Obtaining a proper loss of function approach has been one of the main challenges in long non-coding RNAs (lncRNA) research. The development of CRISPR/Cas9 technology allow permanent editing of any genomic region. Nevertheless, the lack of an open reading frame (ORF) in lncRNAs increases the difficulty of obtaining a long non-coding knock-out line, given that a small alteration of the sequence of a lncRNA will probably not imply a loss of function.
The need of an efficient knockout tool drove us to the development of the system DECKO (Double Excision CRISPR Knockout). This system applies a simple two-step cloning to generate lentiviral vectors expressing two guide RNAs (gRNAs) simultaneously.
On one side, with dual gRNA expression, two excisions are performed resulting in the deletion of the flanked genomic region. Thus, the system can be used to delete any genomic region, including lncRNA promoters, which will lead to an expression knock out.
On the other side, the cloning strategy makes the system suitable not only for single targets but also for high throughput screening libraries.
The main characteristics of the system are:
Each sgRNA is expressed under a different promoter, conferring equal expression levels.
The vector contains two selectable markers: Puromycin and mCherry fluorescent protein for FACS applications.
It can be used for transfection or lentiviral infection.
Adaptable for single clonings and libraries.
See the original paper for further details.
Vectors available here: https://www.addgene.org/Rory_Johnson/
The latest DECKO protocol can be found here.