
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.03.611048
preprint
1
Article
CRISPR-based dissection of miRNA binding sites using isogenic cell lines is hampered by pervasive noise
Prajapat Mahendra K.
Vidigal Joana A.
03 9 2024
2024.09.03.611048https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.09.03.611048
nihpp-2024.09.03.611048.pdf
ABSTRACT

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 making any attempt of assigning function to the binding site essentially impossible. 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.
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