Elimination of camkiiδ autophosphorylation by crispr-cas9 gene editing improves cardiac function in heart failure

Abstract

Provided herein are composition and methods for editing of CaMKIlδ to reduce or prevent stress induction in human cells and mice.

Description

DESCRIPTION ELIMINATION OF CAMKIIδ AUTOPHOSPHORYLATION BY CRISPR-CAS9 GENE EDITING IMPROVES CARDIAC FUNCTION IN HEART FAILURE PRIORITY CLAIM This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 508,703, filed June 16, 2023, the entire contents of which are hereby incorporated 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 XML Sequence Listing, created on June 12, 2024, is named UTFDP4243WO and is 115,453 bytes in size. BACKGROUND 1. Field The present invention relates generally to the fields of molecular biology, medicine, and genetics. More particularly, it concerns compositions and uses thereof for genome editing to eliminate stress responses by CaMKIIδ in cells such as cardiomyocytes. 2. Description of Related Art Cardiac disease represents the leading cause of worldwide morbidity and mortality with the frequency expected to increase in the future (Virani et al., 2021; Collaborators GBDRF, 2020). Despite recent advances in heart failure therapy, there remains a major need for new therapeutic strategies (Hendenreich et al., 2022). CRISPR-Cas9 gene editing technology is an efficient tool for human gene editing with great therapeutic potential (Lui & Olson, 2022; Nishiyama et al., 2022). Whereas conventional gene editing strategies are designed to correct specific disease-causing mutations, the frequency of specific mutations is typically very low, which precludes broad application of individual gene editing strategies (Lui & Olson, 2022; Nishiyama et al., 2022; Chemello et al., 2021). Ca2+ / calmodulin-dependent protein kinase IIδ (CaMKIIδ) is one of many CaMKII isoforms and a key regulator of cardiac physiology and signaling (Beckendorf et al., 2010; Maier, 2009; Lebek et al., 2023). However, chronic overactivation of CaMKIIδ has been implicated in the pathophysiology of heart failure, ischemia / reperfusion injury, arrhythmias,1 4860-0953-0567, v.1alcoholic cardiomyopathy, myocardial hypertrophy, and sleep-disordered breathing (Neef et al., 2010; Backs et al., 2009; Lebek et al., 2020; Zhang et al., 2004; Ling et al., 2013; Luo et al., 2013; Nassal et al., 2020; Pelllicena & Schulman, 2014; Mustroph et al., 2018; Fischer et al., 2014; Toischer et al., 2010; Lebek et al., 2010). Hyperactivation of cardiac CaMKIIδ has been shown to dysregulate cellular Ca2+homeostasis and induce myocardial inflammation, apoptosis, and fibrosis, culminating in loss of cardiac function (Lebek et al., 2023; Neef et al., 2010; Backs et al., 2009; Lebek et al., 2020; Zhang et al., 2004; Ling et al., 2013; Luo et al., 2013; Lebek et al., 2010; Lebek et al., 2018; Pabel et al., 2022). Even though there have been significant efforts to design effective CaMKIIδ inhibitors, they have not yet been successful and there is no clinical drug available for patients (Lebek et al., 2018; Mustroph et al., 2020; Beauverger et al., 2020). Thus, further optimized strategies to target CaMKIIδ are needed. CaMKII autophosphorylation at threonine-287 confers an up to 1,000-fold increased affinity for calmodulin and dramatically increases CaMKII activity by preventing association of the autoinhibitory region with the catalytic domain (Meyer et al., 1992; Simon et al., 2021). Indeed, CaMKII autophosphorylation is increased in numerous cardiac diseases and serves as a measure of total CaMKII activity (Fischer et al., 2014; Toischer et al., 2010; Lebek et al., 2018; Luibojevic-Holzer et al., 2020). However, up to now, it is as yet unproven whether rendering CaMKIIδ resistant to autophosphorylation confers cardioprotection. Thus, the inventors sought to ablate the autophosphorylation site of CaMKIIδ as a potentially generalizable therapeutic concept for multiple cardiac disorders. 2 4860-0953-0567, v.1SUMMARY In accordance with the present disclosure, there is provided a single-guide RNA (sgRNA) comprising a targeting nucleic acid sequence that targets an autophosphorylation site in the regulatory domain of CaMKIIδ. The gRNA may target Thr287 of CaMKIIδ. Sequences of sgRNA2-7 are identical in the human and mouse genome. The sgRNA may comprise the sequence: sgRNA sgRNA-sequence SEQ ID NO PAM Human- AA A A T TA A T T 1TG(A)aMKIIδ autophosphorylation site and a base editor. The base editor may be an adenine base editor (ABE). The sgRNA may be sgRNA as set forth in the preceding table or defined in paragraph

[0004] . The base editor may comprise a CRISPR / Cas nuclease linked to an adenosine deaminase, such as wherein the CRISPR / Cas nuclease is catalytically impaired and / or wherein the CRISPR / Cas nuclease is a Cas9 nuclease, such as wherein the Cas9 nuclease is isolated or derived from Streptococcus pyogenes (spCas9), Streptococcus pyogenes (spRY), Staphylococcus aureus (SaCas9), Staphylococcus auricularis (SauCas9), or Staphylococcus lugdunensis (SlugCas9). Also provided is a nucleic acid comprising a sequence encoding a first sgRNA of as set out in the table above, a sequence encoding a base editor, a sequence encoding a first promoter, wherein the first promoter drives expression of the sequence encoding the base editor, and a sequence encoding a second promoter, wherein the second promoter drives expression of the sequence encoding the first sgRNA. The base editor may be an adenine base editor (ABE). The base editor may comprise a CRISPR / Cas nuclease linked to an adenosine deaminase, such as wherein the CRISPR / Cas nuclease is catalytically impaired. The CRISPR / Cas nuclease may be a Cas9 nuclease, such as wherein the Cas9 nuclease is isolated or derived from Streptococcus pyogenes (spCas9), Streptococcus pyogenes (spRY), Staphylococcus aureus (SaCas9), 3 4860-0953-0567, v.1Staphylococcus auricularis (SauCas9), Staphylococcus lugdunensis (SlugCas9), or Streptococcus pyogenes NGN protospacer (SpCas9-NG). The at least one of the sequences encoding the first promoter and the sequence encoding the second promoter may comprise a cell-type specific promoter, such as a cardiomyocyte- specific promoter, in particular a cardiac troponin T (cTnT) promoter. The sequence encoding the second promoter may comprise a sequence encoding a U6 promoter, an H1 promoter, or a 7SK promoter. The nucleic acid may comprise a DNA sequence, or an RNA sequence, and / or may further comprise a polyadenosine (polyA) sequence, such as a mini polyA sequence. In other embodiments, there are provided: a cell comprising the nucleic acids as defined herein, a composition comprising the nucleic acids as defined herein, a cell comprising such a composition, a composition comprising such a cell. In yet another embodiment, there is provided a vector comprising the nucleic acids as described herein. The vector may further comprise a sequence encoding an inverted terminal repeat (ITR) of a transposable element, such as a transposon (e.g., a Tn7 transposon). The vector may further comprise a sequence encoding a 5’ ITR of a T7 transposon and a sequence encoding a 3’ ITR of a T7 transposon. The vector may be a non-viral vector, such as a plasmid, or a viral vector, such as an adeno-associated viral (AAV) vector or an adenoviral vector. The AAV vector may be replication-defective or conditionally replication defective and / or may be a recombinant AAV vector. The AAV vector may comprise a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6),7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11) or any combination thereof, such as wherein the AAV vector comprises a sequence isolated or derived from an AAV2 and a sequence isolated or derived from an AAV9. The vector may be optimized for expression in mammalian cells, such as optimized for expression in human cells. In a further embodiment, there is provided a composition comprising the vector as defined herein, optionally further comprising a pharmaceutically acceptable carrier. The vectors of the present disclosure, or the compositions comprising such vectors may comprise a trans-splicing intein system. Also provided are cells comprising the vectors as described herein or the compositions comprising the vectors. The cells may be human cells, such as where the cells are cardiomyocytes or human cardiomyocytes, or the cells or human cells may be induced pluripotent stem (iPS) cells. A composition comprising such cells is also provided. In yet a further embodiment, there is provided a method for editing an autophosphorylation site in CaMKIIδ, the method comprising contacting a cell with a 4 4860-0953-0567, v.1vector / composition as described here under conditions suitable for expression of the first sgRNA and the adenine base editor, wherein the first sgRNA forms a complex with the adenine base editor, wherein the complex modifies edits an autophosphorylation site in the regulatory domain of CaMKIIδ thereby blocking sustained overactivation of CaMKIIδ. Also provided is a cell produced by this method, such as a cardiomyocyte, e.g., a stem-cell derived cardiomyocyte. A still further embodiment comprises a method of treating or preventing cardiac injury, such as heart failure, in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition as described herein. The composition may be administered locally, such as directly to cardiac tissue (e.g., by an infusion or injection), or administered systemically (e.g., by an intravenous infusion or injection). Following administration of the composition, the subject may exhibit improved Ca2+transient amplitude, decreased arrhythmia, decreased ventricular dilation, improved cardiac function (improved ejection fraction, fractional shortening), improved survival, decreased fibrosis, decreased apoptosis, or a combination thereof. Also, following administration of the composition, the subject may not exhibit cellular Ca2+dysregulation. The subject may be a neonate, an infant, a child, a young adult, or an adult, a male, or a female. Use of a therapeutically effective amount of a composition as described herein for treating or preventing cardiac injury, such as heart failure, in a subject in need thereof, is provided. In an additional embodiment, there is provided an induced pluripotent stem cell comprising an edited CaMKIIδ gene encoding Ala287. Also provided is an engineered mouse comprising a germline cell having an edited CaMKIIδ gene encoding Ala287 in the germline or Gly286, Ala287, and Val288 in the germline. In one embodiment, provided herein are methods for screening at least one candidate agent in a mouse according to any one of the present embodiments, comprising administering one or more candidate agent to the mouse. The at least one candidate agent may be screened for its ability to improve left ventricular function. The at least one candidate agent may be screened for its ability to rescue cardiac chamber size. The at least one candidate agent may be screened for its ability to increase life span. The candidate agent may comprise a sgRNA of any one of the present embodiments. 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 5 4860-0953-0567, v.1description 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. 6 4860-0953-0567, v.1BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 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-E. Adenine base editing to ablate the autophosphorylation site of CaMKIIδ. (FIG. 1A) Overview of CaMKIIδ with its three domains. Autophosphorylation occurs at threonine-287 in the regulatory domain. Several cardiac stressors have been shown to promote CaMKIIδ autophosphorylation and hyperactivation, subsequently leading to cardiac disease. Using CRISPR-Cas9 adenine base editing, the inventors discovered a strategy to edit c.A859G (p.T287A), thereby ablating the autophosphorylation site of CaMKIIδ (SEQ ID NOs: 83-85). (FIG. 1B) Percentage of adenine (A) to guanine (G) editing in mouse N2a cells at c.A859 (p.T287) for sgRNAs 1-7 combined with either ABEmax or ABE8e that were fused to either SpCas9-NG or SpRY (n=3 transfections). (FIG.1C) DNA sequence showing a segment of the mouse CaMKIIδ gene encoding part of the regulatory domain with alignment of sgRNA2 that has 100% homology to that region. The PAM sequence is in green and the ACT nucleotide triplet encoding the critical threonine-287 is highlighted in yellow. Each adenine along the sequence of sgRNA2 is numbered (counting from the PAM). (FIG. 1D) Percentage of adenine (A) to guanine (G) editing in F0 mice (n=52) for each adenine in sgRNA2 following injection of sgRNA2 and ABE8e fused to SpCas9-NG in zygotes and subsequent transfer into the oviducts of pseudo-pregnant female mice. (FIG.1E) Sequencing of cDNA of a homozygous T287A mouse showing the single nucleotide edit c.A859G that makes CaMKIIδ phospho-resistant (SEQ ID NOs: 86-89). Data are presented as individual data points with mean ± SEM and replicates are either independent transfections (FIG. 1B) or individual mice (FIG.1D). FIGS. 2A-K. Ablation of CaMKIIδ autophosphorylation improves survival and cardiac function post-sTAC. (FIG. 2A) Experimental design for subjecting male mice at 10 weeks of age to sTAC surgery as a model for afterload-induced heart failure. Cardiac function was assessed by echocardiography one week before and one week after sTAC. Two weeks after sTAC, mice were sacrificed, and hearts harvested for further histological and molecular 7 4860-0953-0567, v.1analyses (1 – brachiocephalic artery, 2 – left common carotid artery, 3 – left subclavian artery). (FIG. 2B) Survival curves of WT-Sham, WT-sTAC, T287A-Sham, and T287A-sTAC mice (p=8.2x10-3for WT-sTAC vs. WT-Sham, p=2.5x10-2for T287A-sTAC vs. WT-sTAC). (FIG. 2C) Representative M-mode recordings of mouse hearts from all groups 1-week post- sTAC / Sham surgery, as acquired by echocardiography. (FIG.2D) Mean fractional shortening. (FIG. 2E) Mean left ventricular end-diastolic diameter. (FIG. 2F) Mean left ventricular end- diastolic volume. (FIG. 2G) Mean left ventricular mass. (FIG. 2H) Western blot analysis of autophosphorylated CaMKII and total CaMKII (each with GAPDH as housekeeper protein) for all groups. (FIG. 2I) Mean densitometric analyses for autophosphorylated CaMKII normalized to GAPDH for all groups. (FIG.2J) Mean densitometric analyses for total CaMKII normalized to GAPDH for all groups. (FIG. 2K) Mean CaMKII activity for all groups. Data are presented as individual data points with mean ± SEM and all replicates are individual mice. Statistical comparisons are based on log-rank (Mantel-Cox) test with Bonferroni’s post-hoc correction (B) and on two-way ANOVA post-hoc corrected by Holm-Sidak (FIGS.2D-G, 2I- K). FIGS.3A-E. Rendering CaMKIIδ phospho-resistant prevents myocardial fibrosis and apoptosis. (FIG.3A) Macroscopic images of hearts from WT-Sham, WT-sTAC, T287A- Sham, and T287A-sTAC mice (scale bar 1 mm). (FIG. 3B) H&E staining of cardiac sections for each group (scale bar 1 mm). (FIG. 3C) Trichrome staining of cardiac sections for each group (scale bar 1 mm for whole hearts and 100 μm for close-ups). (FIG.3D) Representative heart sections for all groups showing immunohistochemistry of TUNEL (green, arrows), Hoechst 33342 (blue, for nuclei), and cardiac troponin (red; scale bar 20 μm). (FIG.3E) Mean percentage of apoptotic cells for each group. Data are presented as individual data points with mean ± SEM and all replicates are individual mice. Statistical comparisons are based on two- way ANOVA post-hoc corrected by Holm-Sidak. FIGS. 4A-H. Ablation of CaMKIIδ autophosphorylation protects mice from dysregulation of the cardiac transcriptome. (FIG.4A) Principal component analysis (PCA) of the cardiac transcriptome of WT and T287A mice, both subjected to sham and sTAC surgery (n=3 per group). (FIG.4B) Heat map of 1,982 genes that were differentially expressed between WT-sTAC compared to WT-Sham by more than 2-fold. Additionally, the inventors are reporting the same genes for T287A-Sham and T287A-sTAC. (FIG.4C) Volcano plot showing 1,381 and 601 genes that were up- and downregulated, respectively, by at least 2-fold in WT- sTAC compared to WT-Sham mice. The top 5 up- and downregulated genes are labeled, based on both p-value and fold change. (FIG.4D) Gene Ontology terms associated with upregulated 8 4860-0953-0567, v.1genes in WT-sTAC compared to WT-Sham mice. (FIG.4E) Gene Ontology terms associated with downregulated genes in WT-sTAC compared to WT-Sham mice. (FIG.4F) Volcano plot showing 264 and 496 genes that were up- and downregulated, respectively, by at least 2-fold in T287A-sTAC compared to WT-sTAC mice. The top 5 up- and downregulated genes are labeled, based on both p-value and fold change. (FIG. 4G) Gene Ontology terms associated with upregulated genes in T287A-sTAC compared to WT-sTAC mice. (FIG. 4H) Gene Ontology terms associated with downregulated genes in T287A-sTAC compared to WT-sTAC mice. Data are presented as individual data points and all replicates are individual mice. FIGS. 5A-G. Analysis of CaMKIIδ editing and potential off-targets in human iPSCs using deep amplicon sequencing. (FIG.5A) Percentage of adenine (A) to guanine (G) editing for each adenine in sgRNA2 (CaMKIIδ) following base editing with sgRNA2 and ABE8e. (FIG. 5B) Sequence of sgRNA2 (CaMKIIδ) (SEQ ID NO: 3) and the corresponding DNA and PAM sequences of CaMKIIα, β, and γ as well as the sequences of the top 8 potential off-target loci, as predicted by CRISPOR (SEQ ID NOs: 90-100). Yellow highlighting indicates bases different from sgRNA2. Each nucleotide within the sequence is numbered (counting from the PAM). (FIG.5C) Percentage of adenine (A) to guanine (G) editing for all adenines within the DNA sequence of CaMKIIα corresponding to sgRNA2, either following base editing with sgRNA2 and ABE8e or for an untreated human WT sample (WT control). (FIG. 5D) Percentage of adenine (A) to guanine (G) editing for all adenines within the DNA sequence of CaMKIIβ corresponding to sgRNA2, either following base editing with sgRNA2 and ABE8e or for an untreated human WT sample (WT control). (FIG. 5E) Percentage of adenine (A) to guanine (G) editing for all adenines within the DNA sequence of CaMKIIγ corresponding to sgRNA2, either following base editing with sgRNA2 and ABE8e or for an untreated human WT sample (WT control). (FIG. 5F) Fold change of percentage of adenine (A) to guanine (G) editing at c.A859G (p.T287A) for CaMKIIδ compared to CaMKIIα, β, and γ. (FIG. 5G) Percentage of adenine (A) to guanine (G) editing for all adenines (ordered from 5’ to 3’) within the DNA sequences of the top 8 predicted off-target loci, beginning with the potential off-target #1. Data are presented as individual data points with mean ± SEM and all replicates are human iPSCs following three independent nucleofections with sgRNA2 and ABE8e fused to SpCas9-NG. As a negative control, the inventors analyzed one human WT iPSC sample that was not treated with sgRNA2 and ABE8e fused to SpCas9-NG (WT control). FIGS. 6A-K. ABE with sgRNA2 protects human iPSC-CMs from ISO-induced dysregulation of cellular Ca2+homeostasis. (FIG.6A) Sequencing of cDNA of human iPSC- CMs showing the editing pattern of sgRNA2 (SEQ ID NOs: 86, 87, 89, 101 and 102). (FIG. 9 4860-0953-0567, v.16B) Western blot analysis of autophosphorylated CaMKII and total CaMKII (each with GAPDH as housekeeper protein) in human wild-type (WT) and T287A iPSC-CMs for both control and 10 days of exposure to isoproterenol (ISO). (FIG.6C) Mean densitometric analysis for autophosphorylated CaMKII normalized to GAPDH. (FIG. 6D) Mean densitometric analysis for total CaMKII normalized to GAPDH. (FIG. 6E) Mean CaMKII activity in WT and T287A iPSC-CMs both upon control and ISO. (FIG. 6F) Representative Ca2+transients for WT and T287A human iPSC-CMs upon control and 10 days of chronic ISO, measured by epifluorescence microscopy. (FIG. 6G) Mean diastolic Ca2+levels for all groups. (FIG. 6H) Mean Ca2+transient amplitude for each group. (FIG.6I) Mean relaxation time to 50% baseline. (FIG.6J) Mean relaxation time to 80% baseline. (FIG.6K) Percentage of iPSC-CMs showing arrhythmias. Data are presented as individual data points with mean ± SEM and all replicates are either 5 independent differentiations into iPSC-CMs (C-E) or individual iPSC-CMs (G-K). Statistical comparisons were performed with two-way ANOVA post-hoc corrected by Holm- Sidak (FIGS.6C-E, 6G-J) and with Fisher’s exact test (K). FIGS. 7A-C. The autophosphorylation site is not ablated in other CaMKII isoforms. (FIG. 7A) DNA sequencing chromatogram of the CaMKIIα gene from the founder mouse of the T287A line, showing that the adenine within the threonine-encoding nucleotide triplet ACC was not edited (SEQ ID NOs: 86 and 103-106). (FIG. 7B) DNA sequencing chromatogram of the CaMKIIβ gene from the founder mouse of the T287A line, showing that the adenine within the threonine-encoding nucleotide triplet ACT was not edited. (FIG. 7C) DNA sequencing chromatogram of the CaMKIIγ gene from the founder mouse of the T287A line, showing that the adenine within the threonine-encoding nucleotide triplet ACG was not edited. FIGS. 8A-D. Analysis of basal cardiac function one week before surgery using echocardiography. (FIG.8A) Mean fractional shortening for WT and T287A mice, both one week before either sham or sTAC surgery. (FIG. 8B) Mean left ventricular end-diastolic diameter for all groups. (FIG.8C) Mean left ventricular end-diastolic volume. (FIG.8D) Mean left ventricular mass. Data are presented as individual data points with mean ± SEM and all replicates are individual mice. Statistical comparisons are based on two-way ANOVA. FIGS. 9A-E. T287A mice respond to acute β-adrenergic stimulation. (FIG. 9A) Representative electrocardiograms of wild-type (WT) and T287A mouse hearts before and after acute treatment with isoproterenol (ISO, 3 mg / kg bodyweight). (FIG.9B) Mean heart rate before and after ISO for both groups. (FIG. 9C) Mean P-wave duration before and after ISO for both groups. (FIG.9D) Mean PR interval before and after ISO for both groups. (FIG.9E) 10 4860-0953-0567, v.1Mean QT interval before and after ISO for both groups. Data are presented as individual data points with mean ± SEM and all replicates are individual mice (before and after ISO). Statistical comparisons are based on repeated measures two-way ANOVA with Holm-Sidak’s post-hoc correction. FIGS. 10A-H. Exercise performance is preserved in T287A mice. (FIG. 10A) Experimental design to test the exercise performance in wild-type (WT) and T287A mice. All mice were subjected to echocardiography immediately after exhaustion. (FIG. 10B) Mean maximal velocity on the treadmill prior to exhaustion for both groups. C, Mean total distance achieved on the treadmill for both groups. (FIG. 10D) Representative M-mode recordings of WT and T287A mouse hearts, as acquired by echocardiography immediately after exhaustion on the treadmill. (FIG.10E) Mean fractional shortening. (FIG.10F) Mean left ventricular end- diastolic diameter. (FIG.10G) Mean left ventricular end-diastolic volume. (FIG.10H) Mean left ventricular mass. Data are presented as individual data points with mean ± SEM and all replicates are individual mice. Statistical comparisons are based on Mann-Whitney tests. FIGS. 11A-E. Cardiac function is normal in 1-year-old T287A mice. (FIG. 11A) Representative M-mode recordings of 1-year-old wild-type (WT) and T287A mouse hearts, as acquired by echocardiography. (FIG.11B) Mean fractional shortening. (FIG.11C) Mean left ventricular end-diastolic diameter. (FIG. 11D) Mean left ventricular end-diastolic volume. (FIG.11E) Mean left ventricular mass. Data are presented as individual data points with mean ± SEM and all replicates are individual mice. Statistical comparisons are based on Mann- Whitney tests. FIG. 12. Study flowchart for mice subjected to afterload-induced heart failure. Wild-type (WT) and T287A mice were randomly assigned to either sham or sTAC surgery and monitored for two weeks before hearts were harvested for further molecular analyses. FIGS. 13A-C. Body, heart, and lung weight two weeks post-sham / sTAC surgery. (FIG. 13A) Mean body weight of WT-Sham, WT-sTAC, T287A-Sham, and T287A-sTAC mice two weeks post-sham / sTAC surgery. (FIG.13B) Mean heart weight normalized to tibia length for all groups. (FIG.13C) Mean lung weight normalized to tibia length for all groups. Data are presented as individual data points with mean ± SEM and all replicates are individual mice. Statistical comparisons are based on two-way ANOVA post-hoc corrected by Holm- Sidak. FIG.14A-B. Ablation of CaMKIIδ autophosphorylation protects from myocardial fibrosis following sTAC. (FIG. 14A) Picrosirius red staining of cardiac sections from WT- Sham, WT-sTAC, T287A-Sham, and T287A-sTAC mice (scale bar 1,000 μm). (FIG. 14B) 11 4860-0953-0567, v.1Mean quantification of the percentage of fibrotic tissue for each group. Data are presented as individual data points with mean ± SEM and all replicates are individual mice. Statistical comparisons are based on two-way ANOVA post-hoc corrected by Holm-Sidak. FIGS.15A-B. T287A iPSC-CMs respond to acute β-adrenergic stimulation. (FIG. 15A) Representative Ca2+transients for wild-type (WT) and T287A human iPSC-CMs before and after acute exposure to 100 nM isoproterenol (ISO). (FIG. 15B) Mean gain of Ca2+transient amplitude following acute exposure to ISO. Data are presented as individual data points with mean ± SEM and all replicates are individual iPSC-CMs. The statistical comparison is based on a Mann-Whitney test. FIGS. 16A-G. T287A iPSC-CMs exhibit improved Ca2+characteristics post- pause. (FIG. 16A) Representative Ca2+transients before and after 10 s of paused electrical stimulation for wild-type (WT) and T287A human iPSC-CMs upon control and 10 days isoproterenol (ISO). (FIG.16B) Mean cytosolic Ca2+overload post-pause for all groups. (FIG. 16C) Mean gain of Ca2+transient amplitude post-pause. (FIG.16D) Linear regression analysis of cytosolic Ca2+overload and gain of Ca2+transient amplitude post-pause in WT iPSC-CMs. (FIG. 16E) Linear regression analysis of cytosolic Ca2+overload and gain of Ca2+transient amplitude post-pause in T287A iPSC-CMs. (FIG.16F) Linear regression analysis of cytosolic Ca2+overload post-pause and stimulated steady-state Ca2+transient amplitude in WT iPSC- CMs. (FIG. 16G) Linear regression analysis of cytosolic Ca2+overload post-pause and stimulated steady-state Ca2+transient amplitude in T287A iPSC-CMs. Data are presented as individual data points with mean ± SEM and all replicates are individual iPSC-CMs. Statistical comparisons were performed with two-way ANOVA post-hoc corrected by Holm-Sidak(FIGS. 16B-C) and with linear regression analysis (FIGS. 16D-G).DETAILED DESCRIPTION Despite current medical advancements, effective treatment for cardiovascular disease remains challenging. CRISPR-Cas9 adenine base editing (ABE) allows the conversion of adenine to guanine nucleotides. ABEs consist of a deactivated Cas9 or Cas9 nickase fused to a deoxyadenosine deaminase. When combined with appropriate single-guide RNAs (sgRNAs), ABEs can edit adenine to guanine nucleotides within a specific window in relation to the protospacer adjacent motif (PAM) of the sgRNA.4,6,9,29-32Within the CaMKIIδ gene, threonine-287 is encoded by an ACT codon that is potentially amenable to ABE, which would create a GCT codon, thereby 12 4860-0953-0567, v.1replacing this threonine with an alanine and consequent elimination of the autophosphorylation site of CaMKIIδ. In the present study, the inventors used an ABE-mediated gene editing strategy in the mouse germline to ablate the autophosphorylation site of CaMKIIδ, thereby rendering the enzyme insensitive to pathogenic hyperactivation. They show that mice with this mutation (c.A859G, p.T287A) are protected from afterload-induced heart failure. Similarly, using human cardiomyocytes derived from induced pluripotent stem cells (iPSCs), the inventors show that this specific gene editing approach confers protection against chronic β-adrenergic stress. These findings suggest a new strategy for maintenance of cardiac function in the settings of diverse forms of cardiac stress. These and other aspects of the disclosure are set out in detail below. I. Heart Failure Heart failure is a complex syndrome whereby there is inadequate output of the heart to meet the metabolic demands of the body. Abnormal function of different anatomic parts of the heart cause heart failure including the pericardium, the myocardium, the endocardium, the heart valves and the great vessels. Symptoms of heart failure are due to a lack of both forward blood flow to the body, and backward flow into the lungs. Heart failure is characterized by symptoms of dyspnea, edema and fatigue and signs such as rales on physical examination. Heart failure may be classified based on ejection fraction into heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF). LV remodeling is the basic concept for HFpEF pathophysiology. Two models are emerging in HFpEF pathophysiology, the traditional model discussed about ventricular diastolic dysfunction, LV hypertrophy, impaired relaxation, endothelial dysfunction, arterial and ventricular stiffness and their effect on cardiac function. The emerginh model discussed role of systemic microvascular endothelial inflammation due to existing comorbidities such as diabetes, hypertension, obesity, smoking and ischemia in cardiac remodeling and dysfunction. The pathogenesis of HFrEF is related largely to cellular proliferation and metabolism. Pathological processes that result in progression of HF and are common to both HFrEF and HFpEF are altered excitation-contraction coupling, epigenetic modifications, changes in sarcomeric coupling proteins, increased adrenergic drive, increased activity of renin- angiotensin aldosterone axis, nitric oxide insensitivity, adenosine triphosphate (ATP) depletion, reactive oxygen species production and an elevated cell death rate. 13 4860-0953-0567, v.1The causes of heart failure can be broadly divided into cardiac and non-cardiac. Cardiac causes include aortic regurgitation, aortic stenosis, arrhythmias, arrhythmogenic right ventricular dysplasia, arteriovenous fistula, atrial fibrillation, atrial septal defect, cardiac amyloidosis, cardiac aneurysm, congenital heart disease, constrictive pericarditis, dilated cardiomyopathy, Eisenmenger syndrome, endocarditis, hypertension, hypertrophic cardiomyopathy, hypoplastic left heart syndrome, interferon gamma, ischemic heart disease, malignant hypertension, mitral regurgitation, mitral stenosis, myocardial infarction, oxaprozin, patent ductus arteriosus, pericardial effusion, pericardial tamponade, pericarditis, peripartum cardiomyopathy, pertuzumab, restrictive cardiomyopathy, rheumatic carditis, rupture of the papillary muscles, Takotsubo cardiomyopathy, tricuspid insufficiency, valvular heart disease, ventricular aneurysm, ventricular septal defect. Congestive heart failure should be distinguished from other conditions that cause dyspnea, fatigue and edema. Heart failure affects close to 5 million people in the the United States of America and each year close to 500,000 new cases are diagnosed. Congestive heart failure is responsible for a significant portion of the healthcare budget, and more than 50% of patients seek re-admission within 6 months after treatment and the average duration of hospital stay is 6 days. In 2005 the prevalence among adults aged 20 and older in the United States was 5,300,000 (about 2,650,000 males, and 2,650,000 females). Heart failure is associated with significantly reduced physical and mental health, resulting in a markedly decreased quality of life. Congestive heart failure is also associated with a poor prognosis. If left untreated, heart failure may result in death due to complications associated with the condition. Heart failure resulting from atherosclerotic coronary artery disease has been shown to be associated with a higher incidence of fatal events compared to heart failure that results from other cardiac diseases. Heart failure is a progressive disease with a major impact on the patient's quality of life. With the exception of heart failure caused by reversible conditions, the condition usually worsens with time. Although some people survive many years, progressive disease is associated with an overall annual mortality rate of 10%. In the Framingham experience, 80% of men and 70% of women with heart failure who were under 65 years of age had died within 8 years of the diagnosis. There are several diagnostic criteria / algorithms that are used to diagnose heart failure including an algorithm from the ESC, Framingham study, and Boston. The classic symptoms of heart failure include dyspnea, fatigue, and fluid retention. Patients with heart failure present in different ways. Some patients present with exercise intolerance but show little evidence of congestion or edema. Other patients present with mild symptoms of edema and pulmonary congestion. The ejection fraction is usually below 35% in patients who are symptomatic with 14 4860-0953-0567, v.1systolic heart failure. BNP levels may be useful in the initial establishment of the diagnosis of heart failure in the patient with dyspnea of unclear etiology. Chest x-ray in a patient with heart failure shows cardiomegaly (cardiac enlargement and pulmonary congestion (Kerley B lines, and in some cases pleural effusion). CMR may be used for assessment of LV and RV size and morphology, systolic and diastolic function, and for characterizing myocardial tissue for the purpose of understanding the etiology of LV systolic or diastolic dysfunction. Echocardiography is commonly used to diagnose and monitor the progression of heart failure. This modality uses ultrasound to determine the stroke volume (SV, the amount of blood in the heart that exits the ventricles with each beat), the end-diastolic volume (EDV, the total amount of blood at the end of diastole), and the SV divided by the EDV, a value known as the ejection fraction (EF). In pediatrics, the shortening fraction is the preferred measure of systolic function. Coronary angiography is performed in patients with heart failure in whom there is a suspicion of underlying atherosclerosis as the basis for the heart failure. Patients who have increased levels of troponin or CK-MB, who have dynamic EKG changes or other signs and symptoms of an acute coronary syndrome are revascularization candidates and should undergo coronary angiography. Acute heart failure can occur in the setting of a new onset heart failure or worsening of an existing chronic heart failure (also known as acute decompensated heart failure, flash pulmonary edema, ADHF). ADHF presents with acute shortness of breath due to the development of pulmonary edema (the rapid accumulation of fluid in the lung). Other signs and symptoms of ADHF include hypotension with impaired organ perfusion manifested by worsening of renal function, altered mentation and cold clammy extremities. ADHF is associated with a poor prognosis if not treated aggressively. Like chronic heart failure therapy, the goal is to improve symptoms but unlike chronic therapy the other goals are to improve oxygenation and hemodynamic stability. The mainstays of acute medical treatment in acute decompensated congestive heart failure include oxygen to improve hypoxia, diuresis to reduce both preload and intravascular volume and vasodilators to reduce afterload. Some of the mainstays of chronic heart failure therapy are not initiated acutely (ACE inhibitors, beta blockers and digoxin). End-of-life care in patients with congestive heart failure is focused on making the patient comfortable. Invasive procedures should be minimized, and patients may elect to have their defibrillators inactivated. The growing number of patients with Stage IV heart failure (intractable symptoms of fatigue, shortness of breath or chest pain at rest despite optimal medical therapy) should be considered for palliative care or hospice. 15 4860-0953-0567, v.1In cardiovascular physiology, ejection fraction (Ef) is the fraction of blood pumped out of a ventricle with each heartbeat. The term ejection fraction applies to both the right and left ventricles; one can speak equally of the left ventricular ejection fraction (LVEF) and the right ventricular ejection fraction (RVEF). Without a qualifier, the term ejection fraction refers specifically to that of the left ventricle. By definition, the volume of blood within a ventricle immediately before a contraction is known as the end-diastolic volume (EDV). Similarly, the volume of blood left in a ventricle at the end of contraction is end-systolic volume (ESV). The difference between end-diastolic and end-systolic volumes is the stroke volume, the volume of blood ejected with each beat. Ejection fraction (Ef) is the fraction of the end-diastolic volume that is ejected with each beat; that is the stroke volume (SV) divided by end-diastolic volume (EDV): Ef = ((EDV - ESV) / EDV) x 100 In a healthy 70-kg (154-lb) man, the SV is approximately 70 ml and the left ventricular EDV is 120 ml, giving an ejection fraction of 70 / 120, or 58%. Right ventricular volumes being roughly equal to those of the left ventricle, the ejection fraction of the right ventricle is normally equal to that of the left ventricle within narrow limits. Healthy individuals typically have ejection fractions greater than 0.55. However, normal values depend upon the modality being used to calculate the ejection fraction. Damage to the muscle of the heart (myocardium), such as during myocardial infarction or in cardiomyopathy, impairs the heart's ability to eject blood and therefore reduces ejection fraction. This reduction in ejection fraction can manifest itself clinically as heart failure. The ejection fraction is one of the most important predictors of prognosis; patients with a significantly reduced ejection fraction typically have a poorer prognosis. Ejection fraction is commonly measured by echocardiography, in which the volumes of the heart's chambers are measured during the cardiac cycle. Ejection fraction can then be obtained by dividing stroke volume by end-diastolic volume as described above. Other methods of measuring ejection fraction include cardiac MRI, fast scan cardiac computed axial tomography (CT) imaging, ventriculography, Gated SPECT, and the MUGA scan. A MUGA scan involves the injection of a radioisotope into the blood and detecting its flow through the left ventricle. 16 4860-0953-0567, v.1II. CaMKIIδ Ca2+ / calmodulin-dependent protein kinase II (CaM kinase II or CaMKII) is a group of serine / threonine-specific protein kinases that are regulated by the Ca2+ / calmodulin complex. CaMKII is involved in many signaling cascades and is thought to be an important mediator of learning and memory. CaMKII is also necessary for Ca2+homeostasis and reuptake in cardiomyocytes, chloride transport in epithelia, positive T-cell selection, and CD8 T-cell activation. Dysregulation of CaMKII is linked to Alzheimer's disease, Angelman syndrome, and heart arrhythmia. The four isoforms derive from the alpha, beta, gamma, and delta genes. All of the isoforms of CaMKII have: a catalytic domain, an autoinhibitory domain, a variable segment, and a self-association domain. The catalytic domain has several binding sites for ATP and other substrate anchor proteins. It is responsible for the transfer of phosphate from ATP to Ser or Thr residues in substrates. The autoinhibitory domain features a pseudosubstrate site, which binds to the catalytic domain and blocks its ability to phosphorylate proteins. The structural feature that governs autoinhibition is the Threonine 287 residue. Phosphorylation of this site will permanently activate the CaMKII enzyme. Once the Threonine 287 residue has been phosphorylated, the inhibitory domain is blocked from the pseudosubstrate site. This effectively blocks autoinhibition, allowing for permanent activation of the CaMKII enzyme. This enables CaMKII to be active, even in the absence of calcium and calmodulin. The other two domains in CaMKII are the variable and self-association domains. Differences in these domains contribute to the various CaMKII splicing isoforms. The self- association domain (CaMKII AD) is found at the C terminus, the function of this domain is the assembly of the single proteins into large (8 to 14 subunits) multimers. The sensitivity of the CaMKII enzyme to calcium and calmodulin is governed by the variable and self-associative domains. This sensitivity level of CaMKII will also modulate the different states of activation for the enzyme. Initially, the enzyme is activated; however, autophosphorylation does not occur because there is not enough calcium or calmodulin present to bind to neighboring subunits. As greater amounts of calcium and calmodulin accumulate, autophosphorylation occurs leading to persistent activation of the CaMKII enzyme for a short period of time. However, the Threonine 287 residue eventually becomes dephosphorylated, leading to inactivation of CaMKII. Besides Threonine 287, there are several other amino acids that can undergo posttranslational modification. Among them, Methionines 281 and 282 have 17 4860-0953-0567, v.1been shown to get modified upon oxidative stress and to be critical for CaMKIIδ activation in disease. CaMKIIδ appears in both neuronal and non-neuronal cell types. It is characterized particularly in many tumor cells, such as a variety of pancreatic, leukemic, breast and other tumor cells. Several CaMKIIδ variants are highly abundant in myocardial tissue. Increased CaMKII activity has been observed in patients with heart failure, structural heart disease and arrhythmias and CaMKII inhibition has been shown to be beneficial for cardiac disease in preclinical settings. Even though multiple CaMKII inhibitors have already been developed, they all show several limitations, and no compound has been translated into the clinic. The inventors developed a new strategy to overcome the limitations of current CaMKII inhibitors, by precise modulation of CaMKIIδ autophosphorylation site using CRISPR / Cas9 base editing technology. III. 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 functionally effective. In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing 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. The CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains). 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. 18 4860-0953-0567, v.1The CRISPR system can induce double stranded breaks (DSBs) at the target site, followed by disruptions as discussed herein. In other embodiments, Cas9 variants, deemed “nickases,” are used to nick a single strand at the target site. Paired nickases can be used, e.g., to improve specificity, each directed by a pair of different sgRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a base editing enzyme or a reverse transcriptase. The engineered CRISPR technologies of base editing and prime editing have expanded the toolbox of gene editing strategies to potentially correct genetic mutations by enabling precise edits at individual nucleotides. In base editing, Cas9 nickase (nCas9) or deactivated Cas9 (dCas9) is fused to a deaminase protein, allowing precise single-base pair conversions without DSBs within a defined editing window in relation to the protospacer adjacent motif (PAM) site of a sgRNA. There are two major classes of DNA base editors: cytosine base editors (CBEs), which convert a C:G base pair into a T:A base pair, and adenine base editors (ABEs), which convert an A:T base pair into a G:C base pair. As such, base editors allow efficient installation of single base substitutions in DNA. For example, adenosine deaminases induce adenosine (A) to inosine (I) edits in single-stranded DNA that in turn result in A-to-G transitions after DNA repair or replication. Adenine base editors (ABEs) are fusions of programmable DNA-binding domains (e.g,, catalytically impaired RNA-guided CRISPR / Cas nucleases) linked to an engineered adenosine deaminase. In instances where the programmable DNA-binding domain is a CRISPR / Cas nuclease, targeted adenines lie within an “editing window” in the single-stranded (ss) DNA bubble (R-loop) induced by the CRISPR-Cas RNA- protein complex. The most commonly used ABEs comprise an adenosine deaminase heterodimer consisting of E. coli TadA (wild-type) fused to an engineered E. coli TadA variant (e.g., ABEmax) or a single engineered E. coli TadA variant (e.g., ABE8e, ABE8eV106W, or ABE8.20-m) as well as a nickase Cas9 and nuclear localization sequences (NLS). ABEs have been used successfully for installation of A-to-G substitutions in multiple cell types and organisms and could potentially reverse a large number of mutations known to be associated with human disease. Examples of ABEs include those described in U.S. Pat. Publn. US20200308571, PCT Publn. WO2020214842, and PCT Publn. WO2021025750, which are each incorporated herein by reference in their entirety. Reference is made to International Publication No. WO 2018 / 027078, published August 2, 2018; International Publication No. WO 2019 / 079347 published April 25, 2019; International Publication No. WO 2019 / 226593, published November 28, 2019; U.S. Patent Publication No. 2018 / 0073012, published March 19 4860-0953-0567, v.115, 2018, which issued as U.S. Patent No. 10,113,163, on October 30, 2018; and U.S. Patent Publication No. 2017 / 0121693, published May 4, 2017, which issued as U.S. Patent No. 10,167,457 on January 1, 2019. In some aspects, a Cas nuclease and sgRNA (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 sgRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing. Target sites may be 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides in length. The target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, NG, NRN, NYN, NAG, NNNRRT, or NNGG. 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. 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 an editing template. In some aspects, the recombination is homologous recombination. Typically, in the context of an endogenous CRISPR system, formation of the CRISPR complex (comprising the guide sequence hybridized to the target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. The tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of the CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. The tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of the CRISPR complex, such as at least 20 4860-0953-0567, v.150%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. One or more vectors driving expression of one or more elements of the CRISPR system can be introduced into the 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 also be delivered to cells as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. The sgRNA may be under the control of a constitutive promoter. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. A vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme, such as a Cas protein. 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. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia or S. aureus or S. auricularis or S. lugdunensis). The CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector can encode a CRISPR enzyme that is 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. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I 21 4860-0953-0567, v.1catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR. In some embodiments, a Cas9 polypeptide can be a deactivated (e.g., mutated, dCAs9) Cas9 polypeptide, wherein 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., 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. In an alternative embodiment, the CRIPSR enzyme is a Cas protein, preferably Cas9 (having a nucleotide sequence of Genbank accession no NC_002737.2 and a protein sequence of Genbank accession no NP_269215.1). Again, the Cas9 protein may also be modified to improve activity. For example, the Cas9 protein may comprise the D10A amino acid substitution, this nickase cleaves only the DNA strand that is complementary to and recognized by the crRNA. In an alternative embodiment, the Cas9 protein may alternatively or additionally comprise the H840A amino acid substitution, this nickase cleaves only the DNA strand that does not interact with the sRNA. In this embodiment, Cas9 may be used with a pair (i.e. two) sgRNA molecules (or a construct expressing such a pair) and as a result can cleave the target region on the opposite DNA strand, with the possibility of improving specificity by 100- to 1500-fold. In a further embodiment, the Cas9 protein may comprise a D1135E substitution. The Cas 9 protein may also be the VQR, VRQR or SpRY variant. Alternatively, the Cas9 protein may be xCas9 (a Streptococcus pyogenes variant that can recognize a broad range of PAM sequences including NG, GAA and GAT). In other alternatives, the Cas9 variant is SpCas9-NG (with a relaxed preference to the third nucleotide of the PAM motif, such that the variant can recognize sequences where the PAM motif is NGN rather than NGG), SaCas9 (from S. aureus that can recognize NNGRR(T) PAM sequences; see Ran et al., 2015), SaCas9- KKH (a variant from S. aureus that can recognize NNNRRT PAM sequences), SauCas9 (from 22 4860-0953-0567, v.1S. auricularis that can recognize NNGG PAM sequences; Genbank accession no WP_107392933.1), or SlugCas9 (from S. lugdunensis M23590 that can recognize NNGG PAM sequences; Genbank accession no WP_002460848.1). In some embodiments, an enzyme coding sequence encoding the CRISPR enzyme is 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 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. Each of the guide sequences described herein may further comprise additional nucleotides to form or encode a crRNA, e.g., using any known sequence appropriate for the Cas9 being used. In some embodiments, the crRNA comprises (5’ to 3’) at least a spacer sequence and a first complementarity domain. The first complementary domain is sufficiently complementary to a second complementarity domain, which may be part of the same molecule in the case of an sgRNA or in a tracrRNA in the case of a dual or modular sgRNA, to form a duplex. See, e.g., US 2017 / 0007679 for detailed discussion of crRNA and sgRNA domains, including first and second complementarity domains. 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, 23 4860-0953-0567, v.1a 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 particular embodiments, the disclosure provides for an sgRNA comprising a spacer sequence and a tracrRNA sequence. The guide RNA can be considered to comprise a scaffold sequence necessary for endonuclease binding and a spacer sequence required to bind to the genomic target sequence. An exemplary scaffold sequence suitable for use with SaCas9 to follow the guide sequence at its 3’ end is: GTTTAAGTACTCTGTGCTGGAAACAGCACAGAATCTACTTAAACAAGGCAAAAT GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA (SEQ ID NO: 78) in 5’ to 3’ orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 78, or a sequence that differs from SEQ ID NO: 78 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides. 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). 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 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 24 4860-0953-0567, v.1transcription 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 CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by reference. As an RNA guided protein, Cas9 requires a short RNA to direct the recognition of DNA targets. Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence (e.g., NGG or NG or NNNRRT or NNGG) it can bind here without a protospacer target. However, the Cas9-sgRNA complex requires a close match to the sgRNA to create a double strand break. CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA. Because eukaryotic systems lack some of the proteins required to process CRISPR RNAs, the synthetic construct sgRNA was created to combine the essential pieces of RNA for Cas9 targeting into a single RNA expressed with the RNA polymerase type III promoter U6. Other promoters under the control of RNA Pol III include those for ribosomal 5S rRNA, tRNA and few other small RNAs, RNase P and RNase MRP RNA, 7SL RNA (the RNA component of the signal recognition particles), Vault RNAs, Y RNA, SINEs (short interspersed repetitive elements), 7SK RNA, two microRNAs, several small nucleolar RNAs and several few regulatory antisense RNAs. Synthetic sgRNAs are slightly over 100 bp at the minimum length and contain a portion which targets the 20 or 21 protospacer nucleotides immediately preceding the PAM sequence. The length of the sgRNA can also be shortened at the 5’ with respect to its canonical length to meet specific criteria, e.g., the removal of a stretch of thymines that can inhibit the polymerase type III transcription activity. sgRNAs do not contain the PAM sequence. 25 4860-0953-0567, v.1In some embodiments, the sgRNA targets an autophosphorylation site within the CaMKIIδ gene. In some embodiments, the sgRNA targets a CaMKIIδ regulatory domain. In some embodiments, the sgRNA targets Thr287 of CaMKIIδ. In some embodiments, a nucleic acid may comprise one or more sequences encoding a sgRNA. In some embodiments, a nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 sequences encoding a sgRNA. In some embodiments, all of the sequences encode the same sgRNA. In some embodiments, all of the sequences encode different sgRNAs. In some embodiments, at least 2 of the sequences encode the same sgRNA, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 of the sequences encode the same sgRNA. In some embodiments, nucleotide gene editing may be performed in vitro or ex vivo. In some embodiments, cells are contacted in vitro or ex vivo with a nucleotide editing Cas9 and a sgRNA that targets a CaMKIIδ site. In some embodiments, the cells are contacted with one or more nucleic acids encoding the Cas9 and the guide RNA. In some embodiments, the one or more nucleic acids are introduced into the cells using, for example, lipofection or electroporation. Nucleotide gene editing may also be performed in zygotes. In some embodiments, zygotes may be injected with one or more nucleic acids encoding Cas9 and a sgRNA that targets a CaMKIIδ site. The zygotes may subsequently be injected into a host. In some embodiments, the Cas9 is provided on a vector. In some embodiments, the vector contains a Cas9 derived from S. pyogenes (SpCas9). In some embodiments, the vector contains a Cas9 derived from S. aureus (SaCas9). In some embodiments, the vector contains a Cas9 derived from S. auricularis (SauCas9). In some embodiments, the vector contains a Cas9 derived from S. lugdunensis (SlugCas9). In some embodiments, the Cas9 sequence is codon optimized for expression in human cells or mouse cells. In some embodiments, the vector further contains a sequence encoding a fluorescent protein, such as GFP, which allows Cas 9- expressing cells to be sorted using fluorescence activated cell sorting (FACS). In some embodiments, the vector is a viral vector such as an adeno-associated viral vector. In some embodiments, the sgRNA is provided on a vector. In some embodiments, the vector is a viral vector such as an adeno-associated viral vector. In some embodiments, the Cas9 and the guide RNA are provided on the same vector. In some embodiments, the Cas9 and the guide RNA are provided on different vectors. Any type of vector, such as any of those described herein, may be used. In some embodiments, the vector is a lipid nanoparticle. In some embodiments, the vector is a viral 26 4860-0953-0567, v.1vector. In some embodiments, the viral vector is a non-integrating viral vector (i.e., that does not insert sequence from the vector into a host chromosome). In some embodiments, the viral vector is an adeno-associated virus vector (AAV), a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the vector comprises a cardiomyocyte-specific promoter. In some embodiments, the cardiomyocyte-specific promoter is a cardiac troponin T (cTnT) promoter. In any of the foregoing embodiments, the vector may be an adeno-associated virus vector (AAV). Where a vector is used, it may be a viral vector, such as a non-integrating viral vector. In some embodiments, the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10 (see, e.g., SEQ ID NO: 81 of U.S. Patent 9,790,472, which is incorporated by reference herein in its entirety), AAVrh74 (see, e.g., SEQ ID NO: 1 of U.S. Patent Publication No.2015 / 0111955, which is incorporated by reference herein in its entirety), AAV9 vector, AAV9P vector (also known as AAVMYO, see, Weinmann et al., 2020,), Myo-AAV vectors described in Tabebordbar et al., 2021 (e.g., MyoAAV 1A, 2A, 3A, 4A, 4C, or 4E), and AAV9-rh74-HB-P1, AAV9-AAA-P1-SG vectors described in WO2022053630. wherein the number following AAV indicates the AAV serotype. 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., 2001; Naso et al., 2017; and references cited therein for detailed discussion of various AAV vectors. In some embodiments, the vector is an AAV9 vector. Efficiency of in vitro or ex vivo nucleotide editing Cas9 may be assessed using techniques known to those of skill in the art, such as the T7 E1 assay or sequencing. In some embodiments, in vitro or ex vivo gene editing is performed in a cardiac cell. In some embodiments, gene editing is performed in iPSC or iCM cells. In some embodiments, the iPSC cells are differentiated after gene editing. For example, the iPSC cells may be differentiated into a cardiac cell after editing. In some embodiments, the iPSC cells are differentiated into cardiac muscle cells. In some embodiments, the iPSC cells are differentiated 27 4860-0953-0567, v.1into cardiomyocytes. iPSC cells may be induced to differentiate according to methods known to those of skill in the art. In some embodiments, contacting the cell with the nucleotide editing Cas9 and the sgRNA prevents sustained overactivation of CaMKIIδ following autophosphorylation. In some embodiments, the edited cells, or cells derived therefrom, show reduced or no level of CaMKIIδ activation as compared to non-edited cells. IV. Nucleic Acid Delivery In some embodiments, expression cassettes are employed to express a protein product, either for subsequent purification and delivery to a cell / subject, or for use directly in a genetic- based delivery approach. Provided herein are expression vectors which contain one or more nucleic acids encoding nucleotide editing Cas9 and at least one guide RNA that targets a CaMKIIδ. In some embodiments, a nucleic acid encoding nucleotide editing Cas9 and a nucleic acid encoding at least one guide RNA are provided on the same vector. In further embodiments, a nucleic acid encoding nucleotide editing Cas9 and a nucleic acid encoding least one guide RNA are provided on separate vectors. Expression requires that appropriate signals be provided in the vectors and include various regulatory elements such as enhancers / promoters from both viral and mammalian sources that drive expression of the genes of interest in cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide. A. Regulatory Elements Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one 28 4860-0953-0567, v.1or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites. The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. In some embodiments, the nucleotide editing Cas9 constructs of the disclosure are expressed by a muscle-cell specific promoter. This muscle-cell specific promoter may be constitutively active or may be an inducible promoter. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription. In certain embodiments, viral promotes such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection 29 4860-0953-0567, v.1or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product. Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. Below is a list of promoters / enhancers and inducible promoters / enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. The promoter and / or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and / or DQ β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, t-globin, β-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), α1-antitrypain, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus. In some embodiments, inducible elements may be used. In some embodiments, the inducible element is, for example, MTII, MMTV (mouse mammary tumor virus), β-interferon, adenovirus 5 E2, collagenase, stromelysin, SV40, murine MX gene, GRP78 gene, α-2- macroglobulin, vimentin, MHC class I gene H-2κb, HSP70, proliferin, tumor necrosis factor, and / or thyroid stimulating hormone α gene. In some embodiments, the inducer is phorbol ester 30 4860-0953-0567, v.1(TFA), heavy metals, glucocorticoids, poly(rI)x, poly(rc), ElA, phorbol ester (TPA), interferon, Newcastle Disease Virus, A23187, IL-6, serum, interferon, SV40 large T antigen, PMA, and / or thyroid hormone. Any of the inducible elements described herein may be used with any of the inducers described herein. Of particular interest are cardiomyocyte-specific promoters. In some embodiments, the cardiomyocyte-specific promoter is the cardiac troponin T (cTnT) promoter. Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences. B. 2A Peptide In some embodiments, a 2A-like self-cleaving domain from the insect virus Thosea asigna (TaV 2A peptide) (EGRGSLLTCGDVEENPGP (SEQ ID NO: 9)) is used. These 2A- like domains have been shown to function across eukaryotes and cause cleavage of amino acids to occur co-translationally within the 2A-like peptide domain. Therefore, inclusion of TaV 2A peptide allows the expression of multiple proteins from a single mRNA transcript. Importantly, the domain of TaV when tested in eukaryotic systems has shown greater than 99% cleavage activity. Other acceptable 2A-like peptides include, but are not limited to, equine rhinitis A virus (ERAV) 2A peptide (QCTNYALLKLAGDVESNPGP (SEQ ID NO: 10)), porcine teschovirus-1 (PTV1) 2A peptide (ATNFSLLKQAGDVEENPGP (SEQ ID NO: 11)) and foot and mouth disease virus (FMDV) 2A peptide (PVKQLLNFDLLKLAGDVESNPGP (SEQ ID NO: 12)) or modified versions thereof. In some embodiments, the 2A peptide is used to express a reporter and a nucleotide editing Cas9 simultaneously. The reporter may be, for example, GFP or mCherry. Other self-cleaving peptides that may be used include but are not limited to nuclear inclusion protein a (Nia) protease, a P1 protease, a 3C protease, a L protease, a 3C-like protease, or modified versions thereof. C. Trans-splicing Inteins In some embodiments, trans-splicing inteins are used to permit the covalent splicing of the split nucleotide editing Cas9. Due to delivery size limitation, nucleotide editing Cas9 can 31 4860-0953-0567, v.1be split in N- and C-terminal peptides. Each half of the split nucleotide editing Cas9 when linked to trans-splicing inteins reassemble after translation into a functional nucleotide editing Cas9 that retains similar editing efficiencies compared to its non-split, full-length equivalent. In some embodiments, the N- and C-terminal peptides of nucleotide editing Cas9 are fused to split DnaE intein halves from N. puntiforme (Npu). Other trans-splicing inteins that may be used include but are not limited to Sce VMA, Mtu RecA, Ssp DnaE. D. Delivery of Expression Vectors There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals. One method for in vivo delivery involves the use of an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized. The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans. Adenovirus is particularly suitable for use as a gene transfer vector because of its mid- sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both 32 4860-0953-0567, v.1ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5’-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation. In one system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure. Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E1 proteins. Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the D3 or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector- borne cytotoxicity. Also, the replication deficiency of the E1-deleted virus is incomplete. Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293. 33 4860-0953-0567, v.1The adenoviruses of the disclosure are replication defective, or at least conditionally replication defective. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present disclosure. Retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription. The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5’ and 3’ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells. There are certain limitations to the use of retrovirus vectors in all aspects of the present disclosure. For example, retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes. Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new 34 4860-0953-0567, v.1packaging cell lines are now available that should greatly decrease the likelihood of recombination. Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells. In some embodiments, particular embodiments, the vector is an AAV vector. AAV is a small virus that infects humans and some other primate species. AAV is not currently known to cause disease. The virus causes a very mild immune response, lending further support to its apparent lack of pathogenicity. In many cases, AAV vectors integrate into the host cell genome, which can be important for certain applications, but can also have unwanted consequences. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell, although in the native virus some integration of virally carried genes into the host genome does occur. These features make AAV a very attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. Recent human clinical trials using AAV for gene therapy in the retina have shown promise. AAV belongs to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. The virus is a small (20 nm) replication- defective, non-enveloped virus. Wild-type AAV has attracted considerable interest from gene therapy researchers due to a number of features. Chief amongst these is the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19. This feature makes it somewhat more predictable than retroviruses, which present the threat of a random insertion and of mutagenesis, which is sometimes followed by development of cancer. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. Development of AAVs as gene therapy vectors, however, has eliminated this integrative capacity by removal of the rep and cap from the DNA of the vector. The desired gene together with a promoter to drive transcription of the gene is inserted between the inverted terminal repeats (ITR) that aid in concatemer formation in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact for the life of the host cell. In dividing cells, AAV DNA is lost through cell division, since the 35 4860-0953-0567, v.1episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at very low frequency. AAVs also present very low immunogenicity, seemingly restricted to generation of neutralizing antibodies, while they induce no clearly defined cytotoxic response. This feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for human gene therapy. Use of the AAV does present some disadvantages. The cloning capacity of the vector is relatively limited and most therapeutic genes require the complete replacement of the virus's 4.8 kilobase genome. Large genes are, therefore, not suitable for use in a standard AAV vector. Options are currently being explored to overcome the limited coding capacity. The AAV ITRs of two genomes can anneal to form head to tail concatemers, almost doubling the capacity of the vector. Insertion of splice sites allows for the removal of the ITRs from the transcript. Because of AAV’s specialized gene therapy advantages, researchers have created an altered version of AAV termed self-complementary adeno-associated virus (scAAV). Whereas AAV packages a single strand of DNA and must wait for its second strand to be synthesized, scAAV packages two shorter strands that are complementary to each other. By avoiding second-strand synthesis, scAAV can express more quickly, although as a caveat, scAAV can only encode half of the already limited capacity of AAV. Recent reports suggest that scAAV vectors are more immunogenic than single stranded adenovirus vectors, inducing a stronger activation of cytotoxic T lymphocytes. The humoral immunity instigated by infection with the wild-type is thought to be a very common event. The associated neutralising activity limits the usefulness of the most commonly used serotype AAV2 in certain applications. Accordingly, the majority of clinical trials currently under way involve delivery of AAV2 into the brain, a relatively immunologically privileged organ. In the brain, AAV2 is strongly neuron specific. The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry. The Inverted Terminal Repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of the AAV genome. The feature of these sequences that gives them this property 36 4860-0953-0567, v.1is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease-resistant AAV particles. With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) proteins can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene. However, it was also published that the ITRs are not the only elements required in cis for the effective replication and encapsidation. A few research groups have identified a sequence designated cis-acting Rep- dependent element (CARE) inside the coding sequence of the rep gene. CARE was shown to augment the replication and encapsidation when present in cis. On the “left side” of the genome there are two promoters called p5 and p19, from which two overlapping messenger ribonucleic acids (mRNAs) of different lengths can be produced. Each of these contains an intron which can be either spliced out or not. Given these possibilities, four various mRNAs, and consequently four various Rep proteins with overlapping sequence can be synthesized. Their names depict their sizes in kilodaltons (kDa): Rep78, Rep68, Rep52 and Rep40. Rep78 and 68 can specifically bind the hairpin formed by the ITR in the self- priming act and cleave at a specific region, designated terminal resolution site, within the hairpin. They were also shown to be necessary for the AAVS1-specific integration of the AAV genome. All four Rep proteins were shown to bind ATP and to possess helicase activity. It was also shown that they upregulate the transcription from the p40 promoter (mentioned below) but downregulate both p5 and p19 promoters. The right side of a positive-sensed AAV genome encodes overlapping sequences of three capsid proteins, VP1, VP2 and VP3, which start from one promoter, designated p40. The molecular weights of these proteins are 87, 72 and 62 kiloDaltons, respectively. The AAV capsid is composed of a mixture of VP1, VP2, and VP3 totaling 60 monomers arranged in icosahedral symmetry in a ratio of 1:1:10, with an estimated size of 3.9 MegaDaltons. The cap gene produces an additional, non-structural protein called the Assembly- Activating Protein (AAP). This protein is produced from ORF2 and is essential for the capsid- assembly process. The exact function of this protein in the assembly process and its structure have not been solved to date. 37 4860-0953-0567, v.1All three VPs are translated from one mRNA. After this mRNA is synthesized, it can be spliced in two different manners: either a longer or shorter intron can be excised resulting in the formation of two pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool. Usually, especially in the presence of adenovirus, the longer intron is preferred, so the 2.3-kb-long mRNA represents the so-called “major splice”. In this form the first AUG codon, from which the synthesis of VP1 protein starts, is cut out, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon that remains in the major splice is the initiation codon for VP3 protein. However, upstream of that codon in the same open reading frame lies an ACG sequence (encoding threonine) which is surrounded by an optimal Kozak context. This contributes to a low level of synthesis of VP2 protein, which is actually VP3 protein with additional N terminal residues, as is VP1. Since the bigger intron is preferred to be spliced out, and since in the major splice the ACG codon is a much weaker translation initiation signal, the ratio at which the AAV structural proteins are synthesized in vivo is about 1:1:20, which is the same as in the mature virus particle. The unique fragment at the N terminus of VP1 protein was shown to possess the phospholipase A2 (PLA2) activity, which is probably required for the releasing of AAV particles from late endosomes. Muralidhar et al. reported that VP2 and VP3 are crucial for correct virion assembly. More recently, however, Warrington et al. showed VP2 to be unnecessary for the complete virus particle formation and an efficient infectivity, and also presented that VP2 can tolerate large insertions in its N terminus, while VP1 cannot, probably because of the PLA2 domain presence. The AAV vector may be replication-defective or conditionally replication defective. In some embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, a single viral vector is used to deliver a nucleic acid encoding a nucleotide editing Cas9 and at least one sgRNA to a cell. In some embodiments, nucleotide editing Cas9 is provided to a cell using a first viral vector and at least one sgRNA is provided to the cell using a second viral vector. In some embodiment, the nucleotide editing Cas9 may use a split-intein dual AAV system which reconstitutes the full-length nucleotide editor by protein trans-splicing. In these systems, the Cas9 protein or the base editor is split into two sections, each fused with one part of an intein system (e.g., intein-N and intein-C encoded by dnaEn and dnaEc, respectively). Upon co-expression, the two sections of the Cas9 protein or 38 4860-0953-0567, v.1nucleobase editor are ligated together via intein-mediated protein splicing. See, U.S. Pat. Publn. US20180127780, which is incorporated by reference herein in its entirety. In some embodiments, a single viral vector is used to deliver a nucleic acid encoding nucleotide editing Cas9 and at least one sgRNA to a cell. In some embodiments, nucleotide editing Cas9 is provided to a cell using a first viral vector and at least one sgRNA is provided to the cell using a second viral vector. In some embodiment, the nucleotide editing Cas9 may use a split-intein dual AAV system which reconstitutes the full-length nucleotide editor by protein trans-splicing. In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell. The cell may be a muscle cell, a satellite cell, a mesangioblast, a bone marrow derived cell, a stromal cell or a mesenchymal stem cell. In some embodiments, the cell is a cardiac muscle cell, a skeletal muscle cell, or a smooth muscle cell. In some embodiments, the cell is a cell in the tibialis anterior, quadriceps, soleus, triceps, extensor digitorum longus, diaphragm, or heart. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or inner cell mass cell (iCM). In further embodiments, the cell is a human iPSC or a human iCM. In some embodiments, human iPSCs or human iCMs of the disclosure may be derived from a cultured stem cell line, an adult stem cell, a placental stem cell, or from another source of adult or embryonic stem cells that does not require the destruction of a human embryo. Delivery to a cell may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle. Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present disclosure. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, nanoparticles, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use. Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement), or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences 39 4860-0953-0567, v.1sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed. In yet another embodiment, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product. In still another embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used have comprised biologically inert substances such as tungsten or gold beads. In some embodiments, the expression construct is delivered directly to the liver, skin, and / or muscle tissue of a subject. This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present disclosure. In a further embodiment, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes. Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. A reagent known as Lipofectamine 2000TMis widely used and commercially available. In certain embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ) to facilitate fusion with the cell membrane and promote cell entry of liposome- encapsulated DNA. In other embodiments, the liposome may be complexed or employed in 40 4860-0953-0567, v.1conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present disclosure. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase. Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific. Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) and transferrin. A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells. E. AAV-Cas9 vectors In some embodiments, a Cas9 base editor may be packaged into an AAV vector. In some embodiments, the AAV vector is a wild-type AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. Exemplary AAV-Cas9 vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the Cas9 sequence. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and / or wild-type sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wild-type sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a 41 4860-0953-0567, v.1substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs have a length of 110 ± 10 base pairs. In some embodiments, the ITRs have a length of 120 ± 10 base pairs. In some embodiments, the ITRs have a length of 130 ± 10 base pairs. In some embodiments, the ITRs have a length of 140 ± 10 base pairs. In some embodiments, the ITRs have a length of 150 ± 10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs. In some embodiments, the AAV-Cas9 vector may contain one or more nuclear localization signals (NLS). In some embodiments, the AAV-Cas9 vector contains 1, 2, 3, 4, or 5 nuclear localization signals. Exemplary NLS include the c-myc NLS, the SV40 NLS, the hnRNPAI M9 NLS, the nucleoplasmin NLS, the sequence RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 13) of the IBB domain from importin-alpha, the sequences VSRKRPRP (SEQ ID NO: 14) and PPKKARED (SEQ ID NO: 15) of the myoma T protein, the sequence PQPKKKPL (SEQ ID NO: 16) of human p53, the sequence SALIKKKKKMAP (SEQ ID NO: 17) of mouse c-abl IV, the sequences DRLRR (SEQ ID NO: 18) and PKQKKRK (SEQ ID NO: 19) of the influenza virus NS1, the sequence RKLKKKIKKL (SEQ ID NO: 20) of the Hepatitis virus delta antigen and the sequence REKKKFLKRR (SEQ ID NO: 21) of the mouse Mx1 protein. Further acceptable nuclear localization signals include bipartite nuclear localization sequences such as the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 22) of the human poly(ADP- ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 23) of the steroid hormone receptors (human) glucocorticoid. In some embodiments, the AAV-Cas9 vector may comprise additional elements to facilitate packaging of the vector and expression of the Cas9. In some embodiments, the AAV- Cas9 vector may comprise a polyA sequence. In some embodiments, the polyA sequence may be a mini-polyA sequence. In some embodiments, the AAV-CAs9 vector may comprise a transposable element. In some embodiments, the AAV-Cas9 vector may comprise a regulator element. In some embodiments, the regulator element is an activator or a repressor. 42 4860-0953-0567, v.1In some embodiments, the AAV-Cas9 may contain one or more promoters. In some embodiments, the one or more promoters drive expression of the Cas9. In some embodiments, the one or more promoters are cardiomyocyte-specific promoters. Exemplary cardiac-specific promoters include the cardiac troponin T promoter and α-myosin heavy chain promoter. In some embodiments, the AAV-Cas9 vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a bacculovirus expression system. In some embodiments of the gene editing constructs of the disclosure, the construct comprises or consists of a promoter and a nuclease. In some embodiments, the construct comprises or consists of an cTnT promoter and a Cas9 nuclease. In some embodiments, the construct comprises or consists of an cTnT promoter and a Cas9 nuclease isolated or derived from Staphylococcus pyogenes (“SpCas9”). In some embodiments, the SpCas9 nuclease comprises or consists of a nucleotide sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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: GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCC GTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAAC ACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGC GGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACAC CAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGC CAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGA GGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAG CACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAA GTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGT GGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAA CCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAG CAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGA ATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAA GAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTA CGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCT GTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGA GTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATAC GACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTG CCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGC TACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATC CTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGA CCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCA CCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTG AAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTAC 43 4860-0953-0567, v.1GTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGC GAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCC GCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAG AAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAG CTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGC GGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTG ACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCC GTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCAC GATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAG GACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATG ATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAG CAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATC AACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCC GACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACC TTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCAC GAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAG ACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGA AGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGC AGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAA GCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACT GGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTT CTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCG GGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACT ACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATC TGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCA AGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGG ACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGA AAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTT TTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAA CGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTT CGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGA GCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAA CTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTG ATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTT GCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACC GAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGC GATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTC GACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGC AAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAA AGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAA GAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTG GAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAA CGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTAT GAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAA CAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAG AGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAG CACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACC CTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACC GGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGA 44 4860-0953-0567, v.1GCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC (SEQ ID NO: 24). In some embodiments, the construct comprising a promoter and a nuclease further comprises at least two inverted terminal repeat (ITR) sequences. In some embodiments, the construct comprising a promoter and a nuclease further comprises at least two ITR sequences from isolated or derived from an AAV of serotype 2 (AAV2). In some embodiments, the construct comprising a promoter and a nuclease further comprises at least two ITR sequences each comprising or consisting of a nucleotide sequence of GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGA GAGGGA (SEQ ID NO: 25). In some embodiments, the construct comprising a promoter and a nuclease further comprises at least two ITR sequences, wherein the first ITR sequence comprises or consists of a nucleotide sequence of CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG GGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG AGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 26) and the second ITR sequence comprises or consist of a nucleotide sequence of AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCAC TGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTC AGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID NO: 27). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding an cTnT promoter, a sequence encoding a SpCas9 nuclease and a second AAV2 ITR. In some embodiments, the construct comprising or consisting of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease and a second ITR, further comprises a poly A sequence. In some embodiments, the polyA sequence comprises or consists of a minipolyA sequence. Exemplary minipolyA sequences of the disclosure comprise or consist of a nucleotide sequence of TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTGATCAGG CGCG (SEQ ID NO: 29). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a 45 4860-0953-0567, v.1minipoly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding an cTnT promoter, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, further comprises at least one nuclear localization signal. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, further comprises at least two nuclear localization signals. Exemplary nuclear localization signals of the disclosure comprise or consist of a nucleotide sequence of AAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAA (SEQ ID NO: 28) or a nucleotide sequence of ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC (SEQ ID NO: 30). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a poly A sequence and a second ITR. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a poly A sequence and a second ITR, further comprises a stop codon. The stop codon may have a sequence of TAG, TAA, or TGA. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR. In some embodiments, the construct comprising or consisting of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR, further comprises transposable element inverted repeats. Exemplary transposable element inverted repeats of the disclosure comprise or consist of a nucleotide sequence of TGTGGGCGGACAAAATAGTTGGGAACTGGGAGGGGTGGAAATGGAGTTTTTAAG 46 4860-0953-0567, v.1GATTATTTAGGGAAGAGTGACAAAATAGATGGGAACTGGGTGTAGCGTCGTAAG CTAATACGAAAATTAAAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTG GTT (SEQ ID NO: 31) and / or a nucleotide sequence of GAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAAT TTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCT TTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGA CTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCCGCCCA CA (SEQ ID NO: 32). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprising or consisting of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, and a second transposable element inverted repeat, further comprises a regulatory sequence. Exemplary regulatory sequences of the disclosure comprise or consist of a nucleotide sequence of: CATGCAAGCTGTAGCCAACCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAA CGATGCTCGCCTTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGC ACCACCGGCAAGCGCCGCGACGGCCGAGGTCTTCCGATCTCCTGAAGCCAGGGC AGATCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGA CGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCC TCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATG AAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAG TAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGT AACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTAT GCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTA TGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCG CCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTC GGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGT TCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGA ACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGT TGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAG ATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCA CCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGAT CTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTG GGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAA CAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGT CACAACGCCG (SEQ ID NO: 33). 47 4860-0953-0567, v.1In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, a regulatory sequence and a second transposable element inverted repeat. In some embodiments, the construct may further comprise one or more spacer sequences. Exemplary spacer sequences of the disclosure have length from 1-1500 nucleotides, inclusive of all ranges therebetween. In some embodiments, the spacer sequences may be located either 5’ to or 3’ to an ITR, a promoter, a nuclear localization sequence, a nuclease, a stop codon, a polyA sequence, a transposable element inverted repeat, and / or a regulator element. F. AAV-sgRNA vectors In some embodiments, at least a first sequence encoding a sgRNA and a second sequence encoding a sgRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a sgRNA, a second sequence encoding a sgRNA, and a third sequence encoding a sgRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a sgRNA, a second sequence encoding a sgRNA, a third sequence encoding a sgRNA, and a fourth sequence encoding a sgRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a sgRNA, a second sequence encoding a sgRNA, a third sequence encoding a sgRNA, a fourth sequence encoding a sgRNA, and a fifth sequence encoding a sgRNA may be packaged into an AAV vector. In some embodiments, a plurality of sequences encoding a sgRNA are packaged into an AAV vector. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 sequences encoding a sgRNA may be packaged into an AAV vector. In some embodiments, each sequence encoding a sgRNA is different. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the sequences encoding a sgRNA are the same. In some embodiments, all of the sequences encoding a sgRNA are the same. In some embodiments, the AAV vector is a wild-type AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences. In some 48 4860-0953-0567, v.1embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, the ITRs are isolated or derived from an AAV vector of a first serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a second serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2 and the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2 and the second serotype is AAV9. Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, a first ITR is isolated or derived from an AAV vector of a first serotype, a second ITR is isolated or derived from an AAV vector of a second serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a third serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype, the second serotype, and the third serotype are the same. In some embodiments, the first serotype, the second serotype, and the third serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV9. Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, 49 4860-0953-0567, v.1AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and / or wild-type sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wild-type sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs have a length of 110 ± 10 base pairs. In some embodiments, the ITRs have a length of 120 ± 10 base pairs. In some embodiments, the ITRs have a length of 130 ± 10 base pairs. In some embodiments, the ITRs have a length of 140 ± 10 base pairs. In some embodiments, the ITRs have a length of 150 ± 10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs. In some embodiments, the AAV-sgRNA vector may comprise additional elements to facilitate packaging of the vector and expression of the sgRNA. In some embodiments, the AAV-sgRNA vector may comprise a transposable element. In some embodiments, the AAV- sgRNA vector may comprise a regulatory element. In some embodiments, the regulatory element comprises an activator or a repressor. In some embodiments, the AAV-sgRNA sequence may comprise a non-functional or “stuffer” sequence. Exemplary stuffer sequences of the disclosure may have some (a non-zero percentage of) identity or homology to a genomic sequence of a mammal (including a human). Alternatively, exemplary stuffer sequences of the disclosure may have no identify or homology to a genomic sequence of a mammal (including a human). Exemplary stuffer sequences of the disclosure may comprise or consist of naturally occurring non-coding sequences or sequences that are neither transcribed nor translated following administration of the AAV vector to a subject. In some embodiments, the AAV-sgRNA vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-sgRNA 50 4860-0953-0567, v.1vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a bacculovirus expression system. In some embodiments, the AAV-sgRNA vector comprises at least one promoter. In some embodiments, the AAV-sgRNA vector comprises at least two promoters. In some embodiments, the AAV-sgRNA vector comprises at least three promoters. In some embodiments, the AAV-sgRNA vector comprises at least four promoters. In some embodiments, the AAV-sgRNA vector comprises at least five promoters. Exemplary promoters include, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and / or DQ β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, t- globin, β-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), α1- antitrypain, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus. Further exemplary promoters include the U6 promoter, the H1 promoter, and the 7SK promoter. In some embodiments, the AAV vector comprises a first sequence encoding a sgRNA and a second sequence encoding a sgRNA, a first promoter drives expression of the first sequence encoding a sgRNA and a second promoter drives expression of the second sequence encoding a sgRNA. In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters are different. In some embodiments, the first and second promoters are selected from the H1 promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a sgRNA and the second sequence encoding a sgRNA are identical. In some embodiments, the first sequence encoding a sgRNA and the second sequence encoding a sgRNA are not identical. In some embodiments, the AAV vector comprises a first sequence encoding a sgRNA, a second sequence encoding a sgRNA, and a third sequence encoding a sgRNA, a first promoter drives expression of the first sequence encoding a sgRNA, a second promoter drives expression of the second sequence encoding a sgRNA, and a third promoter drives expression of a third sequence encoding a sgRNA. In some embodiments, at least two of the first, second, and third promoters are the same. In some embodiments, each of the first, second, and third promoters 51 4860-0953-0567, v.1are different. In some embodiments, the first, second, and third promoters are selected from the H1 promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first promoter is the U6 promoter. In some embodiments, the second promoter is the H1 promoter. In some embodiments, the third promoter is the 7SK promoter. In some embodiments, the first promoter is the U6 promoter, the second promoter is the H1 promoter, and the third promoter is the 7SK promoter. In some embodiments, the first sequence encoding a sgRNA, the second sequence encoding a sgRNA, and the third sequence encoding a sgRNA are identical. In some embodiments, the first sequence encoding a sgRNA, the second sequence encoding a sgRNA, and the third sequence encoding a sgRNA are not identical. In some embodiments, the AAV vector comprises a first sequence encoding a sgRNA, a second sequence encoding a sgRNA, a third sequence encoding a sgRNA, and a fourth sequence encoding a sgRNA, a first promoter drives expression of the first sequence encoding a sgRNA, a second promoter drives expression of the second sequence encoding a sgRNA, a third promoter drives expression of the third sequence encoding a sgRNA, and a fourth promoter drives expression of the fourth sequence encoding a sgRNA. In some embodiments, at least two of the first, second, third, and fourth promoters are the same. In some embodiments, each of the first, second, third, and fourth promoters are different. In some embodiments, each of the first, second, third and fourth promoters are selected from the H1 promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a sgRNA, the second sequence encoding a sgRNA, the third sequence encoding a sgRNA, and the fourth sequence encoding a sgRNA are identical. In some embodiments, the first sequence encoding a sgRNA, the second sequence encoding a sgRNA, the third sequence encoding a sgRNA, and the fourth sequence encoding a sgRNA are not identical. In some embodiments, the AAV vector comprises a first sequence encoding a sgRNA, a second sequence encoding a sgRNA, a third sequence encoding a sgRNA, a fourth sequence encoding a sgRNA, and a fifth sequence encoding a sgRNA, a first promoter drives expression of the first sequence encoding a sgRNA, a second promoter drives expression of the second sequence encoding a sgRNA, a third promoter drives expression of the third sequence encoding a sgRNA, a fourth promoter drives expression of the fourth sequence encoding a sgRNA, and a fifth promoter drives expression of the fifth sequence encoding a sgRNA. In some embodiments, at least two of the first, second, third, fourth, and fifth promoters are the same. In some embodiments, each of the first, second, third, fourth, and fifth promoters are different. In some embodiments, each of the first, second, third, and fourth promoters are different. In some embodiments, each of the first, second, third, fourth and fifth promoters are selected from 52 4860-0953-0567, v.1the H1 promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a sgRNA, the second sequence encoding a sgRNA, the third sequence encoding a sgRNA, the fourth sequence encoding a sgRNA, and the fifth sequence encoding a sgRNA are identical. In some embodiments, the first sequence encoding a sgRNA, the second sequence encoding a sgRNA, the third sequence encoding a sgRNA, the fourth sequence encoding a sgRNA, and the fifth sequence encoding a sgRNA are not identical. V. Pharmaceutical Compositions and Delivery Methods For clinical applications, pharmaceutical compositions are prepared in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. Appropriate salts and buffers are used to render drugs, proteins or delivery vectors stable and allow for uptake by target cells. Aqueous compositions of the present disclosure comprise an effective amount of the drug, vector or proteins, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is not incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions may be used. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions. In some embodiments, the active compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route, but generally including systemic administration. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into muscle tissue. Such 53 4860-0953-0567, v.1compositions would normally be administered as pharmaceutically acceptable compositions, as described supra. The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof. 54 4860-0953-0567, v.1In some embodiments, the compositions of the present disclosure are formulated in a neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine) and the like. Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards. In some embodiments, the nucleotide editing Cas9 and sgRNAs described herein may be delivered to the patient using adoptive cell transfer (ACT). In adoptive cell transfer, one or more expression constructs are provided ex vivo to cells which have originated from the patient (autologous) or from one or more individual(s) other than the patient (allogeneic). The cells are subsequently introduced or reintroduced into the patient. Thus, in some embodiments, one or more nucleic acids encoding nucleotide editing Cas9 and a guide RNA that targets a CaMKIIδ splice site are provided to a cell ex vivo before the cell is introduced or reintroduced to a patient. 55 4860-0953-0567, v.1VI. Definitions The term “nucleotide editing Cas9” refers to a Cas9 protein fused to a base editor. Non- limiting examples of Cas9 include SpCas9, SpCas9-NG, SpRY, SaCas9, SaCas9-KKH, SauCas9, and SlugCas9. Non limiting examples of a base editor include ABEmax, ABE8e, ABE8eV106W, ABE8.20-m. 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- 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 56 4860-0953-0567, v.1containing 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 (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), and Myo-AAV vectors described in Tabebordbar et al., 2021 (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., 2001; Naso et al., 2017, 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 57 4860-0953-0567, v.1capsid 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. In general, in the case of a DNA nucleic acid construct encoding a guide RNA, the U residues in any of the RNA sequences described herein may be replaced with T residues, and in the case of a guide RNA construct encoded by any of the DNA sequences described herein, the T residues may be replaced with U residues. Target sequences for Cas9s include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence’s reverse complement), as a nucleic acid substrate for a Cas9 is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a target sequence”, it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence. 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 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 58 4860-0953-0567, v.1may 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. 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 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 59 4860-0953-0567, v.1may 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 an 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”) 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. 60 4860-0953-0567, v.1A “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. 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 61 4860-0953-0567, v.1least 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 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 62 4860-0953-0567, v.1raised 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 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 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. 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. VII. Sequences Mouse CamKIIδ Gene and Protein Sequences 63 4860-0953-0567, v.1See Appendix A (SEQ ID NOs: 79 & 80). Human CaMKIIδ Gene and Protein Sequences See Appendix B (SEQ ID NOs: 81 & 82). VIII. 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. Example 1 - Methods RNA-sequencing data have been uploaded and deposited at Gene Expression Omnibus (world-wide-web at ncbi.nlm.nih.gov / geo / , accession number GSE227720). The authors declare that all other data are available within the article and its online supplementary files. An extended methods section can be found in the online supplemental material. Study design and approval. The aim of the present study was to ablate the autophosphorylation site of CaMKIIδ using CRISPR-Cas9 base editing as a new therapeutic strategy against heart failure. Several sgRNAs were tested in mouse N2a cells to identify the most efficient editing approach. The inventors found a sgRNA that is identical for the mouse and human DNA sequence and used it to generate a germline-edited c.A859G (p.T287A) mouse model that is resistant to CaMKIIδ autophosphorylation. The therapeutic effects of this amino acid mutation were evaluated in adult mice using severe transverse aortic constriction (sTAC) as a model for afterload-induced heart failure. The inventors then applied the same CRISPR-Cas9 base editing system to human induced pluripotent stem cells (iPSCs) to test whether ablation of the autophosphorylation site of CaMKIIδ could confer cardioprotection. The inventors performed all experiments in replicates. Male C57BL / 6 wild-type (WT) and homozygous T287A mice were randomly assigned to either sham or sTAC surgery at 10 weeks of age. Cardiac function was evaluated in each mouse one week before and one week after the surgery. Two weeks after the surgery, all mice were sacrificed. Five mice were 64 4860-0953-0567, v.1dedicated to further molecular and three mice to histological analyses. The sample size was not predetermined by statistical tests. All samples are included in this study with no data excluded. Animal work described in this manuscript has been approved and conducted under the oversight of the UT Southwestern Institutional Animal Care and Use Committee. Mice were housed and bred at the Animal Resource Center at the UT Southwestern Medical Center, a pathogen-free facility with a regular 12-h light / dark cycle, a temperature of 18–24° C, and a humidity of 35–60%. A maximum of 5 mice were housed in one cage with ad libitum access to food and water, and they were monitored daily for potential health problems. Human iPSCs were previously generated and used in the inventors’ laboratory (Chemello et al., 2021; Lebek et al., 2023). All iPSC work was performed in compliance with the UT Southwestern Stem Cell Research Oversight Committee. Statistical analysis. All data are reported as mean ± standard error of the mean (SEM). Shapiro-Wilk normality test was used to test for normal distribution. If a variable was not normally distributed or if the sample size was too small to assess normality, non-parametric tests were applied. Student’s t or Mann-Whitney test were used for the comparison of two groups that were either normally or not normally distributed, respectively. Two-way ANOVA with Holm-Sidak’s post-hoc correction was applied for the comparison of two genotypes (wild- type vs. T287A) that varied on intervention (sham / control vs. sTAC / ISO). Repeated measures two-way ANOVA with Holm-Sidak’s post-hoc correction was used for the comparison of two genotypes (wild-type vs. T287A) with two timepoints (before acute ISO vs. after acute ISO). The inventors refrained from performing post-hoc multiple comparisons when the two-way ANOVA was not significant. Log-rank (Mantel-Cox) test with Bonferroni’s post-hoc correction was used for the comparison of survival curves. Correlations were tested by linear regression analysis and categorial data by Fisher’s exact test. All statistical tests were performed using GraphPad Prism 9. The inventors considered two-sided p-values below 0.05 statistically significant. Plasmids and cloning. A pmCherry_gRNA plasmid with a U6-driven sgRNA scaffold and a cytomegalovirus (CMV)–driven pmCherry fluorescent protein (gift from Ervin Welker, Addgene plasmid #80457) was used to clone the sgRNAs (Table S1). NG-ABEmax (Addgene plasmid #124163) (Huang et al., 2019) and NG-ABE8e (Addgene plasmid #138491) (Richter et al., 2020) were gifts from David Liu. pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP (RTW5025) was a gift from Benjamin Kleinstiver (Addgene plasmid #140003) (Walton et al., 2020) and was adapted to obtain ABE8e-SpRY. Adaptions were performed by cloning oligonucleotides (IDT) or PCR products of appropriate template sequences (PrimeStar GXL 65 4860-0953-0567, v.1Polymerase, Takara) into restriction enzyme-digested vectors using NEBuilder HiFi DNA Assembly (NEB). Cell culture and transfection. Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) with 10% (v / v) fetal bovine serum (GeminiBio) was used to culture N2a cells (ATCC). Approximately 125,000 cells / well were plated onto 24-well plates (Corning) and cells were transfected after 24 h with plasmids expressing the sgRNA and adenine base editor using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were harvested after three days to determine the editing efficiency. Sanger sequencing analysis. Genomic DNA was isolated using DirectPCR cell lysis reagent (Viagen) with proteinase K (1 μg / μL) according to the manufacturer’s recommendations and DNA was amplified using PrimeSTAR GXL DNA polymerase (Takara) and the primers listed in Table S2. ExoSap-IT Express (Thermo Fisher Scientific) was used to clean the PCR product. Analysis of the Sanger chromatograms using the online tool EditR revealed the editing efficiency (Kluesner et al., 2018). Generation of T287A mice. ABE8e (50 ng / μL) and sgRNA2 (50 ng / μL) were injected into the pronucleus and cytoplasm of mouse zygotes to generate c.A859G (p.T287A) mice. NG-ABE8e plasmid and sgRNA2 (cloned into a pmCherry_gRNA plasmid) were transcribed in vitro using T7 RiboMAX™ Express Large Scale RNA Production System (Promega). For NG-ABE8e RNA, poly(A)-tailing was performed using E. coli Poly(A) Polymerase (NEB). RNA was purified using the Monarch® RNA Cleanup Kit (NEB). C57BL / 6 female mice (6 weeks old) were treated for superovulation and mated with C57BL / 6 stud males to induce zygote production. Zygotes were isolated, transferred to M16 (Brinster’s medium for ovum culture with 100 units / mL penicillin and 50 mg / mL streptomycin), and injected in M2 medium (M16 medium and 20 mM HEPES). After incubation in M16 medium for 1 h at 37º C, injected zygotes were transferred into the oviducts of pseudo-pregnant ICR female mice. Ear genomic DNA was extracted from F0 mice, and the edited CaMKIIδ gene segment was PCR-amplified (see primers in Table S2) before confirming the c.A859G (p.T287A) edit by Sanger sequencing. EditR was used to determine the editing efficiency in Sanger chromatograms (Kluesner et al., 2018). The inventors picked one founder mouse of the F0 generation that was homozygous for the c.A859G (p.T287A) edit with a low degree of mosaicism for the c.A857G (p.E286G) bystander edits and mated it to a C57BL / 6 mouse to generate mice heterozygous for the c.A859G (p.T287A) edit without a bystander mutation. A Custom TaqMan™ SNP Genotyping Assay (Thermo Fisher Scientific, Silencer™ Pre-Designed siRNA, catalog number 66 4860-0953-0567, v.1AM16704, siRNA-ID 184038) and KAPA PROBE FAST qPCR Kit - ROX Low (Roche) were used for genotyping. Male homozygous T287A mice were used for the experiments. Electrocardiograms (ECGs). After induction with 5% isoflurane, anesthesia was maintained with 1.5% isoflurane in O2using a face mask. Body temperature was continuously monitored and maintained within 37 ± 0.3° C using a rectal probe. Two alligator clip electrodes were attached at the upper and lower front of the murine chest to record a surface ECG (lead II) using the data acquisition system Power Lab / 4SP (ADInstruments) and Chart software (version 4.2.3). After a basal ECG registration, the inventors injected isoproterenol (ISO, 3 mg / kg bodyweight dissolved in 0.9% NaCl) intraperitoneally in each mouse. ECG signals were analyzed at baseline as well as 60 s after the injection of ISO using LabChart software (version 8.1.25) and its ECG Analysis plugin (version 2.4). All ECG registrations and analyses were performed by an experienced investigator who was blinded to the genotype of the respective mouse. Treadmill exhaustion test. Basal exercise capacity of WT and T287A mice was tested using an Exer-3 / 6 rodent treadmill with 10° inclination (Columbus Instrument), as described previously.9The electric shock grid at the rear end of the treadmill was set at a stimulation intensity of 10 and a frequency of 3 Hz. First, all mice were acclimated to the treadmill by performing 10 min sessions on three consecutive days with a treadmill velocity set to 0, 5, and 10 m / min for the first, second, and third day, respectively. On day 4, mice were subjected to a warm-up of 10 m / min for 2 min before the velocity was set to 15 m / min. The inventors then increased the velocity of the treadmill at a rate of 0.6 m / min per minute until the mouse was exhausted (continuous standing on the electrical shock grid for 5 s). After exhaustion, cardiac function was immediately analyzed by transthoracic echocardiography. Transthoracic echocardiography. Two-dimensional transthoracic echocardiography (Vevo2100 imaging system, VisualSonics) was used to evaluate cardiac function in conscious mice. The analysis is based on M-mode traces and three consecutive heart beats were averaged. Left ventricular end-diastolic (LVIDd) and end-systolic (LVIDs) internal diameter were determined. Fractional shortening (%) was calculated using the equation [(LVIDd – LVIDs) / LVIDd] × 100. Echocardiography was performed and analyzed one week before and one week after the sTAC surgery by the same experienced investigator, who was blinded to the study. Some WT and T287A mice were examined immediately after exhaustion on the treadmill 67 4860-0953-0567, v.1(without prior surgery). The inventors also analyzed basal cardiac function of 1-year-old WT and T287A mice. Severe transverse aortic constriction (sTAC) surgery. The inventors analyzed male mice in the C57BL / 6 background that received standard chow (2916 Teklad Global) and were housed in a standard mouse facility with a regular 12-h light / dark cycle. Since this is the first study investigating the effect of rendering CaMKIIδ phospho-resistant to prevent pathological CaMKIIδ activity, the inventors refrained from including the additional variable of gender at this point. At 10 weeks of age, sTAC surgery was performed in WT (n=26) and T287A (n=9) mice to model afterload-induced heart failure. Due to the heart failure-induced mortality in WT-sTAC, the inventors needed to dedicate a higher number of WT mice for sTAC surgery to obtain enough animals with complete follow-up monitoring for further molecular analyses. Mice were anesthetized with Ketamine / Xylazine mixture and a MiniVent mouse ventilator (Hugo Sachs Elektronik, 105 breaths / min, 250 μL stroke volume) was used after mice were intubated. Body temperature was monitored and kept close to 37.0° C using a rectal probe. Thoracotomy was performed at the second rib left parasternal to display the transversal aorta and a 28-gauge needle was tied against the aorta with a non-absorbable suture. After that, the needle was removed, the skin carefully closed in layers, and the mice kept on a heating plate to recover from anesthesia. Buprenorphine SR was used as post-operative painkiller. For sham surgery, the same procedure was performed in WT (n=8) and T287A (n=8) mice but with no banding of the aorta. Each surgery was performed in a standardized manner by the same experienced surgeon, who was blinded to the genotype of the mouse. Survival was monitored for 14 days before the mouse was euthanized for further molecular and histological analyses. Western blot analysis. Snap-frozen mouse hearts that were pulverized with a tissue crusher and human iPSC-derived cardiomyocytes were used for Western blot analyses. RIPA buffer (Sigma-Aldrich) supplemented with protease- and phosphatase-inhibitors (Roche) was used to isolate proteins and genomic DNA was broken by sonication with a Bioruptor Pico (Diagenode, 10 cycles of 30 s sonication on and 30 s off). After that, samples were centrifuged for 15 min at 10,000 x g at 4º C and the supernatant stored at -80º C. Protein concentration was determined using a BCA assay (Thermo Fisher Scientific) and equal amounts of protein were loaded on a Mini-PROTEAN® TGX™ gel (Bio-Rad). After transferring the proteins onto a polyvinylidene fluoride membrane (Millipore) and blocking in 5% milk with TBS-Tween 0.1%, the membrane was incubated overnight at 4º C with primary antibody: rabbit polyclonal anti-phospho-CaMKII (1:1,000, Invitrogen, catalog number PA5-37833), mouse monoclonal anti-CaMKII (1:1,000, BD Biosciences, catalog number 611293), or mouse monoclonal anti- 68 4860-0953-0567, v.1GAPDH (1:1,000, Sigma-Aldrich, catalog number MAB374). HRP-conjugated goat anti-rabbit (1:10,000, Bio-Rad, catalog number 1706515) and anti-mouse (1:10,000, Bio-Rad, catalog number 1706516) were used as secondary antibodies (incubation for one hour at room temperature). Western Blotting Luminol Reagent (Santa Cruz Biotechnology) was used to facilitate immunodetection on a ChemiDoc MP Imaging System (Bio-Rad) and mean densitometric analysis was performed with ImageJ. CaMKII activity assay. CaMKII activity was measured in snap-frozen hearts that were pulverized with a tissue crusher, and in human iPSC-derived cardiomyocytes. Samples were lysed in 1% (v / v) Triton X-100, 20 mM Tris, 100 mM NaCl supplemented with protease- and phosphatase-inhibitors (Roche) at pH 7.4. Lysates were centrifugated for 15 min at 10,000 x g at 4º C and equal volumes of the supernatant were loaded onto the CycLex® CaM-kinase II assay kit (MBL International Corporation). Absorbance was determined at 450 nm on a CLARIOstar microplate reader (BMG LABTECH). Using a standard curve of the CaM-kinase II Positive Control (MBL International Corporation), the inventors obtained the CaMKII activity of each sample that was normalized to the protein concentration of the lysate (BCA assay, Thermo Fisher Scientific). RNA sequencing. Snap-frozen cardiac mouse samples were homogenized in TRIzol (Thermo Fisher Scientific) with a Precellys Evolution homogenizer (Bertin Instruments, 3 cycles x 20 s at 6,800 rpm). RNA was isolated using the RNeasy Micro Kit (Qiagen). RNA sequencing libraries were prepared using the KAPA mRNA HyperPrep kit (Kapa Biosystems) according to the manufacturer's instructions. High output 75 cycles single-ended sequencing was performed with an Illumina NextSeq500 sequencer by the University of Texas Southwestern Medical Center’s CRI Sequencing Facility. Bioinformatic analysis of RNA sequencing data. The inventors used the FastQC tool (version 0.11.8) for quality control to determine low quality and adaptor portion of the reads for trimming. Trimming of the reads was performed with Trimmomatic (version 0.39) before the trimmed reads were aligned to the mouse reference genome (mm10) using HiSAT2 (version 2.1.0, default settings). Using the program R (version 3.5.1), the raw count matrix for each sample was obtained by counting aligned reads with the tool featureCounts (version 1.6.2). This count matrix was used to identify differentially expressed genes with the DESeq package (version 1.38.0). For the principal component analysis (PCA), the inventors normalized the raw counts of all samples using the rlog function in R, which was then used as input for the prcomp function. This revealed the PC1 and PC2 scores, which were visualized in a scatter plot using the ggplot2 package. For the analysis of enriched gene sets, they used differentially expressed 69 4860-0953-0567, v.1genes with an adjusted p-value below 0.05 (Benjamin-Hochberg correction) and a fold-change of expression of at least 2. Gene Ontology terms indicating up- and downregulated pathways were identified using Metascape (metascape.org) (Zhou et al., 2019). Histology and immunohistochemistry. Mouse hearts were dissected and rinsed in phosphate-buffered saline (PBS) supplemented with cardioplegic 0.2 M KCl before fixating in 10% neutral-buffered formalin (Sigma-Aldrich) at room temperature overnight. Then, 70% ethanol was used to dehydrate samples that were embedded in paraffin. The inventors used 4- chamber-view sections cut at the mitral valve for routine H&E, trichrome, and picrosirius red staining. A BZ-X700 microscope (Keyence) was used to capture images (10x magnification). The percentage of fibrotic tissue was calculated by dividing the collagen positive area by the total area of the heart using ImageJ. To analyze apoptotic cells, terminal deoxynucleotidyltransferase-mediated UTP end label (TUNEL) and immunohistochemical staining for troponin I were performed on the same section. Sections were deparaffinized in xylene. Antigen retrieval was done for 20 min heating in 1 mM EDTA (pH 8.0), before subjecting the sections to 0.3% Triton X-100 with interceding PBS washes throughout the protocol and blocking in 3% normal goat serum. Rabbit polyclonal anti-troponin I was used as primary antibody (1:100, Santa Cruz Biotechnology, catalog number H-170) and incubated at 4° C overnight. Thereafter, sections were incubated with secondary antibody (Cy3-conjugated goat anti-rabbit, 1:50, Jackson ImmunoResearch, catalog number 111-165-144) at room temperature for 30 min. Linked antigen, primary, and secondary antibodies in the sections were crosslinked using 4% paraformaldehyde. Then, DeadEnd™ Fluorometric TUNEL System (Promega) was performed according to the manufacturer's recommendations and sections were further incubated with Hoechst 33342 (1:5,000, Invitrogen, catalog number H3570) at room temperature for 5 min. Images were captured on a LSM 800 confocal microscope (Zeiss) and apoptotic (TUNEL positive) cells were reported as percentage of the total number of cells (Hoechst 33342 positive). Culture and nucleofection of human iPSCs. Matrigel (Corning)-coated 6-well polystyrene culture plates were used to culture human iPSCs (previously generated and used in the inventors’ laboratory) (Chemello et al., 2021; Lebek et al., 2023) in mTeSRTM1 media (STEMCELL). Cells were passaged at 70-80% confluency using Versene (Thermo Fisher Scientific). For nucleofection experiments, the inventors used about 8x105iPSCs that were subjected one hour before to 10 μM ROCK inhibitor (Y-27632, Selleckchem). Accutase (Innovative Cell Technologies) was used to obtain single cell status and iPSCs were mixed with 1.5 μg of pmCherry_gRNA plasmid carrying sgRNA2 and 4.5 μg NG-ABE8e plasmid. 70 4860-0953-0567, v.1Nucleofection was performed using P3 Primary Cell 4D-Nucleofector X Kit (Lonza) according to the manufacturer’s protocol. After that, ROCK inhibitor (10 μM) and Primocin (100 μg / mL) (InvivoGen) were added to the culture media for one day. Cells were clonally expanded to establish an iPSC line corresponding to the editing pattern of sgRNA2. Deep amplicon sequencing analysis. The inventors used deep amplicon sequencing to determine the editing efficiency for the CaMKIIδ gene in human iPSCs nucleofected with ABE8e and sgRNA2. They further assessed potential off-target editing of other CaMKII isoforms (α, β, and γ) and of the top 8 candidate off-target sites, as predicted by the cutting frequency determination (CFD) score of CRISPOR (Table S3) (Concordet & Haeussler, 2018). Genomic DNA was isolated using DNeasy Blood & Tissue Kit (Qiagen). Target sites were PCR-amplified using PrimeStar GXL Polymerase (Takara) with the primers listed in Table S4. Illumina flow cell binding sequences and barcodes were added in a second PCR round. The PCR products were purified using AMPure XP Beads (Beckman Coulter), tested for integrity on a 2200 TapeStation System (Agilent), and DNA concentration was measured by QuBit dsDNA high-sensitivity assay (Invitrogen). The samples were then pooled and analyzed by an Illumina MiSeq. After sequencing, the samples were demultiplexed and the amplicon reads analyzed for editing efficiency using CRISPResso2 (Clement et al., 2019). The formal percentage of adenine to guanine editing was reported for each adenine along the on- and off- target DNA sequences. Differentiation of human iPSCs into cardiomyocytes. Human iPSCs at 70-80% confluency were differentiated into cardiomyocytes by replacing iPSC culture media with RPMI (Thermo Fisher Scientific) supplemented with CHIR99021 (Selleckchem), ascorbic acid (50 µg / mL, Sigma-Aldrich), and B27 without insulin (Invitrogen, RPMI / B27-, day 0). After 24 h, the media was replaced with RPMI / B27- for 2 days and again with RPMI / B27- supplemented with WNT-C59 (Selleckchem) for another 2 days before refreshing with RPMI / B27-. Beginning on day 7, iPSC-cardiomyocytes were cultured in RPMI supplemented with ascorbic acid (50 µg / mL) and B27 (Thermo Fisher Scientific, RPMI / B27). From day 10 onwards, cells were cultured in RPMI without glucose (Thermo Fisher Scientific) but supplemented with 5 mM sodium DL-lactate (Sigma-Aldrich) and CDM3 supplement (500 μg / mL Oryza sativa-derived recombinant human albumin, Sigma-Aldrich) to metabolically select for cardiomyocytes.5 days later, the media was replaced with RPMI / B27. Beginning on day 20, the media was refreshed every day with either RPMI / B27 (control) or RPMI / B27 supplemented with 100 nM isoproterenol (ISO, Sigma-Aldrich). Cardiomyocytes were 71 4860-0953-0567, v.1analyzed after another 10 days, and the inventors collected data from at least three independent differentiations for each experiment. Analysis of cellular Ca2+characteristics. Cellular Ca2+transients were analyzed using epifluorescence microscopy. Therefore, the inventor replated human iPSC-derived cardiomyocytes (iPSC-CMs) on a glass bottom microwell dish (MatTek Corporation) to a single cell density. After 10 days in RPMI / B27 (with or without ISO, see above), cardiomyocytes were loaded with 5 μM Fura-2 AM (20 min at 37º C, Invitrogen) and mounted on an inverted microscope (Motic AE31E). A fluorescence detection system (IonOptix) was used to measure Fura-2 fluorescence emission ratio by switching excitation at 340 nm and 380 nm (switching rate 1,000 Hz). Before the experiment started, cardiomyocytes were incubated for 15 min with Tyrode’s solution (140 mmol / L NaCl, 4 mmol / L KCl, 1 mmol / L MgCl2, 10 mmol / L HEPES, 10 mmol / L glucose, 1.25 mmol / L CaCl2, at pH7.4) to ensure de-esterification of intracellular Fura-2 AM. Ca2+transients were obtained at a steady-state status by electrical field stimulation (0.25 Hz with a 5 ms pulse of 30 V). IonWizard 6.0 analysis software (IonOptix) was used to analyze Ca2+transient characteristics and the occurrence of arrhythmias. To further analyze cellular Ca2+characteristics, the inventors paused the electrical stimulation for 10 s and compared the Ca2+transient after pause with the one before pause. The post-pause cytosolic Ca2+overload was calculated by subtracting the diastolic Ca2+of the last electrically stimulated Ca2+transient before pause from the Ca2+level at the end of the pause. The gain of the post-pause Ca2+transient amplitude was calculated by subtracting the Ca2+transient amplitude from the last electrically stimulated Ca2+transient before pause from the first one after pause. To test the response to acute β-adrenergic stimulation, WT and T287A control iPSC-CMs were acutely subjected to 100 nM ISO. The ISO-induced gain of Ca2+transient amplitude was calculated by subtracting the basal steady-state Ca2+transient amplitude from the post-ISO steady-state Ca2+transient amplitude. Example 2 – Results Development of a gene editing strategy to ablate the autophosphorylation site of CaMKIIδ. Threonine-287, encoded by an ACT codon, is the autophosphorylation site of CaMKIIδ responsible for pathogenic activation of the enzyme. The adenine c.A859 is potentially editable to a guanine using ABE, which would convert the ACT to a GCT codon, resulting in replacement of threonine with alanine, thus rendering CaMKIIδ resistant to autophosphorylation (FIG.1A). 72 4860-0953-0567, v.1To identify an optimal ABE strategy, the inventors tested several editing strategies in mouse N2A cells. As the optimal editing window for a specific nucleotide is often difficult to predict, they tested seven different sgRNAs (Table S1) that place CaMKIIδ c.A859 in protospacer positions 13-19 (with the first nucleotide immediately 5’ of the PAM sequence counted as position 1). The inventors screened different engineered deaminases, including ABEmax, an optimized narrow-windowed ABE7.10 variant (Koblan et al., 2018), and ABE8e, a wide-windowed evolved ABE7.10 variant with a 590-fold increased activity (Richter et al., 2020). The engineered deaminase variants were fused to the engineered SpCas9 variants SpCas9-NG (targeting NG PAMs)40and SpRY (targeting NRN and to a lesser extent NYN PAMs) (Walton et al., 2020). Overall, the deaminase ABE8e displayed higher c.A859G (p.T287A) editing efficiency than ABEmax (FIG. 1B and Tables S1 and S2). Three sgRNAs, sgRNA2, sgRNA3, and sgRNA5, showed similar c.A859G (p.T287A) editing efficiency of ~35-39%. For subsequent studies, the inventors selected sgRNA2 combined with ABE8e fused to SpCas9-NG, which showed a mean editing efficiency of 38.7±0.7% at adenine-16 within the targeted genomic region (FIGS. 1B-C). They chose this editing strategy since sgRNA2 has complete sequence identity for mouse and human genomes. Plus, using the SpCas9-NG variant with more stringent PAM requirements was expected to have less potential off-target editing. Using this gene editing strategy, the inventors injected zygotes of C57BL / 6 wild-type (WT) mice with sgRNA2 combined with ABE8e fused to SpCas9-NG and transferred them into the oviducts of pseudo-pregnant female mice. Genotyping of the founder F0litters (n=52 mice) revealed a mean c.A859G (p.T287A) editing efficiency of 59.8±4.6% (FIG. 1D). The inventors also observed base editing at adenine-18, which would create a glutamic acid to glycine substitution at residue 286 and a silent bystander edit at adenine-11. Sequencing of CaMKIIα, β, and γ genes revealed no editing of these CaMKII isoforms (FIGS. 7A-C). After backcrossing with C57BL / 6 wild-type mice, the inventors obtained heterozygous c.A859G (p.T287A) mice without bystander mutations that were used for further breeding. The homozygous edit was successfully transcribed into cDNA (FIG. 1E). At baseline, cardiac contractility, chamber size, and left ventricular mass were similar in WT and T287A mice with no significant differences between the groups (FIGS. 8A-D). In addition, T287A mice showed ECGs similar to WT mice at baseline (FIGS.9A-F). Following acute β-adrenergic stimulation, they showed an increase in heart rate comparable to WT mice (FIGS. 9A-F). When exercise capacity was evaluated by a treadmill exhaustion test, the inventors found no significant differences between WT and T287A mice with respect to 73 4860-0953-0567, v.1maximal velocity (p=6.9x10-1) or total distance (p=6.9x10-1) achieved on the treadmill (FIGS. 10A-C). All mice were subjected to transthoracic echocardiography immediately after exhaustion on the treadmill, which revealed no significant differences in cardiac function or geometry between both genotypes (FIGS.10D-H). The inventors further analyzed 1-year-old WT and T287A mice and found a similar cardiac function and geometry in both groups (FIGS. 11A-E). T287A mice are protected from afterload-induced heart failure. Male WT and phospho-resistant T287A mice at 10 weeks of age were subjected to severe transverse aortic constriction (sTAC) surgery to test whether ablation of CaMKIIδ autophosphorylation protects mice from afterload-induced heart failure (FIG. 2A and FIG. 12). This severe heart failure model has a high mortality rate in WT mice, which enabled subsequent survival analysis of the mice. WT mice subjected to sTAC (WT-sTAC) showed a dramatically increased mortality compared to WT-Sham mice (FIG.2B). Within the first and second week post-sTAC, 13 of 26 (50.0%) and 4 of 13 (30.8%) WT-sTAC mice died, respectively (FIG.2B). In contrast, T287A- sTAC mice were protected from heart failure-induced mortality as only one of 9 mice (11.1%) died within the two-week follow-up period (FIG. 2B). Accordingly, echocardiography one- week post-sTAC showed an ~75% decrease in fractional shortening from 60.1±0.7% in WT- Sham mice to 14.3±1.5% in WT-sTAC mice (p=8.0x10-12) (FIGS.2C-D). Importantly, T287A- sTAC mice showed improved fractional shortening of 33.9±6.5% (p=8.7x10-5vs. WT-sTAC) (FIG. 2D). The inventors also observed other key features of heart failure in WT-sTAC mice, including increased left ventricular end-diastolic diameter and volume and increased left ventricular mass (FIGS. 2E-G). These pathological responses were significantly attenuated in T287A-sTAC mice in which CaMKIIδ was rendered phospho-resistant. As expected, the inventors observed a >2-fold increase in CaMKII autophosphorylation in hearts of WT-sTAC mice compared to WT-Sham mice (p=8.6x10-4) (FIGS.2H-J). Because the inventors ablated the autophosphorylation site of CaMKIIδ, they only detected a residual signal of autophosphorylated CaMKII in T287A mice. This low signal might either be attributable to other isoforms (e.g., CaMKIIγ) or unspecific background. Accordingly, they found substantially increased CaMKII activity in WT-sTAC mice compared to WT-Sham mice, which was reduced by 5.8-fold in T287A-sTAC mice (p=5.6x10-4vs. WT-sTAC) (FIG. 2K). Mechanisms of cardioprotection conferred by CaMKIIδ editing. Two weeks after sTAC / sham surgery, mice were euthanized, and the hearts were harvested for histological and molecular analyses. As frequently observed in patients with critical illness, WT mice showed 74 4860-0953-0567, v.1a significant reduction in body weight two weeks post-sTAC, which was not observed in T287A mice (FIG.13A). Macroscopically and microscopically, the inventors observed cardiac dilation and hypertrophy in WT-sTAC mice, which was less pronounced in T287A mice following sTAC (FIGS. 3A-C). Accordingly, WT-sTAC mice showed significantly increased heart and lung weights compared to WT-Sham mice, further indicative of cardiac hypertrophy and heart failure (FIG. 13B-C). Notably, T287A-sTAC mice were protected from both pathological alterations and their heart and lung weights were comparable to sham control mice. Additionally, the inventors found cardiac fibrosis and myocardial infiltration of inflammatory cells in WT but not in T287A mice post-sTAC (FIG. 3C and FIGS. 14A-B). Quantification revealed a 1.8-fold increased area of fibrotic tissue in WT-sTAC compared to WT-Sham mice (p=2.3x10-4), while T287A-sTAC mice showed low levels of fibrosis (p=1.9x10-4vs. WT-sTAC) (FIG.14B). In accordance with myocardial fibrosis, the inventors detected substantially increased apoptosis in WT but not in T287A mice post-sTAC (FIGS. 3DE). Compared to WT-Sham mice, they found a 15.7-fold increased percentage of apoptotic cells in WT-sTAC mice (p=2.6x10-5) (FIG. 3E). In contrast, T287A-sTAC mice showed only a slight and non-significant increase in apoptotic cells compared to T287A-Sham mice (FIG. 3E). The inventors then performed RNA sequencing of whole hearts to investigate transcriptomic changes. Principal component analysis (PCA) revealed three distinct transcriptomic profiles. While both WT and T287A sham-treated mice were similar, WT mice subjected to sTAC showed a substantially different cardiac transcriptome (FIG. 4A). Importantly, T287A mice subjected to sTAC formed a third transcriptomic cluster that was closer to the healthy sham control mice than to WT-sTAC mice (FIG. 4A). In WT mice, the inventors found a total of 5,994 genes differentially expressed two weeks after sTAC (3,171 genes up- and 2,823 genes downregulated in WT-sTAC vs. WT-Sham). Compared to WT- sTAC, there were 3,787 differentially expressed genes in T287A-sTAC hearts (1,787 genes up- and 2,000 genes downregulated in T287A-sTAC). For the subsequent analysis of highly enriched gene sets, the inventors only considered at least 2-fold differentially expressed genes (FIG. 4B). Gene Ontology analysis of the 1,381 highly upregulated genes in WT-sTAC compared to WT-Sham hearts revealed pathways related to pathological remodeling and inflammation (FIGS. 4C-D). Additionally, the 601 substantially (fold-change >2) downregulated genes in WT-sTAC hearts were mainly linked to cardiac performance and metabolism (FIGS. 4C and 4E). Importantly, rendering CaMKIIδ phospho-resistant protected hearts from these pathological transcriptomic changes. Compared to WT-sTAC mice, the 264 75 4860-0953-0567, v.1highly upregulated (fold-change >2) genes in T287A-sTAC hearts were mainly linked to cardiac function, performance, and metabolism (FIGS. 4F-G). In contrast, the 496 genes that were substantially (fold-change >2) downregulated in T287A-sTAC vs. WT-sTAC hearts were mainly related to cardiac remodeling and inflammation (FIG.4F and 4H). Editing efficiency and analysis of potential off-target DNA editing in human iPSCs. The inventors next tested whether this editing approach was also feasible in the human genome and could thus potentially be therapeutically applicable. Therefore, human iPSCs were nucleofected with sgRNA2 and ABE8e fused to SpCas9-NG. This revealed a similar editing pattern as observed in the mouse genome with a mean c.A859G (p.T287A) editing efficiency of 48.4±1.0% (FIG.5A). Since other CaMKII isoforms are highly expressed in several organs other than the heart, where they are involved in important processes such as brain and skeletal muscle function, specificity of the gene editing approach for the CaMKIIδ isoform is important (Nassal et al., 2020; Pellicena & Schulman, 2014; Wang et al., 2021). Notably, the inventors observed no significant editing of any other CaMKII isoforms, as determined by deep amplicon sequencing (FIGS. 5B-E). They found a ~2306-, ~3267-, ~2254-fold increased c.A859G (p.T287A) editing efficiency for CaMKIIδ compared to CaMKIIα, β, and γ, respectively (FIG. 5F). Furthermore, the inventors analyzed the top 8 predicted genomic sites for potential off- target editing, as predicted by the bioinformatic tool CRISPOR (FIG. 5B and Tables S3 and S4) (Concordet & Haeussler, 2018). They detected minimal to no off-target editing (less than 1.2%) at any adenines within the top 8 predicted off-target sites (FIG.5G). None of these sites represent a coding region in the genome, making even minimal off-target editing of these sites less likely to be detrimental. ABE-treated iPSC-CMs are protected from chronic β-adrenergic stress. To test whether treatment with sgRNA2 and ABE8e fused to SpCas9-NG confers cardioprotection in human cardiomyocytes, the inventors established an iPSC-line using the same ABE strategy as in mice (FIG. 5A) and differentiated them into cardiomyocytes (iPSC-CMs). Sequencing of the cDNA revealed successful transcription of the editing pattern of sgRNA2 (homozygous for c.A859G (p.T287A) and heterozygous for both c.A857G (p.E286G) and c.A864G (p.V288V)) (FIG.6A). Notably, T287A iPSC-CMs were still responsive to acute β-adrenergic stimulation (FIGS. S9A-G). To test for potential cardioprotection, WT and T287A iPSC-CMs were treated with either normal control medium or with 100 nM isoproterenol for 10 days (ISO) to mimic chronic β-adrenergic stress as it occurs in heart failure. As anticipated, Western blot analyses revealed 76 4860-0953-0567, v.1a 2.9-fold increase in autophosphorylated CaMKII in WT iPSC-CMs upon chronic treatment with ISO (0.60±0.18) compared to control (0.21±0.05, p=8.9x10-3) (FIGS. 6B-D). In T287A- edited iPSC-CMs, ISO treatment did not increase the level of CaMKII autophosphorylation and only minimal autophosphorylation signal was detected. This residual signal may result from non-edited CaMKIIγ (which is also expressed in cardiomyocytes) or unspecific background. In accordance with an expected increase in CaMKII autophosphorylation with ISO treatment, CaMKII activity increased from (in nmol / min / mg) 4.8±0.5 in WT-Control iPSC-CMs to 12.5±3.8 upon treatment with ISO (p=1.2x10-2, FIG. 6E). Compared to WT iPSC-CMs, the T287A iPSC-CMs showed a 4.3-fold lower CaMKII activity upon ISO stimulation (p=5.5x10-3) (FIG.6E). Stimulated Ca2+transients were measured to assess cellular Ca2+homeostasis in WT and T287A iPSC-CMs upon control and chronic ISO treatment (FIG. 6F). The inventors detected no differences in diastolic Ca2+levels (FIG. 6G). However, chronic ISO treatment decreased Ca2+transient amplitude from (in ΔF340 / F380) 0.55±0.03 to 0.37±0.03 in WT iPSC- CMs (p=2.0x10-5) (FIG. 6H). Compared to WT iPSC-CMs, the T287A iPSC-CMs showed an increased Ca2+transient amplitude of 0.75±0.03 upon control conditions (p=1.4x10-6vs. WT- Control) and were resistant to ISO-induced deterioration of cellular Ca2+homeostasis. Similarly, relaxation times to both 50% and 80% of diastolic Ca2+levels were decreased in WT-ISO iPSC-CMs but not in T287A-ISO iPSC-CMs (FIGS. 6I-J). ISO treatment of WT iPSC-CMs increased the risk for arrhythmias by ~7.2-fold, resulting in 68.4% of iPSC-CMs showing arrhythmias (p=2.0x10-4) (FIG.6K). The proarrhythmic risk upon ISO treatment was reduced by ~6.5-fold when CaMKIIδ was rendered phospho-resistant (p=6.3x10-4for T287A- ISO vs. WT-ISO). Analyses of Ca2+characteristics after paused electrical stimulation have previously been used to estimate diastolic sarcoplasmic reticulum Ca2+leak, a key feature of cardiac dysfunction (Lebek et al., 2020; 2018). After 10 s of paused stimulation, the inventors found increased cytosolic Ca2+overload in WT iPSC-CMs upon chronic ISO (p=5.3x10-6vs. WT- Control), but not when the CaMKIIδ autophosphorylation site was ablated (FIG.16A-B). This cytosolic Ca2+overload indicates that there might be less Ca2+in the sarcoplasmic reticulum and thus less Ca2+available for the next contraction (Neef et al., 2010; Lebek et al., 2020; Fischer et al., 2014; Lebek et al., 2018). Indeed, the gain of post-pause Ca2+transient amplitude was impaired by ~1.9-fold in WT iPSC-CMs upon chronic ISO compared to control iPSC- CMs (p=8.6x10-6) (FIG. 16C). Upon control conditions, T287A iPSC-CMs showed an increased gain of post-pause Ca2+transient amplitude compared to WT (p=8.4x10-3), which 77 4860-0953-0567, v.1was resistant to ISO-induced impairment (FIG. 16C). The post-pause cytosolic Ca2+overload correlated significantly negatively with the gain of post-pause Ca2+transient amplitude (FIGS. 16D-E). Moreover, the inventors also found a negative correlation between the post-pause cytosolic Ca2+overload and the stimulated steady-state Ca2+transient amplitude (FIGS. 16F- G), further validating the inventors’ experimental approach. Interestingly, there was a clear separation between control- and chronic ISO-treatment only in WT but not in T287A iPSC- CMs, further indicating that ablating CaMKIIδ autophosphorylation renders cardiomyocytes resistant to chronic β-adrenergic stress. Table S1. Sequences of sgRNAs tested in this study. Sequences of sgRNAs 2- 7 are identical in the human and mouse genome. sgRNA SequenceSEQID NOPAMTable S2. Primers used for Sanger sequencing analyses. Target Primer SequenceSEQ ID NO78 4860-0953-0567, v.1Table S3. Analysis of potential off-target editing sites in the human genome, as predicted by CRISPOR. #Gene Locus Sequence SEQCFD- ID NOPAMScore79 4860-0953-0567, v.1Table S4. Primers for deep amplicon sequencing in the human genome. SE Target Primer Sequence Q D O 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 480 4860-0953-0567, v.1Revers GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTCTGTCCCGTGA 75 e TGTTGA F 76 781 4860-0953-0567, v.1Example 3 – Discussion In the present study, the inventors screened several sgRNAs and ABEs to identify an optimal base editing strategy capable of rendering mouse and human CaMKIIδ resistant to autophosphorylation. Using this cross-species editing strategy, they created a mouse model lacking the autophosphorylation site of CaMKIIδ, which conferred cardioprotection against afterload-induced heart failure following sTAC, resulting in improved survival. Additionally, the inventors showed that CaMKIIδ-edited human iPSC-CMs were protected against chronic β-adrenergic stress. CRISPR-Cas9 base editing can precisely convert one nucleotide to another without inducing DNA double-stranded breaks (Liu & Olson, 2022; Chemello et al., 2021; Gaudelli et al., 2017; Komor et al., 2016; Koblan et al., 2018; Olson, 2021). To date, most gene editing strategies have been designed to correct monogenic disease-causing mutations (Liu & Olson, 2022; Chemello et al., 2021; Chai et al., 2023; Richter et al., 2019; Amoasii et al., 2018; 2019; Koblan et al., 2021; Atmanli et al., 2021). However, the frequency of these mutations is typically low, which prevents broad application of this strategy. The autophosphorylation site of CaMKIIδ represents a promising therapeutic target for a wide range of cardiovascular disorders and base editing can be used to ablate this critical site. The concept of blocking pathogenic signaling by CRISPR-Cas9 gene editing could also be extended to other human diseases with other pathomechanisms. CaMKIIδ is a key regulator of cardiac function and signaling, while chronic overactivation promotes cardiac disease and dysfunction (Beckendorf et al., 2018; Maier, 2009; Neef et al., 2010; Backs et al., 2009; Lebek et al., 2020; Zhang et al., 2004; Ling et al., 2013; Luo et al., 2013; Nassal et al., 2020; Pelllicena & Schulman, 2014; Mustroph et al., 2018; Fischer et al., 2014; Toischer et al., 2010). The enzyme consists of three domains, namely the catalytic, regulatory, and association domains (Beckendorf et al., 2018). Ca2+ / calmodulin binding to the regulatory domain induces a conformational change, which exposes the catalytic domain and activates the enzyme (Beckendorf et al., 2018). Besides Ca2+ / calmodulin binding, CaMKIIδ is also subject to posttranslational modification of several amino acids in the regulatory domain. Autophosphorylation of threonine-287 (Meyer et al., 1992; Simon et al., 2021), oxidation of methionine-281 and methionine-282 by reactive oxygen species (Erickson et al., 2008), O-linked N-acetylglucosaminylation of serine-280 during hyperglycemia (Hegyi et al., 2021; Erickson et al., 2013), and nitrosylation of cysteine-290 through nitric oxide-dependent signaling (Erickson et al., 2015) have been identified. 82 4860-0953-0567, v.1Posttranslational modification of these amino acid residues prevents reassociation of the autoinhibitory region with the catalytic domain resulting in sustained CaMKII hyperactivation and cardiac disease. The functional relevance and importance of each individual posttranslational modification site compared with the other sites remains to be determined. CaMKII autophosphorylation of threonine-287 has been linked to several cardiac diseases and has been used as a measure of total CaMKII activity (Fischer et al., 2014; Toischer et al., 2010; Lebek et al., 2018; Meyer et al., 1992; Simon et al., 2021; Liubojevic-Holzer et al., 2020). However, the impact of genetic ablation of the autophosphorylation site of CaMKIIδ on cardiac function has not yet been explored. Analyzing a phospho-resistant CaMKIIδ mouse model allows deeper insights into the mechanisms of CaMKIIδ-dependent signaling in cardiac disease. Previous studies showed a correlative association between CaMKIIδ autophosphorylation and cardiac disorders. Here, the inventors analyzed a phospho-resistant CaMKIIδ mouse model and provide direct evidence for the role of CaMKIIδ-dependent signaling in heart disease (Fischer et al., 2014; Toischer et al., 2010; Lebek et al., 2018; Liubojevic-Holzer et al., 2020). These findings show that mice harboring a phospho-resistant CaMKIIδ display improved survival and cardiac function in heart failure. It has previously been shown that autophosphorylation increases the binding affinity of CaMKIIδ for calmodulin and slows calmodulin dissociation from CaMKIIδ in isolated rabbit cardiomyocytes following adenoviral transduction of either WT, phospho-mimetic (T287D) or phospho-resistant (T287A) CaMKIIδ (Simon et al., 2021).27Intracardiac njection of adeno- associated virus serotype 9 expressing the CaMKIIδ T287A mutant was found to attenuate myocardial injury after ischemia / reperfusion in rats (Lu et al., 2020). However, viral overexpression only resulted in ~60-125% expression over the endogenous CaMKIIδ (Lu et al., 2020; Simon et al., 2021). This indicates the existence of a mixed population retaining ~31- 63% WT CaMKIIδ. Additionally, CaMKII overexpression itself promotes cardiac disease, making these studies difficult to interpret (Maier et al., 2003; Zhang et al., 2003). The inventors show that complete ablation of the CaMKIIδ autophosphorylation site in mice prevents pathogenic activation of the enzyme, but does not affect overall CaMKII expression nor does this mutation have adverse consequences. As enhanced CaMKIIδ signaling is an established driver of cardiac disease, CaMKIIδ inhibition has been identified as a promising therapeutic target (Nassal et al., 2020; Pellicena & Schulman, 2014; Lebek et al., 2018; Neef et al., 2018; 2017). However, to date, there are no clinically available enzyme inhibitors (Nassal et al., 2020). KN93, a small-molecule allosteric inhibitor, has been extensively studied and shown to improve cardiac function in different 83 4860-0953-0567, v.1disease models (Nassal et al., 2020; Pellicena & Schulman, 2014). However, the usefulness of KN93 is limited by relatively low potency and off-target effects on a number of ion channels, such as potassium channels (Hegyi et al., 2015). Other compounds, like autocamtide-2 related inhibitory peptide, have poor bioavailability and ATP-competitive inhibitors also inhibit other kinases (Nassal et al., 2020; Pellicena & Schulman, 2014). Hesperadin, an ATP-competitive CaMKII inhibitor, targets both CaMKIIδ and Aurora B kinase, conferring cardioprotection and antitumor effects (Zhang et al., 2022). Two other ATP-competitive CaMKII inhibitors, RA608 and RA306, have recently been demonstrated to improve cardiac function in vivo upon afterload-induced heart failure and in a mouse model of dilated cardiomyopathy, respectively (Mustroph et al., 2020; Beauverger et al., 2020). However, the IC50of both compounds for CaMKIIδ was only 4-6-fold lower than for CaMKIIα, ~0.5-fold compared to CaMKIIγ, and RA608 showed 70% inhibition of CaMKIIβ (Mustroph et al., 2020; Beauverger et al., 2020). The lack of CaMKIIδ isoform specificity represents a major challenge in the development of CaMKII inhibitors (Nassal et al., 2020; Pellicena & Schulman, 2014). While CaMKIIα and β are highly expressed in the brain, where they are critically involved in learning and memory, CaMKIIγ has been linked to exercise performance in skeletal muscle (Nassal et al., 2020; Pellicena & Schulman, 2014; Wang et al., 2021). This highlights the necessity of specifically targeting CaMKIIδ, which is the main CaMKII isoform involved cardiac pathology (Neef et al., 2010; Backs et al., 2009; Lebek et al., 2020; Zhang et al., 2004; Ling et al., 2013; Luo et al., 2013; Nassal et al., 2020; Pelllicena & Schulman, 2014; Mustroph et al., 2018; Fischer et al., 2014; Toischer et al., 2010; Lebek et al., 2010). The inventors recently developed a CRISPR-Cas9 gene editing strategy to ablate the oxidative activation sites of CaMKIIδ (Lebek et al., 2023). They found that rendering CaMKIIδ resistant to oxidative activation represents a promising therapeutic strategy to ameliorate direct consequences of oxidative stress (as in ischemia / reperfusion injury) (Lebek et al., 2023). In addition to oxidation, autophosphorylation is a major activator of CaMKIIδ and has been shown to promote various cardiac disorders. Future work that compares targeting the different posttranslational modification sites of CaMKIIδ will help to identify the most comprehensive and beneficial therapeutic approach. Therefore, in the present study, the inventors have developed a new ABE strategy to genetically ablate the autophosphorylation site of CaMKIIδ. The optimal sgRNA identified in this study showed ~2306-, ~3267-, and ~2254-fold specificity for c.A859G (p.T287A) editing of CaMKIIδ compared to CaMKIIα, β, and γ, respectively. This sgRNA specificity is higher than the inventors’ previously reported gene editing strategy for the oxidative activation sites of CaMKIIδ, which represents an important safety feature (Lebek 84 4860-0953-0567, v.1et al., 2023). Moreover, here the inventors present a concept that improves survival in heart failure. Rendering CaMKIIδ resistant to autophosphorylation might be applicable to a broader range of cardiac diseases than ablating the oxidative activation sites, but this remains to be tested. Notably, using CRISPR-Cas9 gene editing to ablate the autophosphorylation site of CaMKIIδ only prevents pathological overactivation and does not block basal enzyme activity. Since the Ca2+ / calmodulin binding site is not targeted, CaMKIIδ can still exert its physiological function as a cellular Ca2+sensor. Moreover, the inventors detected no off-target editing within a coding region of the top 8 predicted genomic sites for potential off-target editing, reducing the risk of potential adverse side effects. Permanent modification of an otherwise normal gene also raises questions from an ethical perspective. One may argue that this approach can be justified when traditional and currently available therapeutic strategies are ineffective or not well-tolerated. Permanent modification of a specific pathogenic signaling cascade may be justified especially for chronic disorders (e.g., chronic heart failure), where permanent perturbation of a pathomechanism is sustained for many years causing organ failure. It will be imperative to carefully assess safety to understand the long-term consequences of the editing strategy, as such a modification would be permanent. In this study, the inventors did not detect overt adverse effects in the basal phenotype of mice harboring the T287A mutation. In the context of personalized medicine, it will be critical to identify the optimal gene to edit for each group of patients, which depends on the predominant pathomechanism of each disease, the editability of the respective genes, and their other functions that might not necessarily be related to pathology. For multimorbid patients, simultaneous approaches targeting different pathomechanisms could also be considered. A significant limitation of this study is that the inventors performed gene editing in the germline, which is not acceptable clinically or ethically. Thus, it will be important to test whether ablating CaMKIIδ autophosphorylation is also feasible postnatally and whether it could also reverse or prevent disease progression after its onset (e.g., in human iPSC-CMs after chronic β-adrenergic stress or in mice when early heart failure has already been developed). Delivery of the CRISPR-Cas9 base editing components into heart cells could be achieved with methods used in gene therapy approaches (e.g., using cardiotropic adeno-associated viruses), which have been shown to enable high editing efficiency in vivo (Chemello et al., 2021; Lebek et al., 2023). Efficiency of delivery is another important factor, as the number of cells expressing the gene editing components would limit the overall treatment success. These 85 4860-0953-0567, v.1aspects are the next steps of investigation required to further develop elimination of CaMKIIδ autophosphorylation as a potential broad-based therapeutic concept for human cardiac disease. * * * 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. 86 4860-0953-0567, v.1IX. 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. Sapranauskas et al. Nucleic Acids Res.39:9275-9282, 2011). Ran et al., Nature 520, 186-191, doi:10.1038 / nature14299, 2015). Weinmann et al., Nature Communications, 11:5432, 2020. Tabebordbar et al., Cell, 184:1-20, 2021. McCarty et al., Gene Ther.8:1248–54, 2001. Naso et al., BioDrugs 31:317-334, 2017. PCT Publn. WO2020214842 PCT Publn. WO2021025750 PCT Publn. WO2018027078 PCT Publn. WO2019079347 PCT Publn. WO2019226593 U.S. Patent Publn. US20200308571 U.S. Patent Publn. US20180073012 U.S. Patent Publn. US20170121693 U.S. Patent 10,113,163 U.S. Patent 10,167,457 1. 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Claims

WHAT IS CLAIMED IS:

1. A single-guide RNA (sgRNA) comprising a targeting nucleic acid sequence that targets an autophosphorylation site in the regulatory domain of CaMKIIδ.

2. The sgRNA of claim 1, wherein the sgRNA targets Thr287 (c.A859) of CaMKIIδ.

3. The sgRNA of claim 1 or 2, wherein the gRNA comprises the sequence: sgRNA sgRNA-sequence SEQ ID NO PAM Human-1TG(A)4.ite in a CaMKIIδ regulatory domain and a base editor.

5. The composition of claim 4, wherein the base editor is an adenine base editor (ABE).

6. The composition of claim 4, wherein the sgRNA is the sgRNA of any one of claims 1- 3.

7. The composition of claim 6, wherein the base editor is an adenine base editor (ABE).

8. The composition of any one of claims 4-7, wherein the base editor comprises a CRISPR / Cas nuclease linked to an adenosine deaminase.

9. The composition of claim 8, wherein the CRISPR / Cas nuclease is catalytically impaired.

10. The composition of claim 8 or claim 9, wherein the CRISPR / Cas nuclease is a Cas9 nuclease.

11. The composition of claim 10, wherein the Cas9 nuclease is isolated or derived from Streptococcus pyogenes (spCas9), Streptococcus pyogenes (spRY), Staphylococcus aureus 94 4860-0953-0567, v.1(SaCas9), Staphylococcus auricularis (SauCas9), Staphylococcus lugdunensis (SlugCas9), or Streptococcus pyogenes NGN protospacer (SpCas9-NG).

12. A nucleic acid comprising: a sequence encoding a first sgRNA of any one of claims 1-3, a sequence encoding a base editor, a sequence encoding a first promoter, wherein the first promoter drives expression of the sequence encoding the base editor, and a sequence encoding a second promoter, wherein the second promoter drives expression of the sequence encoding the first sgRNA.

13. The nucleic acid of claim 12, wherein the base editor is an adenine base editor (ABE).

14. The nucleic acid of claim 12 or claim 13, wherein the base editor comprises a CRISPR / Cas nuclease linked to an adenosine deaminase.

15. The nucleic acid of claim 14, wherein the CRISPR / Cas nuclease is catalytically impaired.

16. The nucleic acid of claim 14 or claim 15, wherein the CRISPR / Cas nuclease is a Cas9 nuclease.

17. The nucleic acid of claim 16, wherein the Cas9 nuclease is isolated or derived from Streptococcus pyogenes (spCas9), Staphylococcus aureus (SaCas9), Staphylococcus auricularis (SauCas9), Streptococcus pyogenes (spRY), Streptococcus pyogenes NGN protospacer (SpCas9-NG) or Staphylococcus lugdunensis (SlugCas9).

18. The nucleic acid of any one of claims 12-17, wherein at least one of the sequences encoding the first promoter and the sequence encoding the second promoter comprises a cell- type specific promoter.

19. The nucleic acid of claim 18, wherein the cell-type specific promoter is a cardiomyocyte-specific promoter.

20. The nucleic acid of claim 19, wherein the muscle-specific promoter is a cardiac troponin T (cTnT) promoter. 95 4860-0953-0567, v.

121. The nucleic acid of any one of claims 12-20, wherein the sequence encoding the second promoter comprises a sequence encoding a U6 promoter, an H1 promoter, or a 7SK promoter.

22. The nucleic acid of any one of claims 12-21, wherein the nucleic acid comprises a DNA sequence.

23. The nucleic acid of any one of claims 12-22, wherein the nucleic acid comprises an RNA sequence.

24. The nucleic acid of any one of claims 12-23, wherein the nucleic acid further comprises a polyadenosine (polyA) sequence.

25. The nucleic acid of claim 24, wherein the polyA sequence is a mini polyA sequence.

26. A cell comprising the nucleic acid of any one of claims 12-25.

27. A composition comprising the nucleic acid of any one of claims 12-25.

28. A cell comprising the composition of claim 27.

29. A composition comprising the cell of claim 28.

30. A vector comprising the nucleic acid of any one of claims 12-25.

31. The vector of claim 30, wherein the vector further comprises a sequence encoding an inverted terminal repeat (ITR) of a transposable element.

32. The vector of claim 31, wherein the transposable element is a transposon.

33. The vector of claim 32, wherein the transposon is a Tn7 transposon.

34. The vector of claim 33, wherein the vector further comprises a sequence encoding a 5’ ITR of a T7 transposon and a sequence encoding a 3’ ITR of a T7 transposon.

35. The vector of any one of claims 30-34, wherein the vector is a non-viral vector.

36. The vector of claim 35, wherein the non-viral vector is a plasmid.

37. The vector of any one of claims 30-34, wherein the vector is a viral vector. 96 4860-0953-0567, v.

138. The vector of claim 37, wherein the viral vector is an adeno-associated viral (AAV) vector or an adenoviral vector.

39. The vector of claim 38, wherein the AAV vector is replication-defective or conditionally replication defective.

40. The vector of claim 38 or 39, wherein the AAV vector is a recombinant AAV vector.

41. The vector of any one of claims 38-40, wherein the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6),7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11) or any combination thereof.

42. The vector of any one of claims 38-41, wherein the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 9 (AAV9).

43. The vector of any one of claims 38-42, wherein the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 2 (AAV2).

44. The vector of any one of claims 38-43, wherein the AAV vector comprises a sequence isolated or derived from an AAV2 and a sequence isolated or derived from an AAV9.

45. The vector of any one of claims 30-44, wherein the vector is optimized for expression in mammalian cells.

46. The vector of any one of claims 30-45, wherein the vector is optimized for expression in human cells.

47. A composition comprising the vector of any one of claims 30-46.

48. The composition of claim 47, further comprising a pharmaceutically acceptable carrier.

49. A vector of claims 30-46 or a composition of claims 47 or 48, wherein said vector comprises a trans-splicing intein system.

50. A cell comprising the vector of claims 30-46 or the composition of claims 47 or 48.

51. The cell of claim 50, wherein the cell is a human cell, such as where the cell is a cardiomyocyte or human cardiomyocyte. 97 4860-0953-0567, v.

152. The cell of claim 50 or 51, wherein the cell or human cell is an induced pluripotent stem (iPS) cell.

53. A composition comprising the cell of any one of claims 50-52.

54. A method for editing an autophosphorylation site in the regulatory domain of CaMKIIδ, the method comprising contacting a cell with a composition or vector of any one of claims 47- 49 under conditions suitable for expression of the first sgRNA and the adenine base editor, wherein the first sgRNA forms a complex with the adenine base editor, wherein the complex modifies or edits an autophosphorylation site in the regulatory domain of CaMKIIδ thereby blocking a stress response of CaMKIIδ.

55. A cell produced by the method of claim 54, such as a cardiomyocyte, e.g., a stem-cell derived cardiomyocyte.

56. A method of treating or preventing cardiac disease, such as heart failure, in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 47 or 48.

57. The method of claim 56, wherein the composition is administered locally.

58. The method of claims 56 or 57, wherein the composition is administered directly to cardiac tissue.

59. The method of any one of claims 56-58, wherein the composition is administered by an infusion or injection.

60. The method of claim 56, wherein the composition is administered systemically.

61. The method of claim 60, wherein the composition is administered by an intravenous infusion or injection.

62. The method of any one of claims 56-61, wherein, following administration of the composition, the subject exhibits improved Ca2+homeostasis, decreased arrhythmia, decreased ventricular dilation, improved cardiac function (improved ejection fraction, fractional shortening), improved survival, decreased fibrosis, decreased apoptosis, improved transcriptome, or a combination thereof. 98 4860-0953-0567, v.

163. The method of any one of claims 56-62, wherein, following administration of the composition, the subject does not exhibit cellular Ca2+dysregulation.

64. The method of any one of claims 56-63, wherein the subject is a neonate, an infant, a child, a young adult, or an adult.

65. The method of any one of claims 56-64, wherein the subject is male.

66. The method of any one of claims 56-64, wherein the subject is female.

67. Use of a therapeutically effective amount of a composition of any one of claims 47 or 48 for treating or preventing cardiac disease, such as heart failure, in a subject in need thereof.

68. An induced pluripotent stem cell comprising an edited CaMKIIδ gene encoding Ala287.

69. An induced pluripotent stem cell comprising an edited CaMKIIδ gene encoding Gly286, Ala287, and Val288.

70. An engineered mouse comprising a germline cell having an edited CaMKIIδ gene encoding Ala287 in the germline.

71. An engineered mouse comprising a germline cell having an edited CaMKIIδ gene encoding Gly286, Ala287, and Val288 in the germline. 99 4860-0953-0567, v.1