Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington's disease

An expanded CAG repeat in the huntingtin gene (HTT) causes Huntington's disease (HD). Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat. Here, we developed base editing strategies to convert CAG in the repeat to CAA and determined their molecular outcomes and effects on relevant disease phenotypes. Base editing strategies employing combinations of cytosine base editors and gRNAs efficiently converted CAG to CAA at various sites in the CAG repeat without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity. Candidate BE strategies converted CAG to CAA on both expanded and non-expanded CAG repeats without altering HTT mRNA and protein levels. In addition, somatic CAG repeat expansion, which is the major disease driver in HD, was significantly decrea..., To determine the molecular consequences of candidate BE strategies, we performed RNAseq analysis. We transfected HEK293 cells with BE4max+empty vector, BE4max+gRNA 1, or  BE4max+gRNA2 for 72hours. Subsequently, genomic DNA for MiSeq analysis and cell pellets for RNAseq analysis were generated from replica plates Genome-wide RNAseq analysis (Tru-Seq strand specific large insert RNA sequencing) was performed by the Broad Institute. Sequence data were processed by STAR aligner (Dobin 2013, 23104886) as part of the Broad Institute's standard RNAseq analysis pipeline. For differential gene expression (DGE) analysis, we used transcripts per million (TPM) data computed by the TPMCalculator (https://github.com/ncbi/TPMCalculator) (Alvarez 2019, 30379987). Expression levels in approximately 19,000 protein-coding genes based on Ensembl (ftp://ftp.ensembl.org/pub/release-75/gtf/homo_sapiens/) were normalized The DGE analysis was performed by the generalized linear model using a library of “glm” in..., Expression data can be opened by text editors, Microsoft Excel, and R. Metadata can be opened by the same programs. README.md file can be opened by text editors., # Base editing strategies to convert CAG to CAA in Huntington's disease

### HD.BE.RNAseq.Meta.Data.230116.csv: Sample characteristics and meta-data

Description of columns in the metadata file

* Sample: Name of the sample for each RNAseq sample
* Cell: HEK293 cells that were used for RNAseq analysis
* Group: experimental group including empty vector-treated controls (n=4), gRNA 1-tretaed samples (n=4), and gRNA2-treated samples (n=4)
* Replicate: replicate number
* PC1: principal component 1 value
* PC2: principal component 2 value
* PC3: principal component 3 value
* PC4: principal component 4 value
* PC5: principal component 5 value
* PC6: principal component 6 value
* PC7: principal component 7 value
* PC8: principal component 8 value
* PC9: principal component 9 value
* PC10: principal component 10 value
* PC11: principal component 11 value
* PC12: principal component 12 value

### HD.BE.RNAseq.12.Sample.230116.txt: RNAseq expression data

亨廷顿基因（huntingtin gene, HTT）中扩展的CAG重复序列会导致亨廷顿舞蹈症（Huntington's disease, HD）。由于决定亨廷顿舞蹈症发病年龄的是连续CAG重复序列的长度，而非多聚谷氨酰胺（polyglutamine），因此将CAG转换为CAA的碱基编辑（base editing）策略有望通过缩短连续CAG重复序列来延缓发病。本研究开发了将重复序列中的CAG转换为CAA的碱基编辑策略，并对其分子效应以及相关疾病表型的影响进行了探究。联合使用胞嘧啶碱基编辑器与向导RNA（guide RNA, gRNA）的碱基编辑策略，可在CAG重复序列的多个位点高效实现CAG向CAA的转换，且未产生显著的插入缺失（insertions/deletions, indels）、脱靶编辑或转录组改变，证实了该策略的可行性与特异性。候选碱基编辑策略可在扩增型与非扩增型CAG重复序列上均完成CAG向CAA的编辑，且不会改变HTT mRNA与蛋白的表达水平。此外，作为亨廷顿舞蹈症主要疾病驱动因素的体细胞CAG重复扩增现象也得到了显著抑制……为明确候选碱基编辑策略的分子效应，我们开展了RNA测序（RNA sequencing, RNAseq）分析。我们将转染了BE4max+空载体、BE4max+gRNA 1或BE4max+gRNA2的HEK293细胞培养72小时。随后，我们从复孔板中制备了用于MiSeq分析的基因组DNA以及用于RNAseq分析的细胞沉淀。全转录组RNA测序分析（Tru-Seq链特异性大片段插入RNA测序）由博德研究所（Broad Institute）完成。序列数据通过STAR比对工具（STAR aligner, Dobin 2013, 23104886）进行处理，作为博德研究所标准RNAseq分析流程的一部分。差异基因表达（differential gene expression, DGE）分析采用TPMCalculator（https://github.com/ncbi/TPMCalculator）计算的每百万转录本（transcripts per million, TPM）数据，相关文献引用为Alvarez 2019, 30379987。基于Ensembl数据库（Ensembl, ftp://ftp.ensembl.org/pub/release-75/gtf/homo_sapiens/）注释的约19000个蛋白质编码基因的表达水平已完成标准化。差异基因表达分析采用R语言中‘glm’程序包的广义线性模型完成。表达数据可通过文本编辑器、Microsoft Excel及R语言打开。元数据可通过相同程序打开。README.md文件可通过文本编辑器打开。
# 亨廷顿舞蹈症中CAG向CAA转换的碱基编辑策略

### HD.BE.RNAseq.Meta.Data.230116.csv：样本特征与元数据
元数据文件各列说明如下：
* Sample：每个RNAseq样本的名称
* Cell：用于RNAseq分析的HEK293细胞
* Group：实验组分组，包括空载体处理对照组（n=4）、gRNA1处理组（n=4）与gRNA2处理组（n=4）
* Replicate：重复样本编号
* PC1：主成分1值
* PC2：主成分2值
* PC3：主成分3值
* PC4：主成分4值
* PC5：主成分5值
* PC6：主成分6值
* PC7：主成分7值
* PC8：主成分8值
* PC9：主成分9值
* PC10：主成分10值
* PC11：主成分11值
* PC12：主成分12值

### HD.BE.RNAseq.12.Sample.230116.txt：RNAseq表达数据