Efficient precise in vivo base editing in adult dystrophic mice

Recent advances in base editing have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) nearby the mutation site further limits the targeting feasibility. Here we modify the NG-targeting adenine base editor (iABE-NGA) to overcome these challenges and demonstrate the high efficiency to precisely edit a Duchenne muscular dystrophy (DMD) mutation in adult mice. Systemic delivery of AAV9-iABE-NGA results in dystrophin restoration and functional improvement. At 10 months after AAV9-iABE-NGA treatment, a near complete rescue of dystrophin is measured in mdx4cv mouse hearts with up to 15% rescue in skeletal muscle fibers. The off-target activities remains low and no obvious toxicity is detected. This study highlights the promise of permanent base editing using iABE-NGA for the treatment of monogenic diseases.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability

Renzhi Han
Apr 5, 2021 NIS-Elements AR version 4.50 was used to collect fluorescence images; ChemiDoc XRS+ system was used to collect Western blot data; DMA v5.501 was used to collect muscle contractility data; Flow cytometry was carried out on Becton Dickinson LSR II with BD FACSDiva software (version 8.0.1).
Graphpad prism 8.0.1 was used to analyze the data. FlowJo 10.4 was used to analyze FACS data collected with the BD FACSDiva software (version 8.0.1). Adobe Photoshop 2020 was used to assemble figures. Western blots were imaged and quantified using Image Lab 6.0.1 software. Sanger sequencing trace data were analyzed by using BEAT v1.0, which is published and available at https://github.com/ HanLab-OSU/Beat. Next generation sequencing data were analyzed by using CRISPResso2. RNAseq data were analyzed by following the GATK Best Practices for RNA-seq variant calling with the following softwares: STAR version 1. Sample size is estimated with G-power software 3.1 by the significance level (0.05), effect size (based on the data in our previous studies) and a given power (usually 0.8). We also conducted power analysis after the experiment and found enough power value (>0.8) in each statistical analysis.

No data exclusion
All attempts for replication were successful. A minimum of three independent experiments (or animals) were included for all FACS, ELISA, immunofluorescence staining, Western blot and contractile measurements.
Mice (only males as DMD primarily affects boys) and cells were assigned randomly into experimental groups and processed in an arbitrary order.
All the enrolled mice or subsequent samples were labeled only with mouse ID numbers without genotype or type of treatment information. Genotype or treatment type were decoded after the data acquisition and quantification analysis were complete. The culture studies (transfection, DNA/RNA isolation, PCR, sequencing, FACS) were carried out identically through standard procedures that should not bias outcomes. All antibodies have been tested for reactivity against the appropriate species on the specification sheets on the providers' websites or in published articles. The dystrophin antibody (ab15277, 1:100, Abcam, Cambridge, MA) used for immunofluorescence staining detected sarcolemmal dystrophin in wild-type muscles but not in mdx4cv muscles. The dystrophin antibody (E2660, 1:500, Spring Bioscience, Pleasanton, CA) used for Western blot detected bands at the expected molecular weight in WT mice but not in mdx4cv mice. The laminin-!2 antibody (ALX804-190-C100, 1:100, Enzo Life Sciences Inc, Farmingdale, NY) recognizes an N-terminal portion of the !2 chain that is deleted in congenital muscular dystrophies and reacts with human and mouse. According to the respective datasheet or the manufacturer's website, the anti-SpCas9 antibody C15310258, Diagenode, Denville, NJ) was raised against Cas9 from Streptococcus pyogenes, and validated for Western blotting,