CRISPR/Cas9 has been widely utilized to facilitate the functional knockout of protein-coding genes. However, a single site of gene editing may not be sufficient to disrupt a protein’s structure (for example, miRNA stem-loop) and function. To overcome this potential limitation, paired guide RNAs targeting both 5’ and 3’ of the target region have been shown to result in larger deletions, thus resulting in loss of function completely.
Dual sgRNA-directed large gene deletion mediated by CRISPR/Cas9 system is a robust tool for generating deletions of long non-coding RNAs (lncRNAs), MicroRNAs (miRNAs), and regulatory sequences [1,2]. Several recent studies have reported the CRISPR/ Cas9 system-induced large genomic deletion, ranging from 23kb to 1Mb, in mice [3], C. elegans [4], rabbit [5], and human cell lines [6].
