Background: Derived from an adaptive bacterial immune system, the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has shown great potential in high-throughput functional genomic screening, especially for protein-coding genes. However, it is still challenging to apply the similar strategy to study non-coding genomic elements such as long non-coding RNAs (lncRNAs) or clusters of microRNAs, because short insertions or deletions may not be sufficient to generate loss-of-function phenotypes. Methods: Here, we presented a systematic strategy for designing a CRISPR-based paired-sgRNA library for highthroughput screening in non-coding regions. Due to the abundance of lncRNAs and their diverse regulatory roles in vivo, we repurposed microarray datasets to select 600 highly expressed lncRNAs in non-small-cell lung cancer and designed two schemes for lncRNA deletion with~20 paired-sgRNAs for each lncRNA. Through Golden-Gate assembly, we generated a pooled CRISPR-based library with a total of 12,878 sgRNA pairs. Results: Over 80% of paired-sgRNAs were recovered from final pooled library with a relatively even distribution. Cleavage efficiency of sgRNA pairs was validated through experiments of transient transfection and viral infection. Moreover, randomly selected paired-sgRNAs showed that efficient deletion of genomic DNA could be achieved with a deletion size within the range of 500 to 3000 bp. Conclusions: In summary, we have demonstrated a strategy to design and construct a pooled paired-sgRNA library to generate genomic deletion in the lncRNA regions, validated their deletion efficiency and explored the relationship of deletion efficiency with respect to deletion size. This method would be also suitable for investigation of other uncharacterized non-coding genomic regions in mammalian cells in an efficient and cost-effective manner.Author summary: The CRISPR/Cas9 system has shown great potential in functional genomic screening, especially for protein-coding genes. However, it is challenging to apply the similar strategy to study non-coding genomic elements, because short insertions or deletions may not be sufficient to generate loss-of-function phenotypes. In this paper, we proposed a strategy to design and construct a CRISPR-based paired-sgRNA library for chromosomal deletions of lncRNA loci in mammalian cells and confirm the cleavage efficiency through experiments. This approach demonstrates a simple and scalable tool for genome-wide functional study of non-coding elements in mammalian cells. # Present address: Zhejiang
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.