Dcas9-KRAB with increased nuclear localization and stability in neurons

Abstract

Provided herein are fusion proteins comprising an enzymatically inactive CRISPR enzyme fused to a modulator of gene expression, where the fusion protein comprises a neuron-specific nuclear localization signal (NLS), and where the modulator of gene expression comprises a Kruppel associated box (KRAB) domain. Also provided herein are method for modulating the expression of a gene in a neuron using a CRISPR system using the fusion proteins.

Description

[0001] DESCRIPTION dCAS9-KRAB WITH INCREASED NUCLEAR LOCALIZATION AND STABILITY IN NEURONS REFERENCE TO RELATED APPLICATIONS The present application claims the priority benefit of United States provisional application number 63 / 706,828, filed October 14, 2024, the entire contents of which are incorporated herein by reference. REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on October 9, 2025, is named CHOPP0085WO.xml and is 38,649 bytes in size. BACKGROUND 1. Field The present disclosure relates generally to the field of molecular biology. More particularly, it concerns compositions and methods for genome editing in neurons. 2. Description of Related Art The ability to differentiate induced pluripotent stem cells (iPSCs) directly into human neurons introduced a powerful and easily accessible cell model to study the molecular biology of neurological diseases (Pang et al. 2011). Efficient implementation of tools for genetic manipulation, such as CRISPR-associated enzymes for gene knockout and knockdown, is essential for this model to realize its full potential, yet such implementation is frequently associated with unique challenges compared to immortalized cell lines. For example, loss-of-function screens using double strand break induced frameshifts are widespread and have led to numerous discoveries in immortalized cell lines, yet their implementation in iPSCs has been challenging due to increased sensitivity to DNA damage (Ihry et al. 2018). CRISPR inhibition and activation has emerged as a powerful and orthogonal approach to CRISPR knockout, which could be utilized in iPSCs as it works by recruiting repression or activation proteins to gene promoters and does not require the induction of DNA damage for its activity (Tian et al.2019, 2021). CRISPR inhibition and activation screens in iPSC-derived neurons have been successful in uncovering genes associated with several neuronal phenotypes including, survival (Tian et al.2019), sensitivity to stress (Tian et al.2021), tau aggregation(Parra Bravo et al. 2024) and the regulation of alpha-synuclein protein levels (Santhosh Kumar et al. 2024). In all of these cases, the screens have relied on introduction of genetic perturbations at the iPSC or early neuronal precursor stage, followed by evaluation of phenotypes after differentiation into mature neurons. This can confound phenotypes related to neuronal maintenance with defects in differentiation and maturation. It may also limit our ability to study genes that are essential for iPSC survival and growth but with distinct roles in post- mitotic neurons. However, developing screening systems where genetic perturbations are performed directly in neurons faces several biological and technical hurdles. Differential stability of proteins and RNA and differential chromatin organization can make it challenging to predict and evaluate the effectiveness of perturbations. And the efficiency of these perturbations is dependent upon ensuring high nuclear expression levels of CRISPR machinery at the time of their introduction. SUMMARY Provided herein are fusion proteins comprising an enzymatically inactive CRISPR enzyme and a neuron-specific nuclear localization signal (NLS). The enzymatically inactive CRISPR enzyme may be an enzymatically inactive Cas9 (dCas9; SEQ ID NO: 1) or a nickase Cas9 (nCas9; SEQ ID NO: 7). The dCas9 enzyme may comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1 in which position 10 is alanine and / or position 840 is alanine. The nCas9 enzyme may comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 7. The neuron-specific nuclear localization signal (NLS) may be a MeCP2 NLS. The neuron-specific nuclear localization signal (NLS) may comprise an amino acid sequence

[0002] 24922-8547-0065, v. 1 at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2. The fusion protein may comprise two copies of the neuron-specific nuclear localization signal (NLS). The neuron-specific nuclear localization signal (NLS) may be positioned at the N-terminal end of the fusion protein. The fusion protein may further comprise a hRNPD NLS, which may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4. The fusion protein may further comprise a cMyc NLS, which may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3. The fusion protein may further comprise a nucleoplasmin NLS and / or SV40 NLS. The fusion protein may further comprise a modulator of gene expression. The modulator of gene expression comprises a Kruppel associated box (KRAB) domain. The KRAB domain may be from a KOX1 gene or a zinc finger imprinted 3 (Zim3) gene. Any two domains or regions of the fusion protein may be connected by an amino acid linker, such as, for example, the linker in SEQ ID NO: 8. The fusion protein may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of any of SEQ ID NOs: 9- 14. SEQ ID NO: 9 provides the sequence of the MeCP2 NLS dCas9-KRAB construct. SEQ ID NO: 10 provides the sequence of the cMyc NLS+MeCP2 NLS dCas9-KRAB construct. SEQ ID NO: 11 provides the sequence of the hRNPD NLS+MeCP2 NLS dCas9-KRAB construct. SEQ ID NO: 12 provides the sequence of the 2xMeCP2 NLS dCas9-KRAB construct. SEQ ID NO: 13 provides the sequence of the 2xMeCP2 NLS Zim3-dCas9 construct. SEQ ID NO: 14 provides the sequence of the 2xMeCP2 NLS nCas9 construct. Provided herein are nucleic acid molecules comprising a nucleotide sequence that encoding any of the fusion proteins described herein. The nucleic acid molecule may be a DNA molecule or an RNA molecule.

[0003] 34922-8547-0065, v. 1 Provided herein are vectors comprise any of the nucleic acid molecules described herein. The vector may be a viral vector. Provided herein are neurons comprising any of the fusion proteins or any of the nucleic acids described herein. The neuron may be an iPSC-derived neuron or a primary neuron. The neuron may be in vitro, ex vivo, or in vivo. Provided herein are methods for modulating the expression of a gene in a neuron, the methods comprising transfecting or transducing the cell with (i) any of the fusion proteins described herein are any of the nucleic acid molecules described herein and (ii) a gRNA which targets the fusion molecule to the gene or a nucleic acid that encodes a gRNA which targets the fusion molecule to the gene. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIGS. 1A-1D. Mislocalization and differential protein stability of nCas9 and dCas9KRAB in iPSC-derived neurons post-differentiation. (FIG. 1A) Illustrations of transposase donor plasmids for making stable dCas9 and nCas9 lines in KOLF2.1J_NGN2 iPSCs (drawn to scale). (FIG. 1B) Flow cytometry plots measuring dCas9-KRAB or nCas9 expression via tagBFP fluorescence in stable iPSCs (top) vs expression in neurons 14 days post induction of differentiation with dox (bottom). Red line indicates the approximate cutoff for positive cells based on parental line autofluorescence. dCas9-KRAB and nCas9 expression are comparable at the iPSC stage; however, dCas9-KRAB neurons appear to lose almost all expression whereas the expression distribution of nCas9 neurons mirrors that of

[0004] 44922-8547-0065, v. 1 nCas9 iPSCs. Plots represent a minimum of 10,000 analyzed single cells for iPSCs and 3000 analyzed single cells for neurons. (FIG. 1C) Quantification of dCas9-KRAB and nCas9 mRNA via RT-qPCR in Day 14 neurons shows no significant difference in gene expression across cell lines, suggesting that loss of dCas9-KRAB in neurons is independent of gene silencing. Values are shown relative to GAPDH expression after being normalized to no-RT samples. Data represents 2-3 independent wells per line. Error bars represent mean ± SD. Unpaired T-test with Welch’s correction was used to compare RNA levels. ns P=0.1694. (FIG. 1D) Representative images of dCas9-KRAB and nCas9 protein localization in iPSCs (left) and Day 14 neurons (right) with ICC using a Cas9 antibody. Cells are stained for actin (phalloidin) or with a MAP2 antibody, and nuclei with NucSpot 750 / 780. Nuclei are outlined for reference. In iPSCs, both versions show localization to the nucleus (dCas9-KRAB is more diffuse, whereas Cas9 appears more nucleolar), along with bright cytoplasmic accumulations. In D14 neurons, both versions show almost exclusively punctate cytoplasmic staining, similar to the cytoplasmic staining in iPSCs. Images are Max IPs of 3-plane (iPSCs) or 4-plane (neurons) Z-stacks (0.6 µm) taken at 40x with a spinning disk confocal. All images within cell type are adjusted to the same LUTS, based on background Cas9 antibody staining in the Parental line. iPSCs and neurons are shown at the same scale; scale bars represent 20 µm. FIGS.2A-2G. Alternative NLSs improve dCas9-KRAB nuclear localization in iPSC-derived neurons and stabilize protein levels during differentiation. (FIG. 2A) Illustration of modified transposase donor plasmids to test the effect of alternative NLSs on dCas9-KRAB localization and stability in neurons (drawn to scale). (FIG. 2B) Flow cytometry plots measuring expression of modified dCas9-KRAB constructs or nCas9 via tagBFP fluorescence in neurons 14 days post induction of differentiation with dox from stably integrated iPSCs. Red line indicates the approximate cutoff for positive cells based on parental line autofluorescence. Plots represent a minimum of 3000 analyzed single cells. (FIG. 2C) Quantification of flow cytometry data showing the mean fluorescence intensity of tagBFP normalized to autofluorescence of dCas9-KRAB constructs and nCas9 in Day 14 neurons (Parental autofluorescence=1). Alternative NLS sequences rescue dCas9-KRAB expression to levels comparable to nCas9, with the 2xMeCP2 NLS construct (SEQ ID NO: 12) having the highest expression with a mean population intensity of 4.931x the parental, compared to a mean of 4.586x for the MeCP2 NLS construct (SEQ ID NO: 9) and 4.636x for the cMyc NLS+MeCP2 NLS construct (SEQ ID NO: 10). The hRNPD NLS+MeCP2 NLS construct is SEQ ID NO: 11. Data represent the mean fluorescence intensity of 2-3 wells per

[0005] 54922-8547-0065, v. 1 line with a minimum of 2000 analyzed single cells per sample. Error bars represent mean ± SD. (FIG. 2D) Representative Western blots (left) and quantification (right) of total protein from Day 14 neurons probed for Cas9 showing significant rescue of total protein levels of 2xMeCPNLS-dCas-KRAB compared to the original dCas9-KRAB, with levels comparable to nCas9. Cas9 antibody signal was normalized to TUBB3 levels. Data represents 3 independent wells per line. Error bars represent mean ± SD. Protein levels between lines were compared with One-way ANOVA with Tukey’s multiple comparisons test. **P=0.0050 for dCas9- KRAB vs Cas9, *P=0.0150 for dCas9-KRAB vs 2xMeCP2 NLS-dCas9-KRAB, ns P=0.5719 for nCas9 vs 2xMeCP NLS-dCas9-KRAB. (FIG. 2E) Representative Western blots (left) and quantifications (right) of dCas9-KRAB or nCas9 protein levels in nuclear and cytoplasmic protein fractions from Day 14 neurons. The original dCas9-KRAB and nCas9 are both predominantly cytoplasmic with nearly identical distributions despite much lower dCas9- KRAB levels. The 2xMeCP2NLS_dCas9-KRAB construct shows a significant shift into the nuclear fraction which corresponds with the rescue of total protein levels. LaminB1 and GAPDH were used as nuclear and cytoplasmic controls, respectively, and were probed on replicate sample blots in parallel with Cas9. Quantifications for each protein represent the intensity of the signal in the respective fraction divided by the sum of intensities across both fractions. Cas9 quantifications were performed on respective LaminB1 and GAPDH blots. Data represent 3 independent wells per line. Error bars represent mean ± SD. Fractionation control and Cas9 antibody signals across cell lines were compared with 2-way ANOVA with Tukey’s multiple comparisons test. For Cas9 on LaminB1 blots: ns P=0.1236 for dCas9- KRAB vs nCas9, **** P<0.0001 for dCas9-KRAB vs 2xMeCP2 NLS-dCas9-KRAB, *** P<0.0001 for nCas9 vs 2xMeCP2 NLS-dCas9-KRAB. For Cas9 on GAPDH blots: ns P=0.6805 for dCas9-KRAB vs nCas9, **** P<0.0001 for dCas9-KRAB vs 2xMeCP2 NLS- dCas9-KRAB, **** P<0.0001 for nCas9 vs 2xMeCP2 NLS-dCas9-KRAB. (FIG. 2F) Representative images of dCas9-KRAB protein localization in Day 14 neurons with ICC using a Cas9 antibody. Cells are stained with a MAP2 antibody and nuclei with NucSpot 750 / 780. Nuclei are outlined for reference. 2xMeCP2NLS-dCas9-KRAB shows improved nuclear localization over original nCas9 and dCas9-KRAB constructs and higher intensity staining than the original dCas9-KRAB, consistent with the results from biochemistry assays. Images are Max IPs of 4-plane Z-stacks (0.6 µm) taken at 40x with a spinning disk confocal. All images are adjusted to the same LUTS, based on background Cas9 antibody staining in the Parental line. Scale bars represent 20 µm. (FIG. 2G) Quantification of RT-qPCR data showing significantly improved knockdown of PSAP (top) and SNCA (bottom) in neurons

[0006] 64922-8547-0065, v. 1 expressing the 2xMeCP2NLS-dCas9-KRAB vs the original dCas9-KRAB. Neurons were transduced with lentivirus expressing an mScarlet marker with either a targeting or non- targeting sgRNA at Day 14 (PSAP) or Day 21 (SNCA) post-differentiation and RNA harvested 7-days post-transduction. Gene expression levels were normalized to GAPDH and ACTB and are shown relative to the non-targeting average. Data represent 3 independent wells per transduction per line. 2-way ANOVA with uncorrected Fisher’s LSD was used to compare targeting guide with non-targeting guide within cell lines and targeting guides across cell lines. For PSAP KDs: ns P=0.0745 for dCas9-KRAB+NTCg vs +PSAPg, *** P=0.0004 for 2xMeCP2 NLS-dCas9-KRAB+NTCg vs +PSAPg, ** P=0.006 for dCas9-KRAB+PSAPg vs 2xMeCP2 NLS-dCas9-KRAB+PSAPg. For SNCA KDs: * P=0.0152 for dCas9- KRAB+NTCg vs +SNCAg, **** P<0.0001 for 2xMeCP2 NLS-dCas9-KRAB+NTCg vs +SNCAg, ** P=0.0035 for dCas9-KRAB+SNCAg vs 2xMeCP2 NLS-dCas9- KRAB+SNCAg. FIGS. 3A-3C. NLS-mediated improvements in dCas9-KRAB expression are not neuron-specific and allow for improved knockdown efficiency after differentiation. (FIG. 3A) Flow cytometry histograms of stable iPSCs expressing the original dCas9-KRAB and nCas9 constructs and the dCas9-KRAB constructs with alternative NLSs. The MeCP2 NLS increased dCas9-KRAB expression and the 2xMeCP2 NLS construct further boosted levels to match nCas9 levels. Other NLSs in combination with a single MeCP2 NLS provided no additive benefit to dCas9-KRAB expression. (FIGS. 3B-3C) Flow cytometry histograms of neurons 7 days after lentiviral transduction with either a non-targeting control guide and a guide targeting PSAP at Day 14 (FIG. 3B) or a non-targeting control guide and a guide targeting SNCA at Day 21 (FIG. 3C). Based on mScarlet fluorescence, almost all neurons were transduced. Expression distributions for each guide at respective timepoints were comparable between dCas9-KRAB and 2xMeCP2 NLS-dCas9-KRAB neurons, so differential knockdown efficiencies between lines are likely not due to differences in transduction and sgRNA expression. FIGS. 4A-4C. Nuclear localization defects are common to SV40NLS- dependent CRISPR constructs while mislocalization-dependent expression loss is KRAB domain-specific. (FIG.4A) Representative images of Zim3-dCas9 protein localization in Day 14 neurons with ICC using a Cas9 antibody. Cells are stained with a MAP2 antibody and nuclei with NucSpot 750 / 780. Nuclei are outlined for reference. Despite showing stable

[0007] 74922-8547-0065, v. 1 protein expression, this alternative construct, which also relies onSV40 NLSs, shows a similar defect in nuclear localization in neurons. Images are a single widefield plane taken at 40x. All images are adjusted to the same LUTS. Scale bars represent 20 µm. (FIG. 4B) Representative Western blots of total protein from Day 14 neurons probed for Cas9 showing rescue of total protein levels with the addition of an MeCP2 NLS. We also show that an alternative dCas9 using the Zim3 domain instead of the KOX1 KRAB domain also retains stable protein expression during differentiation. (FIG. 4C) Quantification of dCas9 levels from western blots. Cas9 antibody signal was normalized to TUBB3 levels. Data represents 3 independent wells per line. Error bars represent mean ± SD. DETAILED DESCRIPTION High-throughput gene perturbation screening in iPSC-derived neurons holds significant promise for understanding the mechanisms underlying various neurological diseases and for identifying potential therapeutic targets. CRISPR-based screens have already been successful in relating gene function to neuronal phenotypes; however, their broad implementation and specific applications have been limited by the technical challenges of ensuring all components can be efficiently expressed in iPSC-derived neurons. Surprisingly, while the commonly used dCas9-KRAB (using the KOX1 domain; the sequence of dCas9- KRAB is provided in SEQ ID NO: 5) displays dramatic reduction in protein expression levels following neuronal differentiation, nCas9 constructs retained comparable protein levels between iPSCs and neurons. These constructs, primarily relying on SV40 Nuclear Localization Signal (NLS), failed to efficiently localize to the nuclei of neurons, despite having robust nuclear levels in iPSCs. By adding a neuronal specific NLS, neuronal nuclear localization and protein expression were corrected, confirming the contribution of mislocalization to the instability of dCas9-KRAB in neurons. I. Aspects of the Present Disclosure Recent advances in applying high throughput CRISPR-based screening methods in iPSC-derived neurons and glia cells have been successful in the elucidation of both disease and basic biology, but have also been limited by the constraints of ensuring efficient expression of sgRNAs and CRISPR machinery post-differentiation. To limit the inefficiencies associated with the delivery of either or both components at the neuron-stage, these studies have relied on delivery of these components in iPSCs followed by

[0008] 84922-8547-0065, v. 1 differentiation. To move towards a system for inducible gene perturbations post- differentiation, the inventors developed and tested piggybac vectors to generate polyclonal iPSC lines and evaluated the stability of expression of CRISPR machinery between iPSCs and neurons. A surprising discrepancy in the stability of nCas9 and dCas9-KRAB expression in iPSCs versus iPSC-derived neurons was found. After generating polyclonal stable lines in iPSCs, both nCas9 and dCas9-KRAB maintained robust expression. However, after differentiation into neurons, a specific loss in dCas9-KRAB expression was observed, whereas nCas9 remained stable, despite retaining comparable mRNA levels. Further investigation identified another unexpected hurdle: the fact that both dCas9 and nCas9 failed to efficiently localize to the nucleus in neurons despite having a robust nuclear presence in iPSCs. This mislocalization may be responsible for the observed dCas9-KRAB-specific repression. To test this, alternative NLSs were tested for the ability to rescue these dCas9- KRAB deficits in neurons. The addition of two tandem MeCP2 NLSs was able to rescue both neuronal dCas9-KRAB protein levels and drastically improve nuclear localization. In addition, this rescue was sufficient to improve the knockdown efficacy of guides delivered directly to mature neurons. While the exact mechanism of the KRAB-domain mediated loss of dCas9- KRAB protein in neurons remains unclear, it seems likely that its mislocalization to the cytoplasm engages targeted protein degradation mechanisms. Given that the KRAB domain is derived from a human transcription factor, specifically here KOX1, the most commonly used version, it may be subject to endogenous regulatory pathways that would recognize and degrade the protein when not in the nucleus or not bound to DNA. On the other hand, nCas9 is a totally ectopic protein, so its “mislocalization” to the cytoplasm is unlikely to be recognized by cells. It also appears that this is not a neuron-specific pathway, as the improved NLS constructs provided herein boosted dCas9-KRAB expression in iPSCs as well. Rather, the neuron-specific deficit in nuclear localization amplified the effect to the point of near- total protein loss. Implementation of effective, post-differentiation gene perturbation technologies will require overcoming many additional challenges including optimization of transgene expression, a neuronal specific epigenetic landscape and a wide range of protein stabilities in post mitotic cells. The studies described herein may have implications for other

[0009] 94922-8547-0065, v. 1 differentiated cell types and therapeutic development as well. Given its unique regulatory and epigenetic environment, robust nuclear levels of CRISPR machinery are a necessary prerequisite for all further steps in applying these techniques. II. CRISPR Systems Gene editing is a technology that allows for the modification of target genes within living cells. Recently, harnessing the bacterial immune system of CRISPR to perform on demand gene editing revolutionized the way scientists approach genomic editing. The Cas9 protein of the CRISPR system, which is an RNA-guided DNA endonuclease, can be engineered to target new sites with relative ease by altering its guide RNA sequence. This discovery has made sequence-specific gene editing and gene expression regulation functionally effective. In general, “CRISPR system” refers collectively to elements involved in the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and / or other sequences and transcripts from a CRISPR locus. One or more elements of a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. The CRISPR system can induce double-stranded breaks (DSBs) at the target site, followed by disruptions. The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia or S. aureus or S. auricularis or S. lugdunensis). Additional non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. For example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2.

[0010] 104922-8547-0065, v. 1 The CRISPR enzyme may be mutated with respect to a corresponding wild- type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. The deactivated Cas9 does not comprise HNH and / or RuvC nickase activities. The HNH and RuvC motifs have been characterized in S. thermophilus (see, e.g., Sapranauskas et al. Nucleic Acids Res. 39:9275- 9282 (2011)) and one of skill would be able to identify and mutate these motifs in Cas9 polypeptides from other organisms. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9. Notably, a Cas9 polypeptide in which the HNH motif and / or RuvC motif is / are specifically mutated so that the nickase activity is reduced, deactivated, and / or absent, can retain one or more of the other known Cas9 functions including DNA, RNA and PAM recognition and binding activities and thus remain functional with regard to these activities, while non-functional with regard to one or both nickase activities. Catalytically inactive Cas9 variants can be fused to a heterologous effector domain, such as a transcriptional repressor (e.g., KRAB), to affect gene expression. The S. pyogenes dCas9 molecule may have an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTA RRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD (SEQ ID NO: 1) The CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. A CRISPR enzyme fusion protein may comprise any

[0011] 114922-8547-0065, v. 1 additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid binding activity, base editing activity, or reverse transcription activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5- transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A coding sequence encoding a CRISPR enzyme may be codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its

[0012] 124922-8547-0065, v. 1 corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). A single-molecule guide RNA (sgRNA) can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and / or an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins. In some embodiments, an sgRNA can comprise a spacer sequence and a tracrRNA sequence. In some aspects, a Cas nuclease and gRNA (including a fusion of crRNA specific for the target sequence and fixed tracrRNA) are introduced into the cell. In general, target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing. The target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG. In this respect, the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Typically, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.

[0013] 134922-8547-0065, v. 1 The target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of the cell, such as within an organelle of the cell. Generally, a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template” or “editing polynucleotide” or “editing sequence.” In some aspects, an exogenous template polynucleotide may be referred to as a DNA template. In some aspects, the recombination is homologous recombination. The elements of the CRISPR system can be introduced into a cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites. Components can be delivered to cells as proteins and / or RNA. For example, a Cas enzyme can be delivered as nucleic acid (e.g., an mRNA) encoding the Cas enzyme and the guide RNA can be delivered as an sgRNA. III. Definitions The terms “polynucleotide,” “nucleic acid” and “transgene” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and polymers thereof. Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides can include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., variant nucleic acid). Polynucleotides can be single stranded, double stranded, or triplex, linear or circular, and can be of any suitable length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5ʹ to 3ʹ direction. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2’ methoxy or 2’ halide substitutions. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1- methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N4-

[0014] 144922-8547-0065, v. 1 methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, O6-methylguanine, 4- thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and O4-alkyl- pyrimidines; U.S. Patent 5,378,825 and PCT No. WO 93 / 13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11thed., 1992). Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (U.S. Patent 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2’ methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA. A nucleic acid encoding a polypeptide often comprises an open reading frame that encodes the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions. Nucleic acids can include one or more expression control or regulatory elements operably linked to the open reading frame, where the one or more regulatory elements are configured to direct the transcription and translation of the polypeptide encoded by the open reading frame in a mammalian cell. Non-limiting examples of expression control / regulatory elements include transcription initiation sequences (e.g., promoters, enhancers, a TATA box, and the like), translation initiation sequences, mRNA stability sequences, poly A sequences, secretory sequences, and the like. Expression control / regulatory elements can be obtained from the genome of any suitable organism. As used herein, “AAV” refers to an adeno-associated virus vector. As used herein, “AAV” refers to any AAV serotype and variant, including but not limited to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10 (see, e.g., SEQ ID NO: 81 of US 9,790,472, which is incorporated by reference herein in its entirety), AAVrh74

[0015] 154922-8547-0065, v. 1 (see, e.g., SEQ ID NO: 1 of US 2015 / 0111955, which is incorporated by reference herein in its entirety), AAV9 vector, AAV9P vector (also known as AAVMYO, see, Weinmann et al., 2020, Nature Communications, 11:5432), and Myo-AAV vectors described in Tabebordbar et al., 2021, Cell, 184:1-20 (e.g., MyoAAV 1A, 2A, 3A, 4A, 4C, or 4E), wherein the number following AAV indicates the AAV serotype. The term “AAV” can also refer to any known AAV (vector) system. In some embodiments, the AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the AAV vector is a double-stranded AAV (dsAAV). Any variant of an AAV vector or serotype thereof, such as a self-complementary AAV (scAAV) vector, is encompassed within the general terms AAV vector, AAV1 vector, etc. See, e.g., McCarty et al., Gene Ther. 2001;8:1248–54, Naso et al., BioDrugs 2017; 31:317-334, and references cited therein for detailed discussion of various AAV vectors. Structurally, AAVs are small (25 nm), single-DNA stranded non-enveloped viruses with an icosahedral capsid. Naturally occurring or engineered AAV serotypes and variants that differ in the composition and structure of their capsid protein have varying tropism, i.e., ability to transduce different cell types. When combined with active promoters, this tropism defines the site of gene expression. “Guide RNA”, “guide RNA”, and simply “guide” are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “guide RNA” refers to each type. The trRNA may be a naturally occurring sequence, or a trRNA sequence with modifications or variations compared to naturally occurring sequences. For clarity, the terms “guide RNA” or “guide” as used herein, and unless specifically stated otherwise, may refer to an RNA molecule (comprising A, C, G, and U nucleotides) or to a DNA molecule encoding such an RNA molecule (comprising A, C, G, and T nucleotides) or complementary sequences thereof. A “promoter” refers to a nucleotide sequence, usually upstream (5') of a coding sequence, which directs and / or controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence

[0016] 164922-8547-0065, v. 1 comprised of a TATA-box and optionally other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. An “enhancer” is a DNA sequence that can stimulate transcription activity and may be an innate element of the promoter or a heterologous element that enhances the level or tissue specificity of expression. It is capable of operating in either orientation (5’->3’ or 3’- >5’) and may be capable of functioning even when positioned either upstream or downstream of the promoter. Promoters and / or enhancers may be derived in their entirety from a native gene or be composed of different elements derived from different elements found in nature, or even be comprised of synthetic DNA segments. A promoter or enhancer may comprise DNA sequences that are involved in the binding of protein factors that modulate / control effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions. Non-limiting examples include SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, pol II promoters, pol III promoters, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, will also find use herein. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or “housekeeping” functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, the actin promoter, and other constitutive promoters known to those of skill in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include: the early and late promoters of SV40; the long terminal repeats (LTRs) of Moloney Leukemia Virus and other retroviruses; and the thymidine kinase promoter of Herpes Simplex Virus, among many others. Accordingly, any of the above-referenced constitutive promoters can be used to control transcription of a heterologous gene insert. A “transgene” is used herein to conveniently refer to a nucleic acid sequence / polynucleotide that is intended or has been introduced into a cell or organism.

[0017] 174922-8547-0065, v. 1 Transgenes include any nucleic acid, such as a gene that encodes an inhibitory RNA or polypeptide or protein, and are generally heterologous with respect to naturally occurring AAV genomic sequences. The term “transduce” refers to introduction of a nucleic acid sequence into a cell or host organism by way of a vector (e.g., a viral particle). Introduction of a transgene into a cell by a viral particle is can therefore be referred to as “transduction” of the cell. The transgene may or may not be integrated into genomic nucleic acid of a transduced cell. If an introduced transgene becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism it can be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced transgene may exist in the recipient cell or host organism extra chromosomally, or only transiently. A “transduced cell” is therefore a cell into which the transgene has been introduced by way of transduction. Thus, a “transduced” cell is a cell into which, or a progeny thereof in which a transgene has been introduced. A transduced cell can be propagated, transgene transcribed and the encoded inhibitory RNA or protein expressed. For gene therapy uses and methods, a transduced cell can be in a mammal. A nucleic acid / transgene is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. A nucleic acid / transgene encoding and RNAi or a polypeptide, or a nucleic acid directing expression of a polypeptide may include an inducible promoter, or a tissue-specific promoter for controlling transcription of the encoded polypeptide. A nucleic acid operably linked to an expression control element can also be referred to as an expression cassette. As used herein, the terms “modify” or “variant” and grammatical variations thereof, mean that a nucleic acid, polypeptide or subsequence thereof deviates from a reference sequence. Modified and variant sequences may therefore have substantially the same, greater or less expression, activity or function than a reference sequence, but at least retain partial activity or function of the reference sequence. A particular type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation, e.g., a missense or nonsense mutation. In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”)

[0018] 184922-8547-0065, v. 1 genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and / or other sequences and transcripts from a CRISPR locus. As used herein, a “spacer sequence,” sometimes also referred to herein and in the literature as a “spacer,” “protospacer,” “guide sequence,” or “targeting sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for cleavage by a Cas9. For clarity, the terms “spacer sequence”, “spacer,” “protospacer,” “guide sequence,” or “targeting sequence” as used herein, and unless specifically stated otherwise, may refer to an RNA molecule (comprising A, C, G, and U nucleotides) or to a DNA molecule encoding such an RNA molecule (comprising A, C, G, and T nucleotides) or complementary sequences thereof. A “nucleic acid” or “polynucleotide” variant refers to a modified sequence which has been genetically altered compared to wild-type. The sequence may be genetically modified without altering the encoded protein sequence. Alternatively, the sequence may be genetically modified to encode a variant protein. A nucleic acid or polynucleotide variant can also refer to a combination sequence which has been codon modified to encode a protein that still retains at least partial sequence identity to a reference sequence, such as wild-type protein sequence, and also has been codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without altering the amino acids of a protein encoded thereby, and some codons of the nucleic acid variant will be changed which in turn changes the amino acids of a protein encoded thereby. The terms “protein” and “polypeptide” are used interchangeably herein. The “polypeptides” encoded by a “nucleic acid” or “polynucleotide” or “transgene” disclosed herein include partial or full-length native sequences, as with naturally occurring wild-type and functional polymorphic proteins, functional subsequences (fragments) thereof, and sequence variants thereof, so long as the polypeptide retains some degree of function or activity. Accordingly, in methods and uses of the disclosure, such polypeptides encoded by nucleic acid sequences are not required to be identical to the endogenous protein that is defective, or whose activity, function, or expression is insufficient, deficient or absent in a treated mammal.

[0019] 194922-8547-0065, v. 1 An example of an amino acid modification is a conservative amino acid substitution or a deletion. In particular embodiments, a modified or variant sequence retains at least part of a function or activity of the unmodified sequence (e.g., wild-type sequence). Another example of an amino acid modification is a targeting peptide introduced into a capsid protein of a viral particle. Peptides have been identified that target recombinant viral vectors or nanoparticles to various organs and tissues. A “variant” of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis, which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the disclosure will have at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence. In certain embodiments, the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of activity or function of wild-type). “Conservative variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGT, CGC, CGA, CGG, AGA and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are “silent variations,” which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein that encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill in the art will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be

[0020] 204922-8547-0065, v. 1 modified to yield a functionally identical molecule by standard techniques. Accordingly, each “silent variation” of a nucleic acid that encodes a polypeptide is implicit in each described sequence. The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, at least 80%, 90%, or even at least 95%. The term “substantial identity” in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with antibodies raised against the second polypeptide. Thus, a polypeptide is identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution. The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, inhibit, reduce, or decrease an undesired physiological change or disorder, such as the development, progression or worsening of the disorder. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilizing a (i.e., not worsening or progressing) symptom or adverse effect of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not

[0021] 214922-8547-0065, v. 1 receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those predisposed (e.g., as determined by a genetic assay). As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods. As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more. Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value. IV. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0022] 224922-8547-0065, v. 1 Example 1 – Materials & Methods Cloning. Original piggybac donor vectors were obtained from VectorBuilder. Guide cloning was performed via Golden Gate assembly in a CROPseq_v2 backbone modified to express 2xcMycNLS-mScarlet-P2A / T2A-puromycin. All oligos for cloning were purchased from IDT. PCRs were performed with PrimeSTAR® GXL DNA Polymerase (Takara, R050A). Custom plasmids were generated with NEBuilder® HiFi DNA Assembly Master Mix (NEB, E2621S). iPSC Maintenance. KOLF2.1J_NGN2 iPSCs (The Jackson Laboratory), described previously (Pantazis et al., 2022; Reilly et al., 2023), were cultured in mTesR Plus medium (STEMCELL Tech, 100-0276) on 6-well cell culture plates coated with hESC- Qualified, LDEV-Free, Matrigel Matrix (Corning, 354277) diluted according to manufacturer’s lot number recommendation. Media was replaced every other day until 80%– 90% confluent when cells were then passaged with Versene (ThermoFisher Scientific, 15040066). Media was aspirated and cells washed 2x with DPBS (ThermoFisher Scientific, 14190144) and then incubated with Versene at 37C for 5-7min. Versene was then aspirated, and cells lifted by washing the well with fresh mTesR Plus medium and gently scraping if needed. Colonies were broken up by gently triturating the cell mixture before transferring cells to a new Matrigel-coated plate at desired concentration. Generation of stable iPSC lines. iPSCs were collected using Accutase (ThermoFisher Scientific, A1110501) by aspirating media, washing 2x with DPBS, and then incubating with Accutase at 37C for 10min. Accutase was then diluted with mTesR Plus medium supplemented with 10nM Y-27632 dihydrochloride ROCK inhibitor (Tocris, 125410) and iPSCs pelleted and then resuspended in fresh mTeSR Plus with Y-27632 for counting. iPSCs were plated at 1.5x106 cells per well of a 6-well plate on hESC Matrigel in 1.5mL of media. After at least 1 hour, iPSCs were transfected using Lipofectamine™ Stem Transfection Reagent (ThermoFisher Scientific, STEM00008) at 5 µL per well with 1ug of DNA at a donor to transposase ratio of 2:1. The following day, iPSCs were split again with Accutase and then maintained in mTeSR Plus with Y-27632 and Blasticidin S HCl (ThermoFisher Scientific, A1113903) selection at 10ug / mL for at least 1 week prior to cell sorting, with Accutase passaging as needed. To generate polyclonal stable lines, the top 50% of cells with distinct tagBFP signal were kept with a minimum of 10,000 cells sorted for any given line. Following sorting, iPSCs were allowed to recover in mTeSR Plus with Y-27632,

[0023] 234922-8547-0065, v. 1 blasticidin selection, and Penicillin-Streptomycin (ThermoFisher Scientific, 15070063). Y- 27632 was removed 48-72 hours post-sorting, and iPSCs were maintained as normal with Versene passaging as needed. Blasticidin and Pen-Strep were removed 1 week post-sorting. Neuronal Differentiation. KOLF2.1J_NGN2 iPSCs were differentiated using doxycycline-induced expression of NGN2 based on previously described methods (Pantazis et al., 2022; Reilly et al., 2023). Day 0 iPSCs were collected using Accutase (ThermoFisher Scientific, A1110501) by aspirating media, washing 2x with DPBS, and then incubating with Accutase at 37C for 10min. Accutase was then diluted with mTesR Plus medium supplemented with 10nM Y-27632 dihydrochloride ROCK inhibitor (Tocris, 125410) and iPSCs pelleted and then resuspended in fresh mTeSR Plus with Y-27632 for counting. iPSCs were plated in Pre-Differentiation Medium, comprised of KnockOut™ DMEM / F-12 (ThermoFisher Scientific, 12660012), 1x N2 supplement (ThermoFisher Scientific, A1370701), 1X MEM Non-Essential Amino Acids (ThermoFisher Scientific, 11140-050), and 1X GlutaMAX Supplement (ThermoFisher Scientific, 35050-061), supplemented with 10nM Y-27632 and 2ug / mL doxycycline hydrochloride (Sigma-Aldrich, D3072). iPSCs were plated at a concentration of 1x106 cells / well on 6-well plates coated with Matrigel, Growth Factor Reduced Basement Membrane Matrix, LDEV-free (Corning, 354230), diluted to 0.5 mg / plate. Media was changed daily. After Day 0, Y-27632 was removed. On Day 3, 5-fluoro- 20-deoxyuridine and uridine (Sigma-Aldrich, F0503 and U3003) were added at 10 µM each. On Day 4, pre-differentiated cells were collected with Accutase as above and resuspended for counting and replating in Maturation Media, comprised of Neurobasal Plus Medium (ThermoFisher Scientific, A3582901), 1x B27 Plus supplement (ThermoFisher Scientific, A3582801), 1x CultureONE supplement (ThermoFisher Scientific, A3320201), 1X MEM Non-Essential Amino Acids, and 1X GlutaMAX, supplemented with with 10nM Y-27632, 2ug / mL doxycycline hydrochloride, 10ng / mL BDNF (R&D, 248-BDB / CF), 10ng / mL NT-3 (PeproTech, 450-03), 10ng / mL GDNF (R&D, 212-GD\CF) and 200μM L-ascorbic acid (Sigma-Aldrich, A8960).12-well plates or 24-well glass-bottomed dishes (Cellvis, P24-1.5H- N) were prepared for replating by coating with 100 ug / mL poly-L-ornithine (Sigma-Aldrich, P3655) overnight at 37C, washing 3x with H2O, and drying overnight at room temperature. Prior to coating, glass-bottomed plates were pre-treated with 1.0N HCl (Sigma-Aldrich, H9892) for at least 15min and washed 1x with DPBS and then 2x with H2O. Prior to replating, coated and dried plates were pre-incubated with plain Neurobasal Plus media at 37C while cells were prepared for replating. Pre-incubation media was aspirated, and pre-

[0024] 244922-8547-0065, v. 1 differentiated cells plated at 1x105 cells / well for 12-well plates for flow cytometry and at 5x104 cells / well on 24-well plates for imaging. After 1hr, at which point cells are well- adhered, an additional volume of supplemented Maturation Media was added, but without Y- 27632 and with laminin (ThermoFisher Scientific, 23017015) at 2ug / mL (final concentration per well of 1ug / mL). Thereafter, half media changes were performed 1-2x per week with supplemented Maturation Medium without Y-27632 or doxycycline and with laminin at 1ug / mL. Flow cytometry analysis of neurons and iPSCs. Neurons were dissociated for flow cytometry analysis using papain (Worthington, LK003176) and resuspended in 0.5mL of base Maturation Medium with 10nM Y-27632, while iPSCs were dissociated with Accutase and resuspended in mTeSR Plus with 10nM Y-27632. Cells were kept on ice and passed through a 40μm cell strainer prior to flow analysis and / or sorting for iPSCs. Cellular fluorescence was measured on a BD FACSAria Fusion (BD Biosciences) using an 85μm nozzle. For dCas9 / nCas9 measurements, mtagBFP fluorescence was detected by the 405nm laser and the 450 / 50 filter and autofluorescence was detected by the 561nm laser and the 582 / 15 filter. For viral titering, mScarlet fluorescence was measured by the 561nm laser and filters 600LP and 610 / 20. Data were analyzed using the R package CytoExploreR (v1.1.0). IF and confocal microscopy of neurons and iPSCs. Neurons and iPSCs grown in glass-bottomed dishes were washed 3x with PHEM Buffer (Electron Microscopy Sciences, 11163), and fixed in 4% formaldehyde plus 0.25% glutaraldehyde (Electron Microscopy Sciences, 15710 and 16120) in PHEM buffer. Fixed cells were washed 3x in DPBS and then blocked and permeabilized in 5% goat serum (Cell Signaling Technology, 5425) and either 0.25% TritonX-100 (VWR, 0694) in DPBS for neurons or 0.1% TritonX-100 for iPSCs. Cells were incubated with primary antibodies diluted in blocking buffer overnight at 4C. Cells were then washed 3x with DPBS for 5min each and then incubated with secondary antibodies or cell stains diluted in blocking buffer for 1hr at room temperature. Cells were washed 1x with DPBS for 5min, then incubated with NucSpot 750 / 780 (Biotium, 41038) diluted at 1:10,000 in DPBS for 5min, followed by 1x DPBS wash for 5min. Cells were finally stored in ibidi Mounting Medium (ibidi, 50001) at 4C. Images were acquired on a Leica TCS SP8 confocal microscope. Z-stacks (0.6μm slices) were recorded with 40× Plan- Apochromat lenses, 1.4 NA. The following primary antibodies were used: Cas9 (Takara, 632607; 1:150), MAP2 (Abcam, ab5392; 1:5000). The following secondary antibodies and

[0025] 254922-8547-0065, v. 1 cell stains were used: Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (ThermoFisher Scientific, A32731; 1:2000), Goat anti- Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555 (ThermoFisher Scientific, A21429; 1:2000), Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647 (ThermoFisher Scientific, A32933; 1:1000), Alexa Fluor™ 647 Phalloidin (ThermoFisher Scientific, A22287; 1:1000). Total protein harvesting and neuronal fractionation. For total protein, neurons were washed 3x with cold DPBS+ / + (ThermoFisher Scientific, 14040133) and lysed in-well for 10min at 4C while rocking with 1x RIPA lysis buffer (Cell Signaling, 9806) plus 1x protease inhibitor cocktail (Sigma-Aldrich, P8340). To extract cytoplasmic protein, neurons were washed 3x with cold DPBS+ / + and then treated in-well for 10min at 4C while rocking with 0.05% Digitonin (ThermoFisher Scientific, BN2006) diluted in DPBS with 1x protease inhibitor cocktail (PIC). Cytoplasmic lysate was then collected and RIPA+PIC added to final 1x concentrations. Neurons were then washed 1x with cold DPBS+ / + and finally lysed with 1x RIPA + 1x PIC as for total protein to extract remaining nuclei. After lysis, all samples were sonicated for 30s and then centrifuged for 10min at 14000g at 4C. Supernatants were transferred to fresh tubes and stored at -80C prior to denaturation. Western Blotting. Protein samples were denatured at 95C for 5min in 1x Laemmli buffer (Bio-Rad, 1610747) with 2.5% 2-mercaptoethanol (Sigma-Aldrich, M6250) and then loaded on precast stain-free SDS-PAGE gels (Bio-Rad, 4568094 and 4568096). Immunoblotting followed using standard protocols with Immun-Blot® Low Fluorescence PVDF membranes (BioRad, 1620260). 5% milk in 1x TBS with 0.1% Tween 20 (VWR, 0777) was used for blocking and antibody dilution. SDS (ThermoFisher Scientific, 15553027) was added to secondary antibody dilutions at a final concentration of 0.02%. Imaging of blots was performed on a LI-COR Odyssey instrument. Quantification of blots was performed using densitometry in ImageJ. The following primary antibodies were used: Cas9 (Cell Signaling, 14697; 1:1000), TUBB3 (Abcam, ab18207; 1:800), LaminB1 (Proteintech, 12987-1-AP; 1:2000), GAPDH (Cell Signaling, 2118; 1:500). The following secondary antibodies were used: IRDye 680LT Goat anti-Rabbit (LI-COR, 926-68021; 1:10,000), IRDye 800CW Goat anti-Mouse (LI-COR, 926-32210; 1:10,000). Transfection for lentiviral production. Lentivirus was produced from HEK293T cells seeded into gelatin-coated 6-well tissue culture dishes at 5x105 cells / well. 24

[0026] 264922-8547-0065, v. 1 hours after plating, transfection was performed with PEI (Polysciences, 24765) in Opti-MEM (ThermoFisher Scientific, 31985062). 100 µL of Opti-MEM was incubated with 1) 1.06ug pMDLg 2) 0.57ug pMD2.G 3) 0.4ug pRSV-Rev 4) 1.06ug donor plasmid. 7.35 μl PEI was added dropwise to the DNA+Opti-MEM solution. The transfection mix was incubated for 15 minutes and then added dropwise to cells containing 2mL of antibiotic-free media. Media was removed 4-6 hours post-transfection, cells washed 1x with DPBS, and 2mL of base Maturation Medium added. Virus was collected 48 hours post-transfection and filtered through 0.45 μm cellulose acetate filters (VWR, 76479-040) or 0.45 μm PES filters (ThermoFisher Scientific, 50-607-518). Virus used within three days was stored at 4C; for longer-term storage, aliquots were frozen at -80°C. Lentiviral transduction of neurons. Neurons were transduced at either Day 14 or Day 21 with lentivirus containing sgRNAs against target genes. Transduction was performed at the time of regular half-media changes. Virus prepared in Maturation Medium was added at 10% of final well volume (200 µL per well for a 12-well) to fresh Maturation Medium followed by standard media supplementation. Approximately half of the media volume of each well was removed (accounting for evaporation) and then replenished with the respective media and virus solution. Following transductions, cells were maintained for 7 days before harvesting RNA. RNA isolation and qPCR. Total RNA was isolated from neurons using the Qiagen RNeasy Plus Mini kit (Qiagen, 74134) according to manufacturer’s recommendations, including the addition of 2-mercaptoethanol. cDNA was prepared using either the SuperScript™ IV VILO™ Master Mix with ezDnase (ThermoFisher Scientific, 11766050) for Cas9 gene expression experiments or the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) for knockdown experiments. qPCR analysis was performed with Power SYBR Green master mix (Applied Biosystems, 4367659) on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad). For evaluating dCas9 / nCas9 expression in neurons, background signal from Cas9 primers in no RT controls for each sample was subtracted from respective RT samples, before normalizing RT sample signal to GAPDH. For knockdown experiments, RNA levels for genes of interest were determined using the ΔΔCt method, using the geometric mean of ACTB and GAPDH as control genes. Primers:

[0027] 274922-8547-0065, v. 1 GAPDH F: GGAGCGAGATCCCTCCAAAAT (SEQ ID NO: 15) GAPDH R: GGCTGTTGTCATACTTCTCATGG (SEQ ID NO: 16) ACTB F: ACCTTCTACAATGAGCTGCG (SEQ ID NO: 17) ACTB R: CCTGGATAGCAACGTACATGG (SEQ ID NO: 18) Cas9 F: GGAAGTTCGACAATCTGACCAAGG (SEQ ID NO: 19) Cas9 R: TGCCACGTGCTTTGTGATCTG (SEQ ID NO: 20) PSAP F: CCCGGTCCTTGGACTGAAAG (SEQ ID NO: 21) PSAP R: TATGTCGCAGGGAAGGGATTT (SEQ ID NO: 22) SNCA F: AAGAGGGTGTTCTCTATGTAGGC (SEQ ID NO: 23) SNCA R: GCTCCTCCAACATTTGTCACTT (SEQ ID NO: 24) Example 2 – dCas9-KRAB exhibits neuron-specific cytoplasmic mislocalization and KRAB- dependent protein depletion in iPSC-derived NGN2 neurons To move toward developing efficient systems for inducible gene perturbations later into neuronal differentiation, the expression levels of two CRISPR cassettes expressed were longitudinally tested in stable iPSC lines generated using piggybac integration into KOLF2.1J iPSCs with NGN2 integration at the AAVS1 locus (Pantazis et al. 2022). The two donor vectors were identical apart from one expressing dCas9 with the KOX1 KRAB domain and the other expressing nCas9 (FIG. 1A). Both were based on previously published dCas9- KRAB constructs (Tian et al.2019; Gilbert et al.2014; Qi et al.2013) used to engineer iPSCs with targeted integration. Polyclonal iPSC lines were generated using piggybac transfection and selection with blasticidin, followed by fluorescence cell sorting based on tagBFP expression levels. The expression levels of dCas9-KRAB and nCas9 were compared with flow cytometry in iPSCs and in neurons 14 days post-induction of differentiation with NGN2 expression by treatment with doxycycline (FIG. 1B). Surprisingly, dCas9-KRAB and nCas9 showed dramatic differences in fluorescence post-differentiation. In iPSCs, both constructs demonstrated comparable fluorescence levels, but in Day 14 neurons, dCas9-KRAB fluorescence was essentially undetectable while nCas9 fluorescence remained robust, with a distribution comparable to that in iPSCs. To test if this was due to an effect on the protein or mRNA, RT-qPCR was performed to measure dCas9-KRAB / nCas9 RNA levels in Day 14 neurons, using a primer set

[0028] 284922-8547-0065, v. 1 generic to both versions (FIG. 1C, STAR methods). No significant difference between the mRNA levels of the dCas9-KRAB and nCas9 was observed in neurons suggesting that the observed effect on fluorescence is due to effects on the protein and not due to differential transgene silencing. This is also consistent with the fact that both cassettes had been introduced in the same donor backbone and displayed similar expression distributions in the iPSC state. To determine if there were any other differences in protein behavior between cell types, immunofluorescence against dCas9-KRAB and nCas9 was performed in both iPSCs and neurons using a single antibody against SpCas9 which recognizes both versions (FIG. 1D). Staining in iPSCs showed both dCas9-KRAB and nCas9 in the nucleus. Interestingly, dCas9-KRAB appeared primarily diffuse while nCas9 had a predominantly nucleolar pattern, which could be due to differential interactions mediated by the KRAB domain. Bright accumulations of both proteins were also observed in the cytoplasm, possibly related to effects of overexpressed proteins. In neurons, however, nCas9 was almost completely absent from the nucleus and restricted to the cytoplasm of the soma. Consistent with the flow data, dCas9 protein was almost undetectable by IF as well, but appeared to also be restricted to bright puncta in the cytoplasm. Based on these observations, it was hypothesized that neuronal-specific cytoplasmic mislocalization of the CRISPR machinery is leading to targeted degradation of dCas9-KRAB as the KRAB domain is an endogenous human protein domain derived from the KOX1 transcription factor. Example 3 – Addition of a neuronal NLS improves nuclear localization and knockdown post- differentiation To test the hypothesis that cytoplasmic mislocalization leads to KRAB- dependent degradation of CRISPR machinery in neurons, a series of modified dCas9-KRAB piggybac constructs were generated with an MeCP2 NLS (KRPGRKRKAEADPQAIPKKRGR or RKRKAEADPQAIPKKRGRK (SEQ ID NO: 2)) and in combination with other alternative NLSs. Stable polyclonal iPSC lines were generated with these new constructs and compared with the original dCas9-KRAB and nCas9 lines using flow cytometry in iPSCs (FIG. 3A) and in day 14 neurons (FIG. 2B). The addition of an MeCP2 NLS (construct of SEQ ID NO: 9) was sufficient to rescue expression of dCas9- KRAB to near-nCas9 levels in day 14 neurons and boosted iPSC expression as well. Other NLSs paired with the MeCP2 NLS seemed to provide little or no additive effect; however, the 2xMeCP2 NLS appeared to provide a further boost to expression. Consistent with the

[0029] 294922-8547-0065, v. 1 flow data, western blots of total protein samples from Day 14 neurons showed no significant difference between nCas9 and 2xMeCP2 NLS-dCas9-KRAB protein levels (FIG.2D). Whether the 2xMeCP2 NLS-dCas9-KRAB construct also displayed corrected nuclear localization was tested using both biochemistry and imaging. We isolated cytoplasmic and nuclear protein fractions from Day 14 neurons using digitonin extraction of cytoplasmic protein followed by extraction of nuclear protein with Radioimmunoprecipitation Assay (RIPA) buffer (FIG. 2E). The original dCas9-KRAB and nCas9 constructs, which share the same original NLSs, had nearly identical cytoplasmic / nuclear distributions, with the majority of the signal coming from the cytoplasmic fraction, confirming the previous imaging results (FIG. 1D). The 2xMeCP2 NLS-dCas9-KRAB construct, however, showed a large and significant shift into the nuclear fraction, with a majority of the signal coming from the nuclear fraction. Imaging was performed to corroborate the fractionation results (FIG. 2F). Similarly to before, the original dCas9-KRAB construct was nearly undetectable by IF, with only occasional cytoplasmic puncta visible, and the nCas9 was largely restricted to the cytoplasm as well. The 2xMeCP2 NLS-dCas9-KRAB construct, however, displayed an easily detectable and predominantly diffuse nuclear signal in most cells, at levels comparable to nCas9, suggesting that the corrected nuclear localization was able to rescue the dCas9-KRAB specific degradation. Lastly, whether rescue of localization and expression would improve knockdown efficiency at the neuronal stage was tested (FIG. 2G). sgRNAs were lentivirally transduced in a fluorescent backbone at similar transduction levels in either Day 14 (FIG. 3B) or Day 21 (FIG. 3C) neurons and RNA was harvested 7 days post-transduction. In both cases, increased knockdown in the 2xMeCP2 NLS neurons was observed compared to the original dCas9- KRAB line, suggesting that the corrected construct can improve sgRNA efficiency. To test whether the defects we observed in nuclear localization and protein expression in neurons were construct-specific or common to any variations of CRISPR machinery, we tested an alternative CRISPRi design, iE61 PB-Zim3-XTEN-dCas9-mScarlet- puro-BFP, which uses the ZIM3 domain instead of the KOX1 KRAB domain, but similarly relies on SV40 NLSs for nuclear localization (Alerasool et al. 2020). The sequence of ZIM3- dCas9 is provided in SEQ ID NO: 6. Imaging confirmed that this alternative CRISPRi design similarly failed to effectively localize to the nucleus of neurons, suggesting that SV40 NLSs are generally ineffective in neurons for CRISPR constructs (FIG. 4A). Interestingly, despite

[0030] 304922-8547-0065, v. 1 the lack of nuclear localization, the Zim3-dCas9 appeared to retain stable expression in neurons unlike mislocalized dCas9-KRAB (FIGS. 4B, 4C). This supports the hypothesis that the loss of expression of cytoplasmic-restricted dCas9-KRAB is mediated by the KRAB domain and is specific to the commonly used KOX1 domain. * * * All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0031] 314922-8547-0065, v. 1 REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Gilbert et al., 2014. “Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.” Cell 159 (3): 647–61. Ihry et al., 2018. “p53 Inhibits CRISPR-Cas9 Engineering in Human Pluripotent Stem Cells.” Nature Medicine 24 (7): 939–46. Karasev et al., 2022. “Nuclear Localization Signals for Optimization of Genetically Encoded Tools in Neurons.” Frontiers in Cell and Developmental Biology 10 (July):931237. Pang et al., 2011. “Induction of Human Neuronal Cells by Defined Transcription Factors.” Nature 476 (7359): 220–23. Pantazis et al., 2022. “A Reference Human Induced Pluripotent Stem Cell Line for Large- Scale Collaborative Studies.” Cell Stem Cell 29 (12): 1685–1702.e22. Parra Bravo et al., 2024. “Human iPSC 4R Tauopathy Model Uncovers Modifiers of Tau Propagation.” Cell 187 (10): 2446–64.e22. Qi et al., 2013. “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression.” Cell 152 (5): 1173–83. Santhosh Kumar et al., 2024. “Sequential CRISPR Screening Reveals Partial NatB Inhibition as a Strategy to Mitigate Alpha-Synuclein Levels in Human Neurons.” Science Advances 10 (6): eadj4767. Tian et al., 2021. “Genome-Wide CRISPRi / a Screens in Human Neurons Link Lysosomal Failure to Ferroptosis.” Nature Neuroscience 24 (7): 1020–34. Tian et al., 2019. “CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons.” Neuron 104 (2): 239–55.e12.

[0032] 324922-8547-0065, v. 1

Claims

CLAIMS 1. A fusion protein comprising an enzymatically inactive CRISPR enzyme and a neuron- specific nuclear localization signal (NLS).

2. The fusion protein of claim 1, wherein the enzymatically inactive CRISPR enzyme is an enzymatically inactive Cas9 (dCas9) or a nickase Cas9 (nCas9).

3. The fusion protein of claim 2, wherein the dCas9 enzyme comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 in which position 10 is alanine and / or position 840 is alanine.

4. The fusion protein of any one of claims 1-3, wherein the neuron-specific nuclear localization signal (NLS) is a MeCP2 NLS.

5. The fusion protein of any one of claims 1-4, wherein the neuron-specific nuclear localization signal (NLS) comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:

2.

6. The fusion protein of any one of claims 1-5, wherein the fusion protein comprises two copies of the neuron-specific nuclear localization signal (NLS).

7. The fusion protein of any one of claims 1-6, wherein the neuron-specific nuclear localization signal (NLS) is positioned at the N-terminal end of the fusion protein.

8. The fusion protein of any one of claims 1-7, further comprising a hRNPD NLS (SEQ ID NO: 4), cMyc NLS (SEQ ID NO: 3), nucleoplasmin NLS and / or SV40 NLS.

9. The fusion protein of any one of claims 1-8, wherein the fusion protein further comprises a modulator of gene expression.

10. The fusion protein of claim 9, wherein the modulator of gene expression comprises a Kruppel associated box (KRAB) domain.

11. The fusion protein of claim 10, wherein the KRAB domain is from a KOX1 gene or a zinc finger imprinted 3 (Zim3) gene.334922-8547-0065, v.

112. The fusion protein of any one of claims 1-8, wherein the fusion protein comprises an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOs: 9-14.

13. A nucleic acid molecule comprising a nucleotide sequence that encoding the fusion protein of any one of claims 1-12.

14. The nucleic acid molecule of claim 13, wherein the nucleic acid molecule is a DNA molecule.

15. The nucleic acid molecule of claim 13, wherein the nucleic acid molecule is an RNA molecule.

16. A vector comprising the nucleic acid molecule of claim 13.

17. The vector of claim 16, wherein the vector is a viral vector.

18. A neuron comprising the fusion protein of any one of claims 1-12 or the nucleic acid of any one of claims 13-15.

19. The neuron of claim 18, wherein the neuron is an iPSC-derived neuron.

20. The neuron of claim 18, wherein the neuron is a primary neuron.

21. The neuron of claim 18, wherein the neuron is in vitro or ex vivo.

22. The neuron of claim 18, wherein the neuron is in vivo.

23. A method for modulating the expression of a gene in a neuron, the method comprising transfecting or transducing the cell with (i) the fusion protein of any one of claims 1-12 or the nucleic acid molecule of any one of claims 13-15 and (ii) a gRNA which targets the fusion molecule to the gene or a nucleic acid that encodes a gRNA which targets the fusion molecule to the gene.344922-8547-0065, v. 1

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