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

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. 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. 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. 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.