CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [gDNA]

CRISPR gene editing has revolutionized biomedicine and biotechnology by providing a simple means to engineer genes in vivo by introducing mutations at target sites in the genomic DNA of living cells. However, given the stochasticity of cellular DNA repair mechanisms and the potential for introducing mutations at off-target sites, technologies capable of introducing targeted changes with increased precision, such as cytidine deaminase single-base editors, are preferred. We here present a versatile method termed CRISPR-SKIP that utilizes cytidine deaminase single-base editors to program de-novo exon skipping by mutating target DNA bases within splice acceptor sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology. Overall design: Four exons were targeted by CRISPR-SKIP. For each exon, genomic DNA at the splice acceptor of two control samples and two treated samples were sequenced. Rate of G to A conversion of intronic flanking G was quantified.CRISPR基因编辑（CRISPR gene editing）通过在活细胞基因组DNA的靶位点引入突变，提供了一种简便的体内基因工程操作手段，彻底革新了生物医学与生物技术领域。然而，由于细胞DNA修复机制存在随机性，且存在在脱靶位点（off-target sites）引入突变的潜在风险，因此更青睐具备更高精准度的靶向修饰技术，例如胞苷脱氨酶单碱基编辑器（cytidine deaminase single-base editors）。 本研究提出一种名为CRISPR-SKIP的多功能方法，该方法借助胞苷脱氨酶单碱基编辑器，通过突变剪接接受位点（splice acceptor sites）内的靶标DNA碱基，实现从头诱导外显子剪接跳过。鉴于其简便性与精准性，CRISPR-SKIP将在基因治疗与合成生物学领域获得广泛应用。 实验设计概述：本研究通过CRISPR-SKIP靶向四个外显子；针对每个外显子，对两份对照样本与两份处理样本的剪接接受位点区域的基因组DNA进行测序，并定量检测了内含子侧翼G位点的G→A碱基转化效率。