Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong SAR, China.
Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong SAR, China; Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China.
Cell Syst. 2023 May 17;14(5):392-403.e4. doi: 10.1016/j.cels.2023.03.007. Epub 2023 May 9.
Selecting the most suitable existing base editors and engineering new variants for installing specific base conversions with maximal efficiency and minimal undesired edits are pivotal for precise genome editing applications. Here, we present a platform for creating and analyzing a library of engineered base editor variants to enable head-to-head evaluation of their editing performance at scale. Our comprehensive comparison provides quantitative measures on each variant's editing efficiency, purity, motif preference, and bias in generating single and multiple base conversions, while uncovering undesired higher indel generation rate and noncanonical base conversion for some of the existing base editors. In addition to engineering the base editor protein, we further applied this platform to investigate a hitherto underexplored engineering route and created guide RNA scaffold variants that augment the editor's base-editing activity. With the unknown performance and compatibility of the growing number of engineered parts including deaminase, CRISPR-Cas enzyme, and guide RNA scaffold variants for assembling the expanding collection of base editor systems, our platform addresses the unmet need for an unbiased, scalable method to benchmark their editing outcomes and accelerate the engineering of next-generation precise genome editors.
选择最适合的现有碱基编辑器，并对其进行工程改造，以实现特定碱基转换的高效安装，同时最小化不必要的编辑，这对精确的基因组编辑应用至关重要。在这里，我们提出了一个创建和分析工程碱基编辑器变体库的平台，以能够大规模地对头对头评估它们的编辑性能。我们的综合比较提供了每个变体在编辑效率、纯度、基序偏好以及单碱基和多碱基转换生成中的偏向性方面的定量衡量标准，同时揭示了一些现有碱基编辑器中存在的不必要的更高的插入缺失（indel）生成率和非规范碱基转换。除了对碱基编辑器蛋白进行工程改造外，我们还进一步应用该平台研究了一个迄今为止探索不足的工程途径，并创建了增强编辑器碱基编辑活性的向导 RNA 支架变体。随着越来越多的工程化部分（包括脱氨酶、CRISPR-Cas 酶和向导 RNA 支架变体）的未知性能和兼容性，用于组装不断扩展的碱基编辑器系统集合，我们的平台解决了对无偏见、可扩展的方法来基准测试它们的编辑结果并加速下一代精确基因组编辑器的工程设计的未满足需求。
