverted terminal repeat. (D) Schematic of the dualfluorescence miRNA sensor (lentivirus backbone). TagBFP functions as a transduction reporter and is not regulated by miRNAs. mCherry harbors a miR-124 MRE and functions as a miR-124 sensor. Sequences of the wild-type (WT) and mutated (MUT, mutated bases in red) miR-124 MREs cloned in the 124 and Ctrl sensors. (E) Design and sequence of the different length gRNA spacers targeting the miR-124 MRE in the sensor. (F) Experimental timeline for the sensor experiments. Lentiviral transduction of dCasl3 and the miR-124 (124) or control (Ctrl) sensors were performed at DIV3 and AAV transduction of the gRNA was performed at DIV5, and the cells were fixed for analysis at DIV14. (G) Percentage of maximum possible de-repression achieved by dCasl3 orthologs. Data are mean ± SEM. N=2-3 biological replicates. Each replicate sampled n=20-103 cells per condition.

[0026] FIGS. 4A-4B. dCasRx outperforms TSBs in de-repressing the miR-124 sensor.

[0027] (A) Representative images and quantification of the miR-124 sensor de-repressed by dCasRx with a 23 or 30 nt gRNA from one biological replicate. dCasRx with a 23 nt gRNA increased mCherry / TagBFP by 0.40, which is 40% of 0.99 (see Figure 3G), the difference between the 124 and Ctrl sensor. dCasRx with a 30 nt gRNA achieved 62% of maximum possible de-repression. EGFP and iRFP670 signal indicates cells transduced with the dCasRx lentivirus and gRNA AAV, respectively. Scale bar, 10 pm. N=27-62 cells from two coverslips. (B) Representative images and quantification of the miR-124 sensor derepressed by a TSB. The TSB increased mCherry / TagBFP by 0.25, achieving 35% of 0.7 (see Figure 3G), the difference between the 124 and Ctrl sensor. Scale bar, 10 pm. N=77- 103 cells from two coverslips. Data are median with interquartile range. Statistics: Mann- Whitney test. ***p < 0.001.

[0028] FIGS. 5A-5E. dCasRx can de-repress endogenous miRNA targets. (A) Genome browser view of CLIPseq peaks identified in the Lfng and Nr4al 3' UTRs in P5 and P14 cortical pyramidal neurons. The MREs targeted by dCasRx are indicated.

[0029] (B) Representative immunofluorescence images and quantification of Lfng levels. dCasRx targeted to the miR-125 MRE can de-repress Lfng. Scale bar, 10 pm. N=50-121 cells (C) Representative immunofluorescence images and quantification of Nr4al levels. dCasRx targeted to the miR-124 MRE de-represses Nr4al when assessed at DIV 19 but no DIV14. Scale bar, 10 pm. N=37-77 cells. (D) Genome browser view of CLIPseq peaks identified in the Sptbnl 3' UTR in P5 and P14 cortical pyramidal neurons. The MREs targeted by dCasRx is indicated below. (E) Representative immunofluorescence images and quantification of Sptbnl levels. dCasRx targeted to the miR-132 MRE can de-repress Sptbnl. Scale bar, 10 pm. N=157-293 cells. (F) dCasRx-mediated Sptbnl increase can rescue the axonal defects of Sptbnl+ / - PNs in vitro. Data are mean ± SEM. Statistics: Welch’s t-test. *p < 0.001; ***p < 0.001.

[0030] FIGS. 6A-6D. Manipulating MTIs as a strategy to restore protein levels of haplo- insufficient genes. (A)Genes have a copy (alleles) on each chromosome. Gene transcription produces mRNA that is translated into protein. (B) miRNAs repress translation to maintain protein levels within a physiological range. (C) Haplo-insufficient genes are dosage-sensitive genes where loss of one allele (in red the allele lost) leads to reduced protein levels, abnormal phenotypes and disease. Repression by miRNAs exacerbates the disease. (D) Removing miRNA regulation from mRNA targets is a novel method to restore protein levels of haplo-insufficient genes.

[0031] FIG. 7 provides a list of MRE containing sequences identified on haploinsufficient targets associated with nervous system diseases.

[0032] FIG. 8 provides a list haploinsufficient genes and their associated diseases.

[0033] FIG. 9 provides a list of haploinsufficient microRNA targets identified, their Ensembl IDs and the associated nervous system disease.

[0034] DESCRIPTION OF THE INVENTION

[0035] The use of CRISPR-Cas systems to manipulate miRNA-target interactions (MTIs) to de-repress a target from miRNA regulation and increase its protein levels have several uses, including, but not limited to, basic research, so as to reveal fundamental miRNA- mediated mechanisms by studying the functional consequences of disrupting specific MTIs. This can be done in a cell type-specific manner and even be scaled up to functionally screen many MTIs at once. Also, therapeutic uses, such as the treatment of diseases caused by haplo-insufficiency (loss-of-function of one allele of a gene leading to reduced protein levels and abnormal phenotypes). Cas-mediated de-repression of haplo-insufficient genes (if they are miRNA targets) can restore protein levels and rescue disease-associated phenotypes. Moreover, de-repression from miRNAs can be used in conjunction with treatments that aim to reduce pathological protein levels as a way to ensure that physiological levels are still maintained. With the MTI mapping method provided herein, 149 miRNA targets have been identified that are haplo-insufficient genes known to cause neurological disorders. The specific miRNA / Ago2 binding sites identified on these targets are sequences that can be targeted by the therapeutic strategies described herein. Figure 7 provides a list of all the MRE containing sequences identified on haploinsufficient targets associated with nervous system diseases.

[0036] The CRISPR-dCasl3 system provides advantages to the current standard, TSBs. TSBs are antisense oligonucleotides (ASOs) designed to outcompete specific MTIs. Because they are synthetic oligos, TSBs are costly, and not cell type-specific, making them difficult to scale up and express selectively in highly heterogenous organs like the brain. However, even with these disadvantages, TSBs can also be used in the methods of the invention. dCasl3, or other Cas proteins, can be delivered via viral vectors (AAV, lentivirus), which easily allows for cell type-specific expression using conditional cassettes or cell type-specific promoters. Moreover, viral vector construction and viral production is much less costly compared to the cost of having a single TSB custom-designed and synthesized via commercial vendors like QIAGEN (miRCURY™ LNA TSBs).

[0037] In terms of clinical application, although ASOs are FDA-approved treatments, they can have adverse immune effects and need to be administered repeatedly to maintain proper dosage, a problem that is exacerbated for brain disorders where ASOs must be delivered inside the brain or spinal cord (intrathecally). dCasl3 on the other hand, can be delivered via AAV, an FDA-approved viral vector with relatively low immunogenicity that allows for stable transgene expression with no genome integration, meaning that they would just need to be delivered once. Other viral or non-viral system delivery systems can also be used in the methods described herein.

[0038] Mapping and manipulating MTIs can restore protein levels of haplo-insufficient genes. Even though de-repressing MTIs represents a promising approach to restore protein levels in disease contexts, the first barrier preventing its adoption is identifying if and where a haplo-insufficient gene is targeted by miRNAs. An example of this technical and conceptual bottleneck is the attempt to use ASOs to de-repress Shank3 (a well-known ASD risk gene) in human PSC-derived neurons. Because the researchers had no knowledge of where MTIs occur on the Shank3 3’ UTR (a major site of miRNA regulation), they had to design hundreds of ASOs to tile the entire sequence. This approach is extremely inefficient because ASOs are 20-30 nucleotide long, while 3’ UTRs can be several thousands. Furthermore, ASOs require optimization of their length and chemistry to achieve the most efficient de-repression while remaining safe for the patient. However, provided herein is a high spatial resolution method for mapping MTIs, and as an example, from specific neuronal subtypes in which it was discovered that over 50% of haplo-insufficient genes are strongly targeted by miRNAs, lending to the MTI manipulation approach.

[0039] Once an MTI is identified, next one must outcompete the MTI at that site, which has until now proven to be technically challenging. As demonstrated herein, dCasRx can be used to outcompete the miRNA machinery and upregulate gene targets, offering a method permanently restoring protein levels of haplo-insufficient genes. TSBs are also a promising strategy for outcompeting the MTIs and restoring their protein levels. And since various ASOs are either FDA-approved drugs or being widely tested in clinical studies, TSBs have enormous clinical potential.

[0040] Definitions

[0041] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

[0042] References in the specification to "one embodiment," "an embodiment," etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0043] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0044] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is di -substituted.

[0045] As used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating a listing of items, “and / or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0046] As used herein, the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof, are intended to be inclusive similar to the term “comprising.”

[0047] The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. The term about can also modify the endpoints of a recited range as discuss above in this paragraph.

[0048] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more," and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

[0049] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group.

[0050] Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0051] Generally, throughout the specification, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, single-stranded, double-stranded or partially doublestranded nucleic acid molecules; a nucleic acid molecule comprising one or more free ends, not comprising a free end (e.g., in a loop); a nucleic acid molecule comprising DNA, RNA, or both; and other polynucleotide variants known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA fragments may be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector in which a viral-derived DNA or RNA sequence is present in the vector for packaging into viruses (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by the virus for transfection into a host cell. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". Vectors used for and resulting in expression in eukaryotic cells may be referred to herein as "eukaryotic expression vectors". The general expression vectors used in recombinant DNA technology are typically in the form of plasmids.

[0052] The term "regulatory element" is meant to include promoters, enhancers, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence (e.g., a tissue-specific regulatory sequence) in only certain host cells. Tissue-specific promoters may direct expression primarily in a desired target tissue, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a timedependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1,2, 3, 4, 5 or more pol III promoters), one or more pol II promoters (e.g., 1,2, 3, 4, 5 or more pol II promoters), one or more pol

[0053] I promoters (e.g., 1,2, 3, 4, 5 or more pol I promoters), or a combination thereof. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol

[0054] II promoters include, but are not limited to, the retrovirus Rous Sarcoma Virus (RSV) LTR promoter (optionally with an RSV enhancer), the Cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) (see, e.g., Bosharp et al, Cell, 41 :521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EFl alpha promoter. The term "regulatory element" also includes enhancer elements, such as WPRE; A CMV enhancer; R-U5' fragment in LTR of HTLV-I (mol. Cell. Biol., volume 8 (1), pages 466-472, 1988); the SV40 enhancer; and intron sequences between exons 2 and 3 of rabbit P -globin (proc. Natl. Acad. Sci. USA., vol.78 (3), pp.1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of expression, and the like. The vector can be introduced into a host cell to produce a transcript, protein or peptide encoded by a nucleic acid described herein, including fusion proteins or peptides (e.g., clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

[0055] As used herein, the term "crRNA" or "guide RNA" or “gRNA” or "single guide RNA" or "sgRNA" or "one or more nucleic acid components" of a Cas effector protein comprises any polynucleotide sequence that has sufficient complementarity to a target nucleic acid sequence to hybridize to the target nucleic acid sequence and direct sequencespecific binding of a nucleic acid targeting complex to the target nucleic acid sequence. The sequence may comprise any structure, including but not limited to, a structure of a natural crRNA, such as a bulge, hairpin, or stem loop structure.

[0056] Sequence homology can be generated by any of a number of computer programs known in the art, such as BLAST or FASTA, and the like. A suitable computer program for performing such an alignment is GCG Wisconsin Bestfit software package (University of Wisconsin, U.S.). Examples of other software that may perform sequence comparisons include, but are not limited to, BLAST packages (see Ausubel et al, 1999 supra, chapter 18), FASTA (Atschul et al, 1990, J. Mol. Biol., 403-410), and GENEWORKS comparison kits. Both BLAST and FASTA can be used for both offline and online searches (see Ausubel et al, 1999 supra, pages 7-58 to 7-60). The percent (%) sequence homology can be calculated over consecutive sequences, i.e., one sequence is aligned with another sequence and each amino acid or nucleotide in one sequence is directly compared to the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is referred to as a "vacancy free" alignment. Typically, such vacancy free alignments are performed on only a relatively small number of residues. Although this is a very simple and consistent method, it does not take into account that, for example, in otherwise identical pairs of sequences, an insertion or deletion may cause subsequent amino acid residue alignments to be unsuccessful, which may result in a substantial reduction in the percentage of homology when global alignments are performed. Thus, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without undue penalty for overall homology or identity scores. This is accomplished by inserting "gaps" in the sequence alignment in an attempt to maximize local homology or identity. However, these more complex methods assign a "gap penalty" to each gap that occurs in an alignment, so that for the same number of identical amino acids, sequence alignment with as few gaps as possible (reflecting a higher correlation between the two compared sequences) can achieve a higher score than sequence alignment with many gaps. An "affinity gap cost" (AFFINITY GAP cost) is typically used, which charges a relatively high cost for the presence of a gap, and a small penalty for each subsequent residue in the gap. This is the most commonly used vacancy scoring system. Of course, high gap penalties can result in optimized alignments with fewer gaps. Most alignment programs allow for modification of the gap penalty. But it is preferred to use default values when using such software for sequence comparison. For example, when using the GCG Wisconsin Bestfit software package, the default gap penalty for amino acid sequences is-12 for gaps and-4 for each extension. Thus, calculating the maximum percent homology first requires that an optimal alignment be produced taking into account the gap penalty. Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on a full pairing or no pairing comparison. Instead, a proportional similarity score matrix is typically used that assigns a score to each pair-wise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix, which is the default matrix for the BLAST suite of programs. The GCG Wisconsin program, if provided, typically uses a common default or custom symbol comparison table (see user manual for more details). For some applications it is preferred to use a common default value for the GCG package, or in the case of other software, it is preferred to use a default matrix such as BLOSUM62. Alternatively, the percent homology can be calculated using multiple alignment features in DNASIS ™ (Hitachi Software) based on an algorithm similar to CLUSTAL (HIGGINS DG & Sharp PM (1988), gene 73 (1), 237-244).

[0057] Once the software produces the optimal alignment, the percent homology / percent sequence identity, can be calculated. The software typically takes this as part of the sequence comparison and generates a numerical result. These sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and form a functionally equivalent substance. Intentional amino acid substitutions may be made based on similarity in amino acid properties such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues, and thus grouping amino acids together according to functional groups is useful. Amino acids may be grouped together based solely on the nature of the amino acid side chains. But it may be more useful to include mutation data as well. The resulting collection of amino acids may be conserved for structural reasons.

[0058] As used herein, the term "variant" is understood to mean exhibiting a quality that differs from the naturally occurring pattern. The terms "non-naturally occurring" or "engineered" are used interchangeably to refer to human intervention.

[0059] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, such as a mammal, including a human. Mammals include, but are not limited to, mice, apes, humans, farm animals, sports animals, and pets. Tissues, cells, and progeny thereof of the biological entity obtained in vivo or cultured in vitro are also included.

[0060] The terms "treating," "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and / or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical, therapeutic, and / or prophylactic administration, as appropriate.

[0061] An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art, especially in light of the detailed disclosure provided herein. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect.

[0062] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0063] The use of the word “detect” and its grammatical variants refers to measurement of the species without quantification, whereas use of the word “determine” or “measure” with their grammatical variants are meant to refer to measurement of the species with quantification. The terms “detect” and “identify” are used interchangeably herein.

[0064] The term “standard,” as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered and used for comparing results when administering a test compound, or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or compound on a parameter or function. Standard can also refer to an “internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured. Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker.

[0065] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1- 3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22: 1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. MicroRNAs

[0066] MicroRNAs (miRNAs) are a class of non-coding RNAs, with an average 22 nucleotides in length, that play roles in regulating gene expression. The majority of miRNAs are transcribed from DNA sequences into primary miRNAs and processed into precursor miRNAs, and finally mature miRNAs. In most cases, miRNAs interact with the 3' untranslated region (3' UTR) of target mRNAs to induce mRNA degradation and translational repression. However, interaction of miRNAs with other regions, including the 5' UTR, coding sequence, and gene promoters, have also been reported.

[0067] Under certain conditions, miRNAs can also activate translation or regulate transcription. The interaction of miRNAs with their target genes is dynamic and dependent on many factors, such as subcellular location of miRNAs, the abundancy of miRNAs and target mRNAs, and the affinity of miRNA-mRNA interactions. miRNAs can be secreted into extracellular fluids and transported to target cells via vesicles, such as exosomes, or by binding to proteins, including Argonautes. Extracellular miRNAs function as chemical messengers to mediate cell-cell communication.

[0068] The minimal miRNA-induced silencing complex (miRISC) consists of the guide strand and AGO. The target specificity of miRISC is due to its interaction with complementary sequences on target mRNA, called miRNA response elements (MREs). The degree of MRE complementarity determines whether there is AGO2-dependent slicing of target mRNA or miRISC-mediated translational inhibition and target mRNA decay. A fully complementary miRNA:MRE interaction induces AG02 endonuclease activity and targets mRNA cleavage. However, this interaction destabilizes the association between AGO and the 3' end of the miRNA promoting its degradation.

[0069] In animal cells, the majority of miRNA:MRE interactions are not fully complementary. Most MREs contain at least central mismatches to their guide miRNA, preventing AG02 endonuclease activity. Consequently, AG02 acts as a mediator of RNA interference. In many cases, a functional miRNA:MRE interaction occurs via the 5' seed region (nucleotides 2-8). However, additional paring at the 3' end aids in the stability and specificity of the miRNA-target interaction.

[0070] CRISPR / Cas

[0071] Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR- associated (Cas) systems can be used in the method of the invention. CRISPR / Cas can reduce or activate the expression or functioning of gene products.

[0072] Provided herein, a CRISPR guide RNA can be used that can target an inactive Cas enzyme (e.g., dCas9, dCasl2, dCasl3, dCasl3b, dCasRx, dCas87, dCpfl, and the like) to the desired location in the genome.

[0073] Cas

[0074] It should be understood that the terms "Cas" and "CRISPR enzyme" and "CRISPR protein" and "Cas protein" are generally used interchangeably and all references herein similarly refer to the novel CRISPR effector proteins further described in the present application unless explicitly stated otherwise, such as specific reference to Casl3.

[0075] Cast 3, a single type VI protein effector (class 2), generally associates with a guide RNA (gRNA) containing about a 20-30-nt-long spacer region with a direct repeat (DR) hairpin that anchors Cast 3 proteins. Hybridization of the spacer region to the target RNA activates Cast 3 as an RNase. Cast 3 gRNAs do not require the protospacer adjacent motif (PAM).

[0076] There are four main subtypes according to the Cas proteins currently known: Casl3a, Casl3b, Casl3c and Casl3d.

[0077] In certain embodiments, an effector protein according to the present invention described herein (CRISPR enzyme; casl3; effector protein) is a catalytically inactive or dead Casl3 effector protein (dCasl3). In some embodiments, the dCasl3 effector comprises a mutation in the nuclease domain. In some embodiments, the dCasl3 effector protein has been truncated. In some embodiments, to reduce the size of the fusion protein of the Cas 13 effector and one or more functional domains, the Cas 13 effector may be N- or C-terminally truncated while still retaining its RNA-binding function. Several dCasl3 variants include, but not limited to Leptotrichia wadei Cas 13a (LwaCasl3a and dLwaCasl3a), Prevotella sp. P5-125 Casl3b (PspCasl3b and dPspCasl3b), Porphyromonas gulae Casl3b (PguCasl3b and dPguCasl3b), and Ruminococcus flavefaciens XPD3002 Cas 13d (RfxCasl3d and dRfxCasl3d) (Apostolopoulos, A., Kawamoto, N., Chow, S.Y.A. et al. dCas 13 -mediated translational repression for accurate gene silencing in mammalian cells. Nat Commun 15, 2205 (2024). https: / / doi.org / 10.1038 / s41467-024-46412-7; incorporated herein specifically for inactive Cas 13 proteins).

[0078] Cas proteins include, but are not limited to, dLwaCasl3a (dCasl3a) (see, https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5706658 / which is incorporated herein by reference), dCasRx

[0079] MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSIRSVNEGEA F S AEMADKN AGYKIGNAKF SHPKGYAVVANNPL YTGP VQQDMLGLKETLEKR YFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTV YT YDEFKDPEHHRAAFNNNDKLIN AIK AQYDEFDNFLDNPRLGYFGQAFF SKEG RNYIINYGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTL NYLYDRITNELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGF NITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAA NKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEF RGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLIN KFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADAR RAMYIDAIRILGTNLSYDELKALADTFSLDENGNKLKKGKHGMRNFIINNVISNK RFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCI GKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREKFKKIISLYL TVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVTKLC AGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEEFTR QINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHA YINDIAEVNSYFQLYHYIMQRIIMNERYEI<SSGI<VSEYFDAVNDEI<I<YNDRLLI< LLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS) dCask87

[0080] MKRQKTFAKRIGIKSTVAYGQGKYAITTFGKGSKAEIAVRSADPPEETLP TESDATLSIHAKFAKAGRDGREFKCGDVDETRIHTSRSEYESLISNPAESPREDYL GLKGTLERKFFGDEYPKDNLRIQIIYSILDIQKILGLYVEDILHFVDGLQDEPEDLV GLGLGDEKMQKLLSKALPYMGFFGSTDVFKVTKKREERAAADEHNAKVFRAL GAIRQKLAHFKWKESLAIF GAN ANMPIRFFQGATGGRQLWND VIAPL WKKRIER VRKSFLSNSAKNLWVLYQVFKDDTDEKKKARARQYYHFSVLKEGKNLGFNLT KTREYFLDKFFPIFHSSAPDVKRKVDTFRSKFYAILDFIIYEASVSVANSGQMGKV APWI<GAIDNALVI<LREAPDEEAI<EI<IYNVLAASIRNDSLFLRLI<SACDI<FGAEQ NRPVFPNELRNNRDIRNVRSEWLEATQDVDAAAFVQLIAFLCNFLEGKEINELVT ALIKKFEGIQALIDLLRNLEGVDSIRFENEFALFNDDKGNMAGRIARQLRLLASV GKMKPDMTDAKRVLYKSALEILGAPPDEVSDEWLAENILLDKSNNDYQKAKKT VNPFRNYIAKNVITSRSFYYLVRYAKPTAVRKLMSNPKIVRYVLKRLPEKQVAS YYSAIWTQSESNSNEMVKLIEMIDRLTTEIAGFSFAVLKDKKDSIVSASRESRAV NLEVERLKKLTTLYMSIAYIAVKSLVKVNARYFIAYSALERDLYFFNEKYGEEFR LHFIPYELNGKTCQFEYLAILKYYLARDEETLKRKCEICEEIKVGCEKHKKNANP PYEYDQEWIDI<I<I<ALNSERI<ACERRLHFSTHWAQYATI<RDENMAI<HPQI<WY DILASHYDELLALQATGWLATQARNDAEALNPVNEFDVYIEDLRRYPEGTPKNK DYHIGSYFEIYHYIRQRAYLEEVLAKRKEYRDSGSFTDEQLDKLQKILDDIRARG

[0081] SYDKNLLKLEYLPFAYNLPRYKNLTTEALFDDDSVSGKKRVAEWREREKTREA

[0082] EREQRRQRSR dCas212 MKKKHQSAAEKRQVKKLKNQEKAQKYASEPSPLQSDTAGVECSQKKTV VSHIASSKTLAKAMGLKSTLVMGDKLVITSFAASKAVGGAGYKSANIEKITDLQ GRVIEEHERMFSADVGEKNIELSKNDCHTNVNNPVVTNIGKDYIGLKSRLEQEFF GKTFENDNLHVQLAYNILDIKKILGTYVNNIIYIFYNLNRAGTGRDERMYDDLIG TLYAYKPMEAQQTYLLKGDKDMRRFEEVKQLLQNTSAYYVYYGTLFEKVKAK SI<I<EQRAI<EAEIDACTAHNYDVLRLLSLMRQLCMHSVAGTAFI<LAESALFNIED VLSADLKEILDEAFSGAVNKLNDGFVQHSGNNLYVLQQLYPNETIERIAEKYYR LTVRKEDLNMGVNIKKLRELIVGQYFPEVLDKEYDLSKNGDSVVTYRSKIYTVM NYILLYYLEDHDSSRESMVEALRQNREGDEGKEEIYRQFAKKVWNGVSGLFGV CLNLFKTEKRNKFRSKVALPDVSGAAYMLSSENIDYFVKMLFFVCKFLDGKEIN ELLCALINKFDNIADILDAAAQCGSSVWFVDSYRFFERSRRISAQIRIVKNIASKDF KKSKKDSDESYPEQLYLDALALLGDVISKYKQNRDGSVVIDDQGNAVLTEQYK RFRYEFFEEH<RDESGGH<YI< I<SGI<PEYNHQRRNFILNNVLI<SI<WFFYVVI<YNR PSSCRELMI<NI<EILRFVLRDIPDSQVRRYFI<AVQGEEAYASAEAMRTRLVDALS QFSVTACLDEVGGMTDKEFASQRAVDSKEKLRAIIRLYLTVAYLITKSMVKVNT RFSIAFSVLERDYYLLIDGKKKSSDYTGEDMLALTRKFVGEDAGLYREWKEKNA EAKDKYFDKAERKKVLRQNDKMIRKMHFTPHSLNYVQKNLESVQSNGLAAVIK

[0083] EYRNAVAHLNIINRLDEYIGSARADSYYSLYCYCLQMYLSKNFSVGYLINVQKQ LEEHHTYMI<DLMWLLNIPFAYNLARYI<NLSNEI<LFYDEEAAAEI<ADI<AENER GE dCas!3b

[0084] MNIPALVENQI<I<YFGTYSVMAMLNAQTVLDHIQI<VADIEGEQNENNEN LWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYK QNRVEVNSNDIFEVLKRAFGVLKMYRDLTNAYKTYEEKLNDGCEFLTSTEQPLS GMINNYYTVALRNMNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFFL SLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIR SFGINSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDD HNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQT RVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPY IVDTYTHYILENNKVEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMA FHMFLFGSKKTEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILD LISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQ ISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQF KLMFEKARLIGKGTTEPHPFLYKVFARSIPANAVEFYERYLIERKFYLTGLSNEIK KGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELPRQMFDNEIKSHLKSLP QMEGIDFNNANVTYLIAEYMI<RVLDDDFQTFYQWNRNYRYMDMLI<GEYDRI< GSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSNRQMRNASSEEIETILDK

[0085] RLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDA EKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGS

[0086] DIVSKEDIMEEFNKYDQCRPEISSIVFNLEKWAFDTYPELSARVDREEKVDFKSIL I<ILLNNI<NINI<EQSDILRI<IRNAFDANNYPDI<GVVEII<ALPEIAMSII<I<AFGEYAI

[0087] MK dCas!3a

[0088] MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIKNPDN

[0089] ASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEYD LKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKI

[0090] NENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIE NSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKS QVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFE YQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNII GVSSVAYFSLRNILETENENGITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQ NEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIAHGIVHFNLELEGKDIFAF

[0091] I<NIAPSEISI<I<MFQNEINEI<I<LI<LI<IFI<QLNSANVFNYYEI<DVIII<YLI<NTI<FNFV NKNIPFVPSFTKLYNKIEDLRNTLKFFWSVPKDKEEKDAQIYLLKNIYYGEFLNK FVKNSKVFFKITNEVIKINKQRNQKTGHYKYQKFENIEKTVPVEYLAIIQSREMIN NQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIESNNNNDNNDIFSKIKIKKDNK

[0092] EKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKYTENLNMFYLILKLLNHKE LTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFELEANEIGKFLDFN ENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAKYKIS

[0093] LKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAIGNIQ KYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEE IFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDL

[0094] YIANYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVAT FKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE

[0095] Each dCasl3 or variant thereof can be expressed with or without a nuclear export signal (NES). In certain embodiments of the invention, at least one Nuclear Export Signal (NES) is attached to a nucleic acid sequence encoding a Casl3 effector protein. In some embodiments, at least one or more C-terminal or N-terminal NES are attached (thus, one or more nucleic acid molecules encoding Cast 3b effector protein may comprise encoding one or more NES such that the expressed product is attached or linked to the one or more NES). In one embodiment, C-terminal and / or N-terminal NES are attached for expression and targeting in eukaryotic cells, such human cells. Non-limiting examples of NES include NES sequence LYPERLRRILT

[0096] (CTGTACCCTGAGCGGCTGCGGCGGATCCTGACC) and LQLPPLERLTL (CTTCAACTGCCTCCACTTGAAAGACTGACACTG). gRNAs

[0097] In embodiments of the invention, the terms guide sequence and guide RNA (i.e., RNA capable of directing Cast 3 to the target genomic locus) are used interchangeably. In the case of CRISPR complex formation, "target sequence" refers to a sequence to which a guide sequence (e.g., gRNA) is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes CRISPR complex formation. The target sequence may comprise an RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell.

[0098] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and guide sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. The optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burrows -Wheel er transform-based algorithm (e.g., burrows WHEELER ALIGNER), clustalW, clustal X, BLAT, novoalign (Novocraft Technologies; available from www.novocraft.com), ELAND (Illumina, san Diego, calif.), SOAP (available from SOAP. Genemics. Org. Cn), andMaq (available from maq. Sourceforg. Net).

[0099] In some embodiments, the length of the guide sequence is about or greater than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides. In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or fewer nucleotides in length. For example, the length of the guide sequence is 10-40 nucleotides, such as 20-30 or 20-40 nucleotides or more, such as 30 nucleotides or about 30 nucleotides. In certain embodiments, the guide sequence is 10-30 nucleotides in length, such as 20-30 or 20-40 nucleotides or more, such as 30 nucleotides or about 30 nucleotides.

[0100] In some embodiments, the gRNA is complementary to a sequence on target mRNA expressed from one or more the genes listed in Figure 8, such as one or more MRE sequences (one of skill in the art can locate the one or more MRE sequences, such as through the use of the screening process disclosed herein). In some embodiments, the gRNA is complementary to one or more of the MREs provided in Figure 7 (one can readily design a gRNA of any length complementary to one or more MREs provided in Figure 7, such as a gRNA having 22, 23, 26, or 30 nucleotides or more (for example, a gRNA for an Abcdl gene MRE can have the sequence CCCCAGTGGCAACTGGCAGTAT) or gRNA for an Actb-1 gene MRE can have the sequence TCAACCCCCTCCCAGGGAGACC)).

[0101] In certain embodiments, chemically modified guide RNAs are utilized. Examples of chemical modifications of the guide RNA include, but are not limited to, incorporation of 2' -O-methyl (M), 2' -O-methyl 3' phosphorothioate (MS), or 2' -O-methyl 3' phosphorothioate (MSP) at one or more terminal nucleotides. The stability and activity of such chemically modified guide RNAs can be increased compared to unmodified guide RNAs. (see Hendel, 2015, Nat Biotechnol. 33 (9): 985-9, doi: 10.1038 / nbt.3290). Chemically modified guide RNAs also include, but are not limited to, RNAs with phosphorothioate linkages comprising methylene bridged Locked Nucleic Acid (LNA) nucleotides between the 2 'and 4' carbons of the ribose ring.

[0102] Target site blockers (TSBs)

[0103] A TSB is a polymer of ribose nucleotides or deoxyribose nucleotides generally having more than 13 nucleotides in length. A TSB can include naturally occurring nucleotides; synthetic, modified, or pseudo-nucleotides such as phosphorothiolates; as well as nucleotides having a detectable label such as P32, biotin or digoxigenin. In the present invention, a TSB can reduce the activity of the miRNA by out competing for its binding site, MRE. The TSB may be completely complementary to a segment of an endogenous nucleic acid (e.g., an RNA). Alternatively, some variability is permitted in the TSB nucleic acid sequences relative to the sequences of interest. A TSB can hybridize to a nucleic acid of interest under intracellular conditions or under stringent hybridization conditions and is sufficiently complementary to bind the desired MRE and block the activity of the miRNA. Intracellular conditions refer to conditions such as temperature, pH and salt concentrations typically found inside a cell, e.g., an animal or mammalian cell. One example of such an animal or mammalian cell is a neuronal cell. Another example of such an animal or mammalian cell is a differentiated neural cell derived from an iPSC. Generally, stringent hybridization conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1°C to about 20 °C lower than the thermal melting point of the selected sequence, depending upon the desired degree of stringency as otherwise qualified herein. TSBs that comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides that are precisely complementary to the coding sequence of interest, each separated by a stretch of contiguous nucleotides that are not complementary to adjacent coding sequences, can inhibit the function of one or more miRNAs. In general, each stretch of contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences may be 1, 2, 3, or 4 or more nucleotides in length. One skilled in the art can easily use the calculated melting point of a TSB hybridized to a nucleic acid to estimate the degree of mismatching that will be tolerated for binding to a particular target nucleic acid. TSBs of the invention include, for example, a short hairpin RNA, a small interfering RNA, a ribozyme or an antisense nucleic acid molecule.

[0104] Haploinsufficiency

[0105] Haploinsufficiency in genetics describes a model of dominant gene action in diploid organisms, in which a single copy of the wild-type allele at a locus in heterozygous combination with a variant allele is insufficient to produce the wild-type phenotype. Haploinsufficiency may arise from a de novo or inherited loss-of-function mutation in the variant allele, such that it yields little or no gene product (often a protein). Although the other, standard allele still produces the standard amount of product, the total product is insufficient to produce the standard phenotype. This heterozygous genotype may result in a non- or sub-standard, deleterious, and (or) disease phenotype.

[0106] About 3,000 human genes cannot tolerate loss of one of the two alleles. Examples diseases / conditions caused by haploinsufficiency include, but are not limited to, Williams syndrome (a neurodevelopmental disorder caused by the haploinsufficiency of genes at 7ql 1.23 (Tassabehji et al. 1999. Am. J. Human Genetics. 64(1): 118-125))), telomerase reverse transcriptase (which leads to anticipation in autosomal dominant dyskeratosis congenita; a null mutation in motif D of the reverse transcriptase domain of the telomerase protein, hTERT, leads to this phenotype (Armanios et al. 2004. Genetics. 102(44): 15960- 15964)), several cancers, lq21.1 deletion syndrome, 5q- syndrome in myelodysplastic syndrome (MDS), 22ql l.2 deletion syndrome, CHARGE syndrome, Cleidocranial dysostosis, Ehlers-Danlos syndrome, Frontotemporal dementia caused by mutations in progranulin, GLUT1 deficiency (DeVivo syndrome; Rotstein et al. 2010. Ann Neurol. 68(6):955-958), Haploinsufficiency of A20, Haploinsufficiency of PRR12 (Chowdhury et al. 2021. Genetics in Medicine. 23(7): 1234-1245), Holoprosencephaly caused by haploinsufficiency in the Sonic Hedgehog gene, Holt-Oram syndrome, Marfan syndrome (Robinson et al. 2006. J. Medical Genetics. 43(10):769-787), Phelan-McDermid syndrome, Polydactyly, Dravet Syndrome, ZTTK, FOXP1 Syndrome, NR4A2-related syndrome and those provided in Figure 8.

[0107] Vectors

[0108] In certain aspects, the invention relates to vectors, e.g., for delivering or introducing Cas in a cell and / or RNAs (i.e., guide RNAs) capable of directing Cas to a target locus, and also for propagating these components (e.g., in a prokaryotic cell). Generally, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, single- stranded, double-stranded or partially double-stranded nucleic acid molecules; a nucleic acid molecule comprising one or more free ends, not comprising a free end (e.g., in a loop); a nucleic acid molecule comprising DNA, RNA, or both; and other polynucleotide variants known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNAjfagments may be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector in which a viral-derived DNA or RNA sequence is present in the vector for packaging into viruses (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAV)). Viral vectors also include polynucleotides carried by the virus for transfection into a host cell. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell.upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0109] In some aspects, the vectors are AAV vectors. In some aspects, the vectors are single stranded AAV vectors. In some aspects the AAV is recombinant AAV (rAAV). In some aspects, the rAAV lack rep and cap genes. In some aspects, rAAV are self- complementary (sc) AAV.

[0110] There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC 002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC 001401 and Srivastava et al., J. Virol., 45: 555-564 11983); the complete genome of AAV-3 is provided in GenBank Accession No. NC 1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC 001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC 00 1862; at least portions of AAV- 7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Then, 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-anc80, AAV-PHP.eB, AAV-PHP.eB, AAV-PHP.S, AAV.CAP- B10, AAV.CAP-B22, AAV.CAP-Mac, AAV-MaCPNSl, AAV-MaCPNS2, AAVrh32.33 and AAV rh.74. As set out herein above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0111] Recombinant expression vectors may comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory elements operably linked to the nucleic acid sequence to be expressed, which regulatory elements may be selected according to the host cell to be used for expression. Within a recombinant expression vector, "operably linked" refers to the linkage of a nucleotide sequence of interest to one or more other elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system, or in a host cell when the vector is introduced into the host cell).

[0112] One or more vectors may include one or more regulatory elements, such as one or more promoters. One or more vectors can comprise a Cas coding sequence, and / or a single gRNA, but can also comprise at least 3 or 8 or 16 or 32 or 48 or 50 guide RNA coding sequences, such as 1-2, 1-3, 1-4, 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNAs, optionally each with its own promoter.

[0113] The one or more guide RNA coding sequences and / or Cas coding sequences may be functionally or operably linked to one or more regulatory control elements, which may thus drive expression. The one or more promoters may be one or more constitutive promoters and / or conditional promoters and / or inducible promoters and / or tissue specific promoters. The promoter may be selected from: RNA polymerase pol I, pol II, pol III, T7, U6, Hl retrovirus Rous Sarcoma Virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, CMV early enhancer / chicken P actin (CAG) promoter, SV40 promoter, dihydrofolate reductase promoter, chicken beta-actin promoter, phosphoglycerate kinase (PGK) promoter, EFl alpha promoter, EFl alpha short (EFS) promoter and human synapsin (hSyn) promoter. An advantageous promoter is the promoter U6.

[0114] In some embodiments, one or more vectors driving expression of one or more elements are introduced into the host cell such that expression of the elements of the nucleic acid targeting system directs formation of a nucleic acid targeting complex at one or more target sites. For example, the nucleic acid targeting effector enzyme and the nucleic acid targeting guide RNA may each be operably linked to separate regulatory elements on separate vectors. One or more RNAs of the nucleic acid targeting system can be delivered to a transgenic nucleic acid targeting effector protein animal or mammal, such as an animal or mammal that constitutively or inductively or conditionally expresses the nucleic acid targeting effector protein; or an animal or mammal having cells containing the nucleic acid targeting effector protein by other means of expressing the nucleic acid targeting effector protein or, such as by prior administration of one or more vectors encoding and expressing the nucleic acid targeting effector protein in vivo.

[0115] Alternatively, two or more elements expressed by the same or different regulatory elements may be combined in a single vector, while one or more additional vectors provide any component of the nucleic acid targeting system that is not included in the first vector. The nucleic acid targeting system elements combined in a single vector may be arranged in any suitable orientation, such as one element being located (upstream) 5 'with respect to the second element or (downstream) 3' with respect to the second element. The coding sequences of one element may be located on the same or opposite strands of the coding sequences of a second element and oriented in the same or opposite directions. In some embodiments, a single promoter drives expression of transcripts encoding nucleic acid- targeted effector proteins and nucleic acid-targeted guide RNAs embedded within one or

[0116] T1 more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the nucleic acid targeting effector protein and the nucleic acid targeting guide RNA may be operably linked to and expressed from the same promoter. Delivery vehicles, carriers, particles, nanoparticles, formulations and components thereof for expressing one or more elements of a nucleic acid targeting system are describe in, for example, WO 2014 / 093622 (PCT / US 2013 / 074667). In some embodiments, the vector comprises one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites (e.g., about or greater than about 1, 2, 3, 4,5,6, 7, 8, 9, 10 or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors. In some embodiments, the vector comprises two or more insertion sites to allow insertion of the guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or a combination of these. When multiple different guide sequences are used, a single expression construct can be used to target nucleic acid targeting activity to multiple different corresponding target sequences within a cell. For example, a single vector may comprise about or greater than about 1, 2,3, 4,5,6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or greater than about 1, 2,3, 4,5,6, 7, 8, 9, 10 or more such vectors comprising a guide sequence may be provided and optionally delivered to a cell. In some embodiments, the vector comprises a regulatory element operably linked to the enzyme coding sequence encoding the nucleic acid targeting effector protein. Nucleic acid targeting effector proteins or one or more nucleic acid targeting guide RNAs may be delivered separately (such as via a particle or nanoparticle complex). The nucleic acid targeting effector protein mRNA may be delivered prior to the nucleic acid targeting guide RNA to allow time for expression of the nucleic acid targeting effector protein. Nucleic acid targeting effector protein mRNA may be administered 1-12 hours (such as about 2-6 hours) prior to administration of the nucleic acid targeting guide RNA. Alternatively, the nucleic acid targeting effector mRNA and the nucleic acid targeting guide RNA may be administered together. Advantageously, the second booster dose of guide RNA may be administered 1- 12 hours (such as about 2-6 hours) after the initial administration of the nucleic acid targeting effector mRNA plus guide RNA.

[0117] Treatment / Admini strati on The invention also includes methods for delivering one or more nucleic acid components to a cell and / or a subject. In certain embodiments, the cell is a eukaryotic cell, such as mammalian cell, including a human cell.

[0118] In aspects of the invention, the Cast 3 effector proteins or systems described herein are useful for treating a disease or condition associated with haploinsufficiency in a mammalian subject. Aspects of the invention provide a Cast 3 CRISPR system for treating haploinsufficiency in a subject comprising (a) a Casl3 / CRISPR effector protein and / or a polynucleic acid encoding a Casl3 / CRISPR effector protein, and (b) one or more guide RNAs designed to bind to one or more target molecules / mRNA expressed from the functional allele of a haploinsufficient gene and / or one or more polynucleic acids coding for one or more guide RNAs. Cast 3 effector proteins are defined herein, including with respect to wild-type Cast 3 proteins and preferred derivatives and modifications thereof.

[0119] Vector delivery, e.g., plasmid, viral delivery: any suitable vector (e.g., a plasmid or viral vector, such as an adeno-associated virus (AAV), lentivirus, adenovirus, or other viral vector type, or combination thereof) may be used to deliver the CRISPR enzyme and / or any RNA of the present invention, e.g., guide RNA. Effector proteins and one or more guide RNAs may be packaged into one or more vectors, such as a plasmid or viral vector. In some embodiments, the vector, e.g., plasmid or viral vector, is delivered to the target tissue by, e.g., intramuscular injection, while other times are delivered via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dosage delivered herein may vary greatly depending on a variety of factors, such as carrier selection, target cells, organisms or tissues, general condition of the subject to be treated, degree of transformation / modification sought, route of administration, mode of administration, type of transformation / modification sought, and the like.

[0120] In one embodiment herein, delivery is via an adenovirus, which may be a single booster dose containing at least 1 x 105adenovirus vector particles (also referred to as particle units, pu). In one embodiment herein, the dose is at least about 1 x 106adenovirus vector particles (e.g., about 1 x 106-l * 1012particles), including at least about 1 x 107particles, including at least about 1 x 108particles (e.g., About 1 x 108- I x lO11particles or about 1 x 108-l * 1012particles), including at least about 1 x 100 particles (e.g., about 1 x 109-l x lO10particles or about 1 x 109-l x l012particles), or even at least about 1 x 1010particles (e.g., about 1 x 10lo-l x lO12particles). Alternatively, the dosage comprises no more than about IX 1014particles, such as no more than about IX 1013particles, including no more than about IX 1012particles, including no more than about 1X 1011particles, and including no more than about 1 x 1010particles (e.g., no more than about 1 x 109particles). Thus, a dose may contain a single dose of adenovirus vector having, for example, about IX 106particle units (pu), about 2X 106pu, about 4X 106pu, about IX 107pu, About 2X 107pu, about 4X 107pu, about IX 108pu, about 2X 108pu, About 4X 108pu, about IX 109pu, about 2X 109pu, about 4X 109pu, About IX 1010pu, about 2X 1010pu, about 4X 1010pu, about IX 1011pu, An adenovirus vector of about 2 x 1011pu, about 4 x 1011pu, about 1 x 1012pu, about 2 x 1012pu, or about 4 x 1012pu.

[0121] In one embodiment herein, delivery is via AAV. A therapeutically effective dose for in vivo delivery of AAV to a human is believed to range from about 20 to about 50ml of saline solution containing from about 1 xlO10to about 1 xlO10functional AAV / ml of solution. Dosages may be adjusted to balance therapeutic benefit with any side effects. In one embodiment herein, the concentration of AAV dose is typically in the range of about l x l05to lx105° genomic AAV, about l x l08to lx1020genomic AAV, about 1x1010to about 1x1016genomic, or about 1x1011to about 1x1016genomic AAV. The human dose may be about 1 xlO13genomic AAV. Such concentrations may be delivered in about 0.001ml to about 100ml, about 0.05 to about 50ml, or about 10 to about 25ml of carrier solution. Other effective dosages can be readily determined by one of ordinary skill in the art by routine experimentation to establish a dose response curve.

[0122] Such dosages may also include, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically acceptable carrier (e.g., phosphate buffered saline), a pharmaceutically acceptable excipient, and / or other compounds known in the art. The dose may also comprise one or more pharmaceutically acceptable salts, such as mineral acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, gels or jellifying materials, flavoring agents, coloring agents, microspheres, polymers, suspending agents, and the like may also be present therein. In addition, one or more other conventional pharmaceutical ingredients may also be present, such as preservatives, wetting agents, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, and the like, especially if the dosage form is of a reconfigurable form. Suitable exemplary ingredients include microcrystalline cellulose, sodium carboxymethyl cellulose, polysorbate 80, phenethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, p-chlorophenol, gelatin, albumin, and combinations thereof. A detailed discussion of pharmaceutically acceptable excipients is available in REMINGTON' S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J.1991), which is incorporated herein by reference.

[0123] In one embodiment herein, delivery is via a plasmid. In such plasmid compositions, the dose should be an amount of plasmid sufficient to elicit a response. For example, a suitable amount of plasmid DNA in a plasmid composition may be about 0.1 to about 2mg or about 1 pg to about 10 pg per 70kg individual. The plasmids of the invention generally comprise (i) a promoter; (ii) A sequence encoding a nucleic acid targeting a CRISPR enzyme operably linked to the promoter; (iii) optionally a selectable marker; (iv) optionally an origin of replication; and (v) optionally a transcription terminator downstream of and operably linked to (ii). The plasmid may also encode the RNA component of the CRISPR complex, but one or more components may alternatively be encoded on a different vector.

[0124] In some embodiments, the DNA and / or RNA molecules of the invention are delivered in the form of liposomes or lipofectin formulations and the like, and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. nos. 5,593,972, 5,589,466, and 5,580,859, which are incorporated herein by reference.

[0125] RNA delivery is a useful in vivo delivery method. Liposomes or particles can be used to deliver nucleic acids targeted to Cas proteins and guide RNAs into cells. Thus, delivery of the nucleic acid targeted Cas 13 proteins and / or delivery of guide or crRNAs of the invention can be performed in RNA form and via microbubbles, liposomes, or particles. For example, casl3 mRNA and guide or crRNA can be packaged into liposome particles for in vivo delivery.

[0126] Kits

[0127] Provided herein are kits for use in the treatment of a haploinsufficient disease / disorder. Such kits include at least a first sterile composition comprising any of the nucleic acids described herein above or any of the viral vectors described herein above in a pharmaceutically acceptable carrier. Another component is optionally a second therapeutic agent for the treatment of the disease / disorder along with suitable container and vehicles for administrations of the therapeutic compositions. The kits optionally comprise solutions or buffers for suspending, diluting or effecting the delivery of the first and second compositions.

[0128] In one embodiment, such a kit includes the nucleic acids or vectors in a diluent packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the nucleic acids or vectors.

[0129] In some aspects, the formulation comprises a stabilizer. The term "stabilizer" refers to a substance or excipient which protects the formulation from adverse conditions, such as those which occur during heating or freezing, and / or prolongs the stability or shelflife of the formulation in a stable state. Examples of stabilizers include, but are not limited to, sugars, such as sucrose, lactose and mannose; sugar alcohols, such as mannitol; amino acids, such as glycine or glutamic acid; and proteins, such as human serum albumin or gelatin.

[0130] In some aspects, the formulation comprises an antimicrobial preservative. The term "antimicrobial preservative" refers to any substance which is added to the composition that inhibits the growth of microorganisms that may be introduced upon repeated puncture of the vial or container being used. Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.

[0131] In some aspects, the kit comprises a label and / or instructions that describes use of the reagents provided in the kit. The kits also optionally comprise catheters, syringes or other delivering devices for the delivery of one or more of the compositions used in the methods described herein.

[0132] The invention will be described by the following non-limiting example.

[0133] EXAMPLE

[0134] Introduction

[0135] MicroRNAs (miRNAs) are a class of small noncoding-RNAs that bind to mRNA targets by recognizing a miRNA response element (MRE) via the Argonaute protein (Ago). Ago then recruits the scaffolding protein Tnrc6, which then mediates translational repression. miRNAs and their targets form complex posttranscriptional regulatory networks because a single miRNA can target hundreds of different mRNAs and a target can be cooperatively regulated by multiple miRNAs (Selbach et al., 2008; Cherone et al., 2019). These features make miRNAs ideally suited to coordinate and fine-tune complex gene expression programs underlying neural development. At the same time these features make it technically challenging to pinpoint the exact molecular mechanisms of miRNAs. From previous studies, it was shown that manipulation of a single miRNA can induce significant developmental phenotypes (Schratt et al., 2006; Siegel et al., 2009; Tan et al., 2013; Siegert et al., 2015; Lippi et al., 2016; Dulcis et al., 2017; Taylor et al., 2023; Zolboot etal., 2023). Phenotypes resulting from manipulation of a single miRNA usually represent a compound phenotype caused by simultaneous de-repression of all targets that are regulated by that miRNA. However, there are cases where disruption of a single miRNA- target interaction (MTI) can phenocopy specific aspects (and occasionally all aspects) of miRNA loss-of-function.

[0136] Transient miR-101 loss-of-function during early postnatal life is known to increase the length of primary dendritic branches of hippocampal CAI and CA3 pyramidal neurons as well as increase the number of Vglutl -positive presynaptic boutons on those branches (Lippi et al., 2016). The increased length of dendritic branches could be fully recapitulated by disrupting the interaction between miR-101 and its target Na-K-Cl cotransporter sodium-potassium-chloride cotransporter 1 (Nkccl). The miR-lOl-Nkccl interaction was blocked by using target site blockers (TSB), which are synthetic locked nucleic acid antisense oligonucleotides designed to bind to specific MREs and prevent miRNAs from accessing the site (Figure 3.1 A, top panel). TSBs are the current benchmark technique for disrupting MTIs. The study on miR-101 (Lippi et al., 2016) demonstrated how a single MTI can be critical for proper neuronal development. While TSBs have proven useful, because they are synthesized oligonucleotides, they are costly and not cell type-specific, which makes them difficult to use in heterogenous tissue like the brain and impractical for in vivo studies. Thus, there is a clear need for flexible and scalable alternatives for disrupting individual MTIs. Searching for alternative strategies, the RNA-targeting CRISPR-Casl3 system was re-purposed based on its widespread use in other forms of RNA manipulation (Abudayyeh et al., 2016; Abudayyeh et al., 2017; Cox et al., 2017; Konermann et al., 2018, Yang et al., 2019; Marina et al., 2020; Han et al., 2020; Kannan et al., 2022).

[0137] First discovered in prokaryotes, the CRISPR-Cas system provides a form of acquired immune system to defend against viruses and other mobile genetic elements (Terns, 2018). These systems recognize and store short fragments of foreign nucleic acid into the host genome as CRISPR arrays, which comprise of alternating repeat and spacer sequences (the foreign sequence) (Barrangou et al., 2007). Once transcribed, the array is processed into small mature CRISPRRNAs (crRNAs), consisting of the spacer, which can then recognize the invading foreign nucleic acid by complementarity, and a direct repeat (DR) sequence, which folds into a stem loop structure to interact with the effector Cas protein (Brouns et al., 2008; Hsu et al., 2014).

[0138] The Cas 13 family of effector proteins possesses two conserved Higher Eukaryotic and Prokaryotic Nucleotide-binding (HEPN) domains that catalyze target RNA cleavage. Based on phylogeny, the system is further divided into seven subtypes (VI-A, B, C, D, E, X and Y), each with distinct crRNAs and Cas 13 effectors that exhibit different requirements and patterns for target recognition and cleavage (Kordys et al., 2020, Borrajo et al., 2023). Engineering of CRISPR-Casl3 has already yielded numerous novel molecular tools for programmable RNA manipulation such as targeted RNA degradation (Abudayyeh et al., 2016; Abudayyeh et al., 2017; Cox et al., 2017; Konermann et al., 2018; Kannan et al., 2022), RNA editing (Cox et al., 2017; Abudayyeh et al., 2019; Marina et al., 2020; Kannan et al., 2022), live cell RNA tracking (Abuddayeh et al., 2017; Yang et al., 2019; Han et al., 2020) and pre-mRNA splicing regulation (Konermann et al., 2018, Borrajo et al., 2023). Targeted RNA degradation utilizes the wildtype catalytically active Cas 13 ribonuclease while the applications that require binding of target RNA without cleavage use a catalytically inactive nuclease dead variant of Cas 13 (dCasl3), where the catalytic arginine residues in the HEPN domains are mutated. Konermann et al. (2018) successfully utilized an ortholog of dCasl3d, dRfxCasl3d (dCasRx) for disrupting proper pre-mRNA splicing. dCasRx targeted to exon-intron junctions was found to induce exon exclusion, likely by outcompeting splicing factors (Konermann et al., 2018). Based on this study, it was reasoned that dCasl3s recruited to MREs on a target could sterically outcompete miRNA-Ago complexes, resulting in de-repression of the target (Figure 3 A).

[0139] Provided herein is the use of CRISPR-dCasl3 systems with applications in basic and translational research, as well as clinical applications.

[0140] Results

[0141] Screening dCasl3 effectors for disrupting MTIs in cultured cortical neurons

[0142] Different CRISPR-Cas system orthologs from various prokaryotic species can have variable activity when engineered for mammalian cells (Ran et al., 2015; Abuddayeh et al., 2017). Of the seven subtypes of Cast 3, Cas 13b and Cas 13d have shown to be more efficient for programmable RNA binding and has been used to visualize RNAs and disrupt splicing, respectively (Yang et al., 2019; Han et al., 2020; Konermann et al., 2018). Their activity is also dependent on the composition of their guiding crRNAs, better known as guide RNAs (gRNAs). Casl3 gRNAs consist of a direct repeat sequence that forms a stem loop and is recognized by the effector Casl3 and a spacer sequence that will bind to the target (Figure 3 A). Each Cast 3 ortholog is known to have varying preferences for the length of spacer (Abudayyeh et al., 2016; Abudayyeh et al., 2017; Cox et al., 2017; Konermann et al., 2018; Yang et al., 2019; Marina et al., 2020). Thus, the identification of an efficient Casl3b or Casl3d ortholog and gRNA spacer length for disrupting MTIs was determined (Figure 3B).

[0143] Lentiviral expression plasmids carrying the catalytically inactive effectors of PspCasl3b (Cox et al., 2017), RfxCasl3d, k87Casl3d and 212Casl3d (Konermann et al., 2018) were generated. The strong constitutive CMV promoter was chosen to express the effector to ensure high expression levels and effector was fused to a nuclear export signal (NES) because canonical MTIs mostly occur in the cytoplasm. Each expression plasmid also has an EGFP expression cassette to serve as a transduction reporter (Figure 3C). AAV expression plasmids were then generated with the corresponding gRNA expression cassettes with the human U6 promoter (Figure 3C). The gRNA cassette was positioned at the 3’ end of the AAV genome and inverted the orientation based on previous published studies showing that this design strategy has improved efficiency (Ran et al., 2015, Levy et al., 2020). An iRFP670 expression cassette was added as a transduction reporter (Figure 3C). Viruses were the produced from the expression plasmids and the ability of dCasl3 to disrupt the interaction between miR-124 and a miR-124 sensor (Figure 3. ID) in cultured cortical neurons was assessed. The gRNA spacer sequence position on the miR-124 MRE (Figure 3E) was determined using a computational model that predicts the most effective gRNA designs for Casl3d based on a variety of features including the predicted secondary structures of the gRNA, target site accessibility and RNA-RNA hybridization energies. (Wessels et al., 2020). Lentiviral transduction of dCasl3 and the miR-124 (124) or control (Ctrl) sensors were performed at DIV3 and AAV transduction of the gRNA was performed at DIV5, and the cells were fixed for analysis at DIV14 (Figure 3F).

[0144] The Ctrl sensor provides a way to measure and quantitatively compare the efficiencies of the dCasl3 orthologs while taking experimental variability into account. Because the Ctrl sensor is unable to bind miR-124 (Figure 3D), one can set the difference in mCherry / TagBFP ratio between the 124 and Ctrl sensors as the maximum possible derepression (100%). Then one can represent the de-repression induced by dCasl3 as a percentage of maximum possible de-repression. Of the four orthologs tested, it was found that dRfxCasl3d (dCasRx) and k87Casl3d (dCask87) efficiently de-repressed mCherry from miR-124 and increased its levels compared to a non-targeting (NT) gRNA condition (Figures 3G and 4A). It was further found that dCasRx worked more efficiently with a 30 nucleotide (nt) spacer sequence (compared to 23 nt) and dCask87 works better with a 26 nt spacer sequence (compared to 30 nt) (Figure 3G). dCasRx was the most efficient and was able to achieve 62% of maximum possible de-repression (Figure 3G; see Figure 4A for detailed calculations). Next, dCasRx and dCask87 were compared against the current standard, TSBs. A TSB designed for the miR-124 MRE on the miRNA sensor was able to de-repress the sensor, but not to the extent of dCasRx or dCask87, achieving 35% of maximum possible de-repression (Figure 3G and 4B). Thus, provided herein is a more efficient molecular tool for disrupting individual MTIs that is scalable, less costly and can be modified for cell type-specificity. Because of its higher efficiency, dCasRx was chosen for further experiments.

[0145] Mapping and manipulating endogenous miRNA targets with dCasRx

[0146] To further validate dCasRx, it was asked whether dCasRx could de-repress endogenous miRNA targets. To identify targets amenable to de-repression by dCasRx, their MTIs need to be mapped. The classic approach for mapping MTIs is Ago2 CLIPseq (cross-linking and immunoprecipitation of Argonaute2, followed by sequencing). This technique first uses ultraviolet irradiation to covalently crosslink the Ago2-miRNA-target complex within cells or tissues, followed by RNase digestion to degrade fragments of the target not bound by Ago2. Then the Ago2-miRNA-target complex is immunoprecipitated and the miRNA and target fragments are sequenced, yielding a genome-wide map of functionally relevant MTIs (Chi et al., 2009). Provided herein are methods that allows one to perform CLIPseq with high temporal resolution, allowing one to map MTIs in a cell type-specific manner. Performing CLIPseq in cortical pyramidal neurons, thousands of MTIs corresponding to hundreds of mRNA targets were mapped. For initial proof-of concept, lunatic fringe (Lfng) and nuclear receptor 4A1 (Nr4al) were tested, which display prominent CLIPseq peaks (footprint of Ago2, indicating where MTIs are frequent) that contain binding sites for the neuronal -enriched miR-125b and miR-124, respectively (Figure 5A). Lentiviral transduction of dCasl3 and AAV transduction of the gRNA was performed at DIV 3 and DIV5, respectively. Lfng was first examined, and it was found that dCasRx was able to efficiently de-repress Lfng from miR-125b and increase its protein levels 1.5-fold when assessed at DIV14 (Figure 5B). When Nr4al was examined, however, no increase in Nr4al levels at DIV14 were observed (Figure 5C). Only when assessed at DIV19, was there a 1.2-fold increase in Nr4al (Figure 5C). This could be explained by when the interaction between miR-124 and Nr4al is possibly occurring. The CLIPseq peak observed for Nr4al that corresponds to miR-124 is more prominent at P14 than P5 as opposed to Lfng (Figure 5A), suggesting that Nr4al is more strongly regulated by miR- 124 later in development. Thus, dCasRx may not have been able to de-repress Nr4al by DIV14 because the miR-124-Nr4al interaction was not substantial enough to elicit an increase in protein levels. These results indicate that dCasRx is an efficient RNA-binding module that can be used for disrupting MTIs and increasing protein levels.

[0147] Manipulating MTIs as a strategy to correct gene dosage in disease

[0148] For subsets of genes (-3000 in humans), loss-of-function of one copy can lead to reduction in protein levels that results in abnormal phenotypes or disease, which is termed haploinsufficiency. For around 700 of these genes, there is already sufficient evidence for dosage pathogenicity, meaning that their haploinsufficiency results in severe diseases, from heart, limb, and skeletal abnormalities to neurodevelopmental disorders (see, for example, Figure 8). It is therefore needed for hundreds of diseases to develop novel strategies to restore protein levels. miRNAs can tightly regulate protein levels by repressing translation of their mRNA targets. Intriguingly, of the 700 human haplo- insufficient genes known to cause disease, a majority is strongly targeted by miRNAs, suggesting that miRNAs can exacerbate these diseases (Figures 6A-C). Provided herein is a method to restore pathologically low levels of haplo-insufficient genes by removing miRNA repression from the remaining healthy allele (Figure 6D). This strategy has not yet been explored because the current tools for mapping and manipulating miRNA-target interactions (MTIs) had lacked the necessary spatial resolution. This bottleneck has been tackled with the cell type-specific MTI approach provided herein and the development of dCasRx as a method to manipulate MTIs in a flexible and scalable manner.

[0149] To demonstrate this strategy, we queried the PN CLIPseq dataset for targets linked to haploinsufficiency disorders. One such target - Sptbnl - encodes the neuronal piL spectrin, which is a scaffold protein that links the cell membrane to the actin cytoskeleton (Xu et al., 2012; Lorenzo et al., 2023). Individuals with heterozygous SPTBN1 variants exhibit language and motor delays, intellectual disability and autistic features (Cousin et al., 2021). Mice with heterozygous loss of Sptbnl in neural progenitors (via NestinCre), have axonal growth defects, corpus callosum hypoplasia, which is also observed in some patients, and impaired sociability (Cousin et al., 2021). First it was tested whether dCasRx could de-repress Sptbnl in wildtype cortical neurons. Anticipating future use in vivo, an AAV expression plasmid for dCasRx was constructed. Because of its relatively small size (930 amino acids), the Cast 3d family provides an advantage over other Cas effectors because it is under the packaging size limit of AAV (Konermann et al., 2018). The CLIPseq data showed two prominent peaks in the Sptbnl 3' UTR. The higher peak was focused on - spanning a large footprint that harbors MREs for several brain-enriched miRNAs, miR-132, miR-101, miR-219 and miR-29 (Figure 5D). gRNAs were designed for each MRE (Figure 5D) and it was found that dCasRx guided by the gRNA targeting the miR-132 MRE was able to de-repress Sptbnl and increase its levels by nearly 50% (Figure 5E). Finally, dCasRx was able to upregulate Sptbnl enough to rescue the axonal defects observed in Sptbnl heterozygous (Sptbnl+ / -) PNs (Figure 5F), highlighting the therapeutic potential of dCasRx. This strategy can be applied to any haplo-insufficient gene that is repressed by miRNAs within and outside the brain, representing a tremendously impactful therapeutic strategy that can treat many diseases.

[0150] Discussion

[0151] Dissecting miRNA function at the single MTI level is useful for revealing precise miRNA mechanisms. The ability of dCasl3 effectors to disrupt individual MTIs and derepress miRNA targets was assessed. dCasRx was identified to be the most efficient, outperforming TSBs. Different dCasl3 orthologs varied in their ability to de-repress miRNA targets. Even for dCasRx, the efficiency varied with different targets, gRNA length and gRNA targeting position. It is also possible that other dCasl3 orthologs can outperform dCasRx depending on the target.

[0152] Herein it has been demonstrated that dCasRx can efficiently de-repress endogenous miRNA targets, which has therapeutic use, such as to rescue haploinsufficiency phenotypes. CRISPR-mediated activation (CRISPRa) to increase transcription of the functioning gene copy has been shown to rescue obesity (Matharu et al., 2018) and neurodevelopmental disorders caused by haploinsufficiency (Tamura et al., 2022; Chang et al., 2023). Due to the large size of the effector Cas used in CRISPRa, these studies have relied on transgenic mice or dual AAV strategies. CRISPR-Casl3 stands out because of their conveniently small size, which renders them suitable for all-in-one AAV delivery along with their gRNA expression cassette (Konermann et al., 2018). Moreover, because dCasl3s can process their own arrays of gRNAs, simultaneous delivery of gRNAs targeting different MREs could be utilized to de-repress targets convergently regulated by multiple miRNAs. The results showed that dCasRx could derepress and increase Sptbnl levels in a wildtype background (Figure 5E) and rescue the axonal growth phenotype of Sptbn l + / - neurons in vitro (Figure 5F). Altogether, dCasRx and other dCasl3 variants enable flexible manipulation of MTIs.

[0153] Bibliography

[0154] Abudayyeh, O. O., et al. (2017). RNA targeting with CRISPR-Casl3. Nature, 550(7675), Article 7675.

[0155] Abudayyeh, O. O., et al. (2019). A cytosine deaminase for programmable single-base RNA editing. Science, 365(6451), 382-386.

[0156] Abudayyeh, O. O., et al. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, 353(6299), aaf5573.

[0157] Barrangou, R., et al. (2007). CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science, 315(5819), 1709-1712.

[0158] Borrajo, J., et al. (2023). Programmable multi-kilobase RNA editing using CRISPR- mediated trans-splicing (p. 2023.08.18.553620). bioRxiv.

[0159] Brouns, S. J. J., et al. (2008). Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes. Science, 321(5891), 960-964.

[0160] Challis, R. C., et al. (2019). Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nature Protocols, 14(2), Article 2.

[0161] Chandradoss, S. D., et al. (2015). A Dynamic Search Process Underlies MicroRNA Targeting. Cell, 162(1), 96-107.

[0162] Chang, H.-C., et al. (2023). RAAV-CRISPRa therapy corrects Rail haploinsufflciency and rescues selective disease features in Smith-Magenis syndrome mice. loumal of Biological Chemistry, 299(1).

[0163] Chi, S. W., et al. (2009). Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature, 460(7254), 479-486.

[0164] Choi, S.-Y., et al. (2015). Post-transcriptional regulation of SHANK3 expression by microRNAs related to multiple neuropsychiatric disorders. Molecular Brain, 8(1), 74.

[0165] Cousin, M. A., et al. (2021). Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome. Nature Genetics, 53(7), Article 7.

[0166] Cox, D. B. T., et al. (2017). RNA editing with CRISPR-Casl3. Science, 358(6366), 1019- 1027.

[0167] Dang, V. T., et al. (2008). Identification of human haploinsufficient genes and their genomic proximity to segmental duplications. European lournal of Human Genetics, 16(11), Article 11. Fu, J. M., et al. (2022). Rare coding variation provides insight into the genetic architecture and phenotypic context of autism. Nature Genetics, 54(9), Article 9.

[0168] Gameau, J. E., et al. (2010). The CRISPR / Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 468(7320), Article 7320.

[0169] Gaudelli, N. M., et al. (2017). Programmable base editing of A»T to G»C in genomic DNA without DNA cleavage. Nature, 551(7681), Article 7681.

[0170] Han, S., et al. (2020). RNA-protein interaction mapping via MS2- or Casl3-based APEX targeting. Proceedings of the National Academy of Sciences, 117(36), 22068-22079.

[0171] Hsu, P. D., et al. (2014). Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 157(6), 1262-1278.

[0172] Kannan, S., et al. (2022). Compact RNA editors with small Casl3 proteins. Nature Biotechnology, 40(2), Article 2.

[0173] Komor, A. C., et al. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533(7603), Article 7603.

[0174] Konermann, S., et al. (2018). Transcriptome Engineering with RNA-Targeting Type VI- D CRISPR Effectors. Cell, 173(3), 665-676.el4.

[0175] Kordys, M., Sen, R., & Warkocki, Z. (2022). Applications of the versatile CRISPR-Casl3 RNA targeting system. WIREs RNA, 13(3), el694.

[0176] Levy, J. M., et al. (2020). Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nature Biomedical Engineering, 4(1), Article 1.

[0177] Lippi, G., et al. (2016). MicroRNA-101 Regulates Multiple Developmental Programs to Constrain Excitation in Adult Neural Networks. Neuron, 92(6), 1337-1351.

[0178] Lorenzo, D. N., et al. (2014). A PIK3C3-Ankyrin-B-Dynactin pathway promotes axonal growth and multiorganelle transport. Journal of Cell Biology, 207(6), 735-752.

[0179] Lorenzo, D. N., Edwards, R. J., & Slavutsky, A. L. (2023). Spectrins: Molecular organizers and targets of neurological disorders. Nature Reviews Neuroscience, 24(4), Article 4.

[0180] Marina, R. J., et al. (2020). Evaluation of Engineered CRISPR-Cas-Mediated Systems for Site-Specific RNA Editing. Cell Reports, 33(5), 108350.

[0181] Matharu, N., et al. (2019). CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science, 363(6424), eaau0629.

[0182] Moore, M. J., et al. (2015). MiRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity. Nature Communications, 6(1), Article 1. Nishiyama, J., Mikuni, T., & Yasuda, R. (2017). Virus-Mediated Genome Editing via Homology -Directed Repair in Mitotic and Postmitotic Cells in Mammalian Brain. Neuron, 96(4), 755-768. e5.

[0183] Ran, F. A., et al. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature, 520(7546), Article 7546.

[0184] Schratt, G. M., et al. (2006). A brain-specific microRNA regulates dendritic spine development. Nature, 439(7074), Article 7074.

[0185] Selbach, M., et al. (2008). Widespread changes in protein synthesis induced by microRNAs. Nature, 455(7209), Article 7209.

[0186] Shmakov, S., et al. (2015). Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell, 60(3), 385-397.

[0187] Siegel, G., et al. (2009). A functional screen implicates microRNA- 138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis. Nature Cell Biology, 11(6), Article 6.

[0188] Siegert, S., et al. (2015). The schizophrenia risk gene product miR-137 alters presynaptic plasticity. Nature Neuroscience, 18(7), Article 7.

[0189] Stirmlinger, N., et al. (2023). Elevation of SHANK3 Levels by Antisense Oligonucleotides Directed Against the 3'-UTR of the Human SHANK3 mRNA. Nucleic Acid Therapeutics, 33(1), 58-71.

[0190] Tamura, S., et al. (2022). CRISPR activation rescues abnormalities in SCN2A haploinsufficiency-associated autism spectrum disorder (p. 2022.03.30.486483). bioRxiv.

[0191] Tan, C. L., et al. (2013). MicroRNA-128 governs neuronal excitability and motor behavior in mice. Science, 342(6163), 1254-1258.

[0192] Taylor, S. et al. (2023). MicroRNA-218 instructs proper assembly of hippocampal networks. eLife, 12, e82729.

[0193] Terns, M. P. (2018). CRISPR-Based Technologies: Impact of RNA-Targeting Systems. Molecular Cell, 72(3), 404-412.

[0194] Wessels, H.-H., et al. (2020). Massively parallel Casl3 screens reveal principles for guide RNA design. Nature Biotechnology, 38(6), Article 6.

[0195] Xu, K., Zhong, G., & Zhuang, X. (2013). Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons. Science, 339(6118), 452-456.

[0196] Yang, L.-Z., et al. (2019). Dynamic Imaging of RNA in Living Cells by CRISPR-Casl3 Systems. Molecular Cell, 76(6), 981 -997. e7. Zolboot, N., et al. MicroRNAs are necessary for the emergence of Purkinje cell identity. bioRxiv 2023.09.28.560023 (2023).

[0197] All publications, patents, and patent applications, Genbank sequences, websites and other published materials referred to throughout the disclosure herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application, Genbank sequences, websites and other published materials was specifically and individually indicated to be incorporated by reference. In the event that the definition of a term incorporated by reference conflicts with a term defined herein, this specification shall control.

Claims

WHAT IS CLAIMED IS:

1. A method to treat a disease or condition associated with a haplo-insufficient gene comprising administering to a subject in need thereof a dCas protein and one or more gRNA, wherein the one or more gRNA is complementary to a miRNA response element (MRE) on target mRNA expressed from a functioning allele of the haplo-insufficient gene so as to cause dCas / gRNA binding to the MRE thereby blocking miRNA repression of the functioning allele.

2. The method of claim 1, wherein the dCas protein is a dCasl3d protein, dCasl3b, dCasl3a protein or ortholog or variant thereof.

3. The method of claim 2, wherein the dCasl3d protein variant or dCasl3b protein variant is one or more of dRfxCasl3d (dCasRx), k87Casl3d (dCask87), 212Casl3d (dCas212), dLwaCasl3a (dCasl3a), and / or PspCas 13b (dCasl3b).

4. The method of claim 2 or 3, wherein the dCasl3d protein variant is dCasRx.

5. The method of any one of claims 1 to 4, wherein the dCas protein is fused to at least one localization signal.

6. The method of claim 5, wherein the localization signal is a nuclear export signal (NES).

7. The method of any one of claims 1 to 6, wherein the dCas protein and the one or more gRNA are provided by one or more polynucleotide molecules coding for the dCas protein and / or the one or more gRNA, and wherein the one or more polynucleotide molecules are operably configured to express the dCas protein and / or the one or more gRNA.

8. The method of claim 7, wherein the one or more polynucleotide molecules comprise one or more promoters.

9. The method of claim 8, wherein the one or more promoters comprise one or more inducible promoters or one or more tissue specific promoters.

10. The method of any one of claims 7 to 9, wherein the one or more polynucleotide molecules are contained within one or more vectors.

11. The method of any one of claims 7 to 10, wherein the one or more polynucleotide molecules are contained within a vector.

12. The method of claim 10 or 11, wherein the vector comprises a viral vector.

13. The method of claim 12, wherein the viral vector comprises one or more retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, or herpes simplex viral vectors.

14. The method of claim 13, wherein the adeno-associated viral vector is a recombinant AAV (rAAV).

15. The method of 14, wherein the rAAV is AAV-1, AAV-2, AAV-3, AAV-4, AAV- 5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV- anc80, AAV-PHP.eB, AAV-PHP.eB, AAV-PHP.S, AAV.CAP-B10, AAV.CAP-B22, AAV.CAP-Mac, AAV-MaCPNSl, AAV-MaCPNS2, AAVrh32.33 or AAV rh.74.

16. The method of claim 14 or 15, wherein the rAAV is rAAV-9.

17. The method of any one of claims 1 to 16, wherein target mRNA is coded by one or more genes comprising Abcdl, Actb, Adnp, Ahdcl, Akt3, Ank2, Ankrdl 1, Apls2, Aridla, Arid2, Arx, Ashll, Asxll, Atplal, Atp8a2, Atrx, Auts2, Brwd3, Camtal, Cavl, Cdkl5, Chll, Cic, Cnksr2, Crebbp, Ctcf, Ctnnbl, Ctnnd2, Dex, Ddx3x, Dffb, Dmd, Dnmt3a, Dpp6, Dyrkla, Erf, Fgfrl, Flna, Foxp2, Fus, Gabral, Gatad2b, Gria2, Gria3, Grinl, Grin2a, Grin2b, Gtf2i, Hdac4, Hicl, Hivep2, Hmga2, Hnmpk, Hnrnpu, Huwel, Ids, Iqsec2, Kenai, Kcnab2, Kcnbl, Kcnq2, Kdm6a, Kifla, Kiflb, Kmt2a, Kmt2b, Kmt2c, Kmt2d, Llcam, Magel2, Magtl, Map2kl, Mapt, Mbd5, Mecp2, Medl31, Mef2c, Meis2, Mllt3, Mytll, Nckapl, Nexmif, Nfl, Nf2, Nfia, Nfix, Nrxnl, Nsdl, Ocrl,Pafahlbl, Pak2, Pak3, Pax6, Pcdhl9, Pdhal, Pgapl, Phf6, Phip, Plpl, Pnpla6, Prrt2, Pten, Puml, Pura, Qrichl, Rbfoxl, Rein, Rps6ka3, Rtn4r, Satb2, Senia, Scn2a, Scn8a, Setbpl, Setd5, Shank3, Six3, Slc2al, Slc5a3, Slc6al, Slc6a8, Slc9a6, Smarca2, Smarccl, Smcla, Snca, Son, Soxl l, Sox8, Sptanl, Sptbnl, Sptbn2, Stxbpl, Suzl2, Syngapl, Tbkl, Tbrl, Tcf4, Trio, Tscl, Tspan7, Vampl, Wdfy3, Wdr26, Ywhae, Zdhhc9, Zeb2 or Zic2.

18. A method to increase haplo-insufficient gene protein levels comprising removing miRNA repression from a functioning allele of a haplo-insufficient gene, wherein the miRNA repression is removed by introducing a dCas protein and one or more gRNA into a cell, wherein the one or more gRNA is complementary to a miRNA response element (MRE) on a target mRNA expressed from the functioning allele of the haplo-insufficient gene so as to block miRNA repression from the functioning allele and increase said protein levels.

19. The method of claim 18, wherein the dCas protein is a dCasl3d protein, dCasl3b, dCasl3a protein or ortholog or variant thereof.

20. The method of claim 19, wherein the dCasl3d protein variant or dCasl3b protein variant is one or more of dRfxCasl3d (dCasRx), k87Casl3d (dCask87), 212Casl3d (dCas212) and / or PspCas 13b (dCasl3b).

21. The method of claim 19 or 20, wherein the dCasl3d protein variant is dCasRx.

22. The method of any one of claims 18 to 21, wherein the dCas protein is fused to at least one localization signal.

23. The method of claim 22, wherein the localization signal is a nuclear export signal (NES).

24. The method of any one of claims 18 to 23, wherein the dCas protein and the one or more gRNA are provided by one or more polynucleotide molecules coding for the dCas protein and / or the one or more gRNA, and wherein the one or more polynucleotidemolecules are operably configured to express the dCas protein and / or the one or more gRNA.

25. The method of claim 24, wherein the one or more polynucleotide molecules comprise one or more promoters.

26. The method of claim 25, wherein the one or more promoters comprise one or more inducible promoters or one or more tissue specific promoters.

27. The method of any one of claims 24 to 26, wherein the one or more polynucleotide molecules are contained within one or more vectors.

28. The method of any one of claims 24 to 27, wherein the one or more polynucleotide molecules are contained within a vector.

29. The method of claim 27 or 28, wherein the vector comprises a viral vector.

30. The method of claim 29, wherein the viral vector comprises one or more retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, or herpes simplex viral vectors.

31. The method of claim 30, wherein the adeno-associated viral vector is a recombinant AAV (rAAV).

32. The method of 31, wherein the rAAV is AAV-1, AAV-2, AAV-3, AAV-4, AAV- 5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV- anc80, AAV-PHP.eB, AAV-PHP.eB, AAV-PHP.S, AAV.CAP-B10, AAV.CAP-B22, AAV.CAP-Mac, AAV-MaCPNSl, AAV-MaCPNS2, AAVrh32.33 or AAV rh.74.

33. The method of claim 31 or 32, wherein the rAAV is rAAV-9.

34. The method of any one of claims 18 to 33, wherein target mRNA is coded by one or more genes comprising Abcdl, Actb, Adnp, Ahdcl, Akt3, Ank2, Ankrdl 1, Apls2, Aridla, Arid2, Arx, Ashll, Asxll, Atplal, Atp8a2, Atrx, Auts2, Brwd3, Camtal, Cavl,Cdkl5, Chll, Cic, Cnksr2, Crebbp, Ctcf, Ctnnbl, Ctnnd2, Dex, Ddx3x, Dffb, Dmd, Dnmt3a, Dpp6, Dyrkla, Erf, Fgfrl, Flna, Foxp2, Fus, Gabral, Gatad2b, Gria2, Gria3, Grinl, Grin2a, Grin2b, Gtf2i, Hdac4, Hicl, Hivep2, Hmga2, Hnmpk, Hnrnpu, Huwel, Ids, Iqsec2, Kenai, Kcnab2, Kcnbl, Kcnq2, Kdm6a, Kifla, Kiflb, Kmt2a, Kmt2b, Kmt2c, Kmt2d, Llcam, Magel2, Magtl, Map2kl, Mapt, Mbd5, Mecp2, Medl31, Mef2c, Meis2, Mllt3, Mytll, Nckapl, Nexmif, Nfl, Nf2, Nfia, Nfix, Nrxnl, Nsdl, Ocrl, Pafahlbl, Pak2, Pak3, Pax6, Pcdhl9, Pdhal, Pgapl, Phf6, Phip, Plpl, Pnpla6, Prrt2, Pten, Puml, Pura, Qrichl, Rbfoxl, Rein, Rps6ka3, Rtn4r, Satb2, Senia, Scn2a, Scn8a, Setbpl, Setd5, Shank3, Six3, Slc2al, Slc5a3, Slc6al, Slc6a8, Slc9a6, Smarca2, Smarccl, Smcla, Snca, Son, Soxl l, Sox8, Sptanl, Sptbnl, Sptbn2, Stxbpl, Suzl2, Syngapl, Tbkl, Tbrl, Tcf4, Trio, Tscl, Tspan7, Vampl, Wdfy3, Wdr26, Ywhae, Zdhhc9, Zeb2 or Zic2.

35. A method to prevent miRNA repression of their target mRNAs comprising introducing dCas protein and one or more gRNA into a cell, wherein the one or more gRNA are complementary to a miRNA response element (MRE) on the target mRNA expressed by the functioning allele of a haplo-insufficient gene so as to block miRNA repression of the target mRNA.

36. The method of claim 35, wherein the dCas protein is a dCasl3d protein, dCasl3b, dCasl3a protein or ortholog or variant thereof.

37. The method of claim 36, wherein the dCasl3d protein variant or dCasl3b protein variant is one or more of dRfxCasl3d (dCasRx), k87Casl3d (dCask87), 212Casl3d (dCas212) and / or PspCas 13b (dCasl3b).

38. The method of claim 36 or 37, wherein the dCasl3d protein variant is dCasRx.

39. The method of any one of claims 35 to 38, wherein the dCas protein is fused to at least one localization signal.

40. The method of claim 39, wherein the localization signal is a nuclear export signal (NES).

41. The method of any one of claim 35 to 40, wherein the dCas protein and the one or more gRNA are provided by one or more polynucleotide molecules coding for the dCas protein and / or the one or more gRNA, and wherein the one or more polynucleotide molecules are operably configured to express the dCas protein and / or the one or more gRNA.

42. The method of claim 41, wherein the one or more polynucleotide molecules comprise one or more promoters.

43. The method of claim 42, wherein the one or more promoters comprise one or more inducible promoters or one or more tissue specific promoters.

44. The method of any one of claims 41 to 43, wherein the one or more polynucleotide molecules are contained within one or more vectors.

45. The method of any one of claims 41 to 44, wherein the one or more polynucleotide molecules are contained within a vector.

46. The method of claim 44 or 45, wherein the vector comprises a viral vector.

47. The method of claim 46, wherein the viral vector comprises one or more retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, or herpes simplex viral vectors.

48. The method of claim 47, wherein the adeno-associated viral vector is a recombinant AAV (rAAV).

49. The method of 48, wherein the rAAV is AAV-1, AAV-2, AAV-3, AAV-4, AAV- 5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV- anc80, AAV-PHP.eB, AAV-PHP.eB, AAV-PHP.S, AAV.CAP-B10, AAV.CAP-B22, AAV.CAP-Mac, AAV-MaCPNSl, AAV-MaCPNS2, AAVrh32.33 or AAV rh.74.

50. The method of claim 48 or 49, wherein the rAAV is rAAV-9.

51. The method of any one of claim 35 to 50, wherein target mRNA is coded by one or more genes comprising Abcdl, Actb, Adnp, Ahdcl, Akt3, Ank2, Ankrdl 1, Apls2, Aridla, Arid2, Arx, Ashll, Asxll, Atplal, Atp8a2, Atrx, Auts2, Brwd3, Camtal, Cavl, Cdkl5, Chll, Cic, Cnksr2, Crebbp, Ctcf, Ctnnbl, Ctnnd2, Dex, Ddx3x, Dffb, Dmd, Dnmt3a, Dpp6, Dyrkla, Erf, Fgfrl, Flna, Foxp2, Fus, Gabral, Gatad2b, Gria2, Gria3, Grinl, Grin2a, Grin2b, Gtf2i, Hdac4, Hicl, Hivep2, Hmga2, Hnmpk, Hnrnpu, Huwel, Ids, Iqsec2, Kenai, Kcnab2, Kcnbl, Kcnq2, Kdm6a, Kifla, Kiflb, Kmt2a, Kmt2b, Kmt2c, Kmt2d, Llcam, Magel2, Magtl, Map2kl, Mapt, Mbd5, Mecp2, Medl31, Mef2c, Meis2, Mllt3, Mytll, Nckapl, Nexmif, Nfl, Nf2, Nfia, Nfix, Nrxnl, Nsdl, Ocrl, Pafahlbl, Pak2, Pak3, Pax6, Pcdhl9, Pdhal, Pgapl, Phf6, Phip, Plpl, Pnpla6, Prrt2, Pten, Puml, Pura, Qrichl, Rbfoxl, Rein, Rps6ka3, Rtn4r, Satb2, Senia, Scn2a, Scn8a, Setbpl, Setd5, Shank3, Six3, Slc2al, Slc5a3, Slc6al, Slc6a8, Slc9a6, Smarca2, Smarccl, Smcla, Snca, Son, Soxl l, Sox8, Sptanl, Sptbnl, Sptbn2, Stxbpl, Suzl2, Syngapl, Tbkl, Tbrl, Tcf4, Trio, Tscl, Tspan7, Vampl, Wdfy3, Wdr26, Ywhae, Zdhhc9, Zeb2 or Zic2.

52. A method to de-repress at least one target RNA of the functioning allele of a haplo-insufficient gene expressing comprising introducing a dCas protein and one or more gRNA into a cell, wherein the one or more gRNA are complementary to at least one miRNA response element (MRE) on the at least one target RNA so as to cause the dCas / gRNA to bind to the at least one MRE thereby de-repressing miRNA repression of the at least one target RNA of the functioning allele of a haplo-insufficient gene expressing.

53. The method of claim 52, wherein the dCas protein is a dCasl3d protein, dCasl3b, dCasl3a protein or ortholog or variant thereof.

54. The method of claim 53, wherein the dCasl3d protein variant or dCasl3b protein variant is one or more of dRfxCasl3d (dCasRx), k87Casl3d (dCask87), 212Casl3d (dCas212) and / or PspCas 13b (dCasl3b).

55. The method of claim 53 or 54, wherein the dCasl3d protein variant is dCasRx.

56. The method of any one of claims 52 to 55, wherein the dCas protein is fused to at least one localization signal.

57. The method of claim 56, wherein the localization signal is a nuclear export signal (NES).

58. The method of any one of claim 52 to 57, wherein the dCas protein and the one or more gRNA are provided by one or more polynucleotide molecules coding for the dCas protein and / or the one or more gRNA, and wherein the one or more polynucleotide molecules are operably configured to express the dCas protein and / or the one or more gRNA.

59. The method of claim 58, wherein the one or more polynucleotide molecules comprise one or more promoters.

60. The method of claim 59, wherein the one or more promoters comprise one or more inducible promoters or one or more tissue specific promoters.

61. The method of any one of claims 58 to 60, wherein the one or more polynucleotide molecules are contained within one or more vectors.

62. The method of any one of claims 58 to 61, wherein the one or more polynucleotide molecules are contained within a vector.

63. The method of claim 61 or 62, wherein the vector comprises a viral vector.

64. The method of claim 63, wherein the viral vector comprises one or more retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, or herpes simplex viral vectors.

65. The method of claim 64, wherein the adeno-associated viral vector is a recombinant AAV (rAAV).

66. The method of 65, wherein the rAAV is AAV-1, AAV-2, AAV-3, AAV-4, AAV- 5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV- anc80, AAV-PHP.eB, AAV-PHP.eB, AAV-PHP.S, AAV.CAP-B10, AAV.CAP-B22, AAV.CAP-Mac, AAV-MaCPNSl, AAV-MaCPNS2, AAVrh32.33 or AAV rh.74.

67. The method of claim 65 or 66, wherein the rAAV is rAAV-9.

68. The method of any one of claims 52 to 67, wherein target mRNA is coded by one or more genes comprising Abcdl, Actb, Adnp, Ahdcl, Akt3, Ank2, Ankrdl 1, Apls2, Aridla, Arid2, Arx, Ashll, Asxll, Atplal, Atp8a2, Atrx, Auts2, Brwd3, Camtal, Cavl, Cdkl5, Chll, Cic, Cnksr2, Crebbp, Ctcf, Ctnnbl, Ctnnd2, Dex, Ddx3x, Dffb, Dmd, Dnmt3a, Dpp6, Dyrkla, Erf, Fgfrl, Flna, Foxp2, Fus, Gabral, Gatad2b, Gria2, Gria3, Grinl, Grin2a, Grin2b, Gtf2i, Hdac4, Hicl, Hivep2, Hmga2, Hnmpk, Hnrnpu, Huwel, Ids, Iqsec2, Kenai, Kcnab2, Kcnbl, Kcnq2, Kdm6a, Kifla, Kiflb, Kmt2a, Kmt2b, Kmt2c, Kmt2d, Llcam, Magel2, Magtl, Map2kl, Mapt, Mbd5, Mecp2, Medl31, Mef2c, Meis2, Mllt3, Mytll, Nckapl, Nexmif, Nfl, Nf2, Nfia, Nfix, Nrxnl, Nsdl, Ocrl, Pafahlbl, Pak2, Pak3, Pax6, Pcdhl9, Pdhal, Pgapl, Phf6, Phip, Plpl, Pnpla6, Prrt2, Pten, Puml, Pura, Qrichl, Rbfoxl, Rein, Rps6ka3, Rtn4r, Satb2, Senia, Scn2a, Scn8a, Setbpl, Setd5, Shank3, Six3, Slc2al, Slc5a3, Slc6al, Slc6a8, Slc9a6, Smarca2, Smarccl, Smcla, Snca, Son, Soxl l, Sox8, Sptanl, Sptbnl, Sptbn2, Stxbpl, Suzl2, Syngapl, Tbkl, Tbrl, Tcf4, Trio, Tscl, Tspan7, Vampl, Wdfy3, Wdr26, Ywhae, Zdhhc9, Zeb2 or Zic2.

Images