Prajapat Mahendra K, Maria Andrea G, Vidigal Joana A
Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, The National Institutes of Health, 37 Convent Dr, Bethesda, MD 20892, USA.
Nucleic Acids Res. 2025 Jan 7;53(1). doi: 10.1093/nar/gkae1138.
Non-coding regulatory sequences play essential roles in adjusting gene output to cellular needs and are thus critical to animal development and health. Numerous such sequences have been identified in mammalian genomes ranging from transcription factors binding motifs to recognition sites for RNA-binding proteins and non-coding RNAs. The advent of CRISPR has raised the possibility of assigning functionality to individual endogenous regulatory sites by facilitating the generation of isogenic cell lines that differ by a defined set of genetic modifications. Here we investigate the usefulness of this approach to assign function to individual miRNA binding sites. We find that the process of generating isogenic pairs of mammalian cell lines with CRISPR-mediated mutations introduces extensive molecular and phenotypic variability between biological replicates confounding attempts at assigning function to the binding site. Our work highlights an important consideration when employing CRISPR editing to characterize non-coding regulatory sequences in cell lines and calls for the development and adoption of alternative strategies to address this question in the future.
非编码调控序列在根据细胞需求调整基因输出方面发挥着重要作用，因此对动物发育和健康至关重要。在哺乳动物基因组中已鉴定出许多此类序列，从转录因子结合基序到RNA结合蛋白和非编码RNA的识别位点。CRISPR的出现增加了通过促进产生因一组定义明确的基因修饰而不同的同基因细胞系来赋予单个内源性调控位点功能的可能性。在这里，我们研究了这种方法用于赋予单个miRNA结合位点功能的实用性。我们发现，利用CRISPR介导的突变产生哺乳动物细胞系同基因对的过程在生物学重复之间引入了广泛的分子和表型变异性，这使得在为结合位点赋予功能时的尝试变得复杂。我们的工作突出了在利用CRISPR编辑来表征细胞系中的非编码调控序列时的一个重要考虑因素，并呼吁未来开发和采用替代策略来解决这个问题。
