Construction of a CRISPR‐based paired‐sgRNA library for chromosomal deletion of long non‐coding RNAs

Abstract

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

CRISPR-Based Approaches for the High-Throughput Characterization of Long Non-Coding RNAs

“…While effective, their screen only targeted approximately 700 lncRNAs with over 12,000 sgRNA pairs, making it useful only for small-scale screening. A comparable pgRNA screening strategy was also attempted recently by Tao et al [41], who screened approximately 600 lncRNAs with a library of nearly 13,000 sgRNA pairs; their approach was similarly difficult to scale up. Subsequently, the Wei group removed the need for pgRNAs by designing a more comprehensive, efficient, and high-throughput screen with a library of sgRNAs that were targeted to either the 5 splice donor or 3 splice acceptor sites of lncRNAs to induce exon skipping or intron retention, respectively [42].…”

Integration of transcription regulation and functional genomic data reveals lncRNA SNHG6’s role in hematopoietic differentiation and leukemia

“…While CRISPR has revolutionized reverse genetics by increasing accuracy for the study of PCGs, Cas9-based knockout is not effective for lncRNAs, which are minimally sequence-dependent. Nevertheless, several library screens have been performed using this method, typically by targeting splice sites [ 10 ] or with paired gRNAs designed to remove entire exons, introns, promoters, or transcription start sites (TSSs) from the genome [ 1 , 11 , 12 ]. Alternative CRISPR-based tools have been developed to modulate gene expression without modifying the genome by using a nuclease-dead Cas9 (dCas9) that binds the target sequence without cleaving the DNA.…”