College of Life Science, Tianjin Normal University, Tianjin, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Nucleic Acids Res. 2022 Apr 22;50(7):4161-4170. doi: 10.1093/nar/gkac201.
CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increased the single-base editing fraction relative to normal gRNA editing with increased editing efficiencies. Similar results were obtained with an adenine base editor (ABE). At loci such as DNMT3B, NSD1, PSMB2, VIATA hs267 and ANO5, near-perfect single-base editing was achieved. Normally an igRNA with good single-base editing efficiency could be selected from a set of a few igRNAs, with a simple protocol. As a proof-of-concept, igRNAs were used in the research to construct cell lines of disease-associated SNP causing primary hyperoxaluria construction research. This work provides a simple strategy to achieve single-base base editing with both ABEs and CBEs and overcomes a key obstacle that limits the use of base editors in treating SNP-associated diseases or creating disease-associated SNP-harboring cell lines and animal models.
CRISPR 碱基编辑技术往往会在靶向区域编辑多个碱基，这对于精确逆转与疾病相关的单核苷酸多态性（SNP）是一个限制。我们设计了一种不完美的 gRNA（igRNA）编辑方法，该方法利用一个与靶位点不完全互补的 gRNA 来引导碱基编辑，以产生单碱基编辑产物。碱基编辑实验表明，与正常 gRNA 编辑相比，CBEs 的 igRNA 编辑大大增加了单碱基编辑分数，同时提高了编辑效率。腺嘌呤碱基编辑器（ABE）也得到了类似的结果。在 DNMT3B、NSD1、PSMB2、VIATA hs267 和 ANO5 等基因座，几乎可以实现完美的单碱基编辑。通常，可以通过一个简单的方案，从一组 igRNA 中选择具有良好单碱基编辑效率的 igRNA。作为概念验证，igRNA 用于构建与疾病相关 SNP 相关的原发性高草酸尿症研究的细胞系。这项工作为使用 ABE 和 CBE 实现单碱基碱基编辑提供了一种简单的策略，克服了限制碱基编辑在治疗 SNP 相关疾病或创建携带疾病相关 SNP 的细胞系和动物模型中的应用的关键障碍。
