CRISPR/Cas9 genome engineering tools to discover lncRNAs in cancer
Our challenge is to discover a small group of cancer-related lncRNAs amongst tens of thousands of potential candidates. The newly-discovered genome-engineering technology, CRISPR/Cas9, represents a uniquely powerful tool for achieving this. Not only can CRISPR/Cas9 be used to manipulate lncRNAs in their endogenous environment, it can also be used at high throughputs.
Our lab recently developed a tool, called DECKO (Aparicio-Prat et al.), which enables one to delete virtually any desired genomic region using pairs of CRISPR/Cas9 complexes. We use DECKO to study lncRNAs, either silencing them by deleting their promoter, or else by leaving expression intact and removing fragments of their exons. The power of DECKO is that it is highly scalable: we can use it equally to target a single lncRNA, or thousands of lncRNAs in parallel. This enables us to screen for new cancer lncRNAs.
DECKO creates the need for bioinformatic design of CRISPR/Cas9 targeting constructs. We have created a design pipeline for this, called CRISPETa (Pulido-Quetglas et al.).
We are presently using the DECKO and CRISPETa tool kit to discover new lncRNAs in various human cancers. The lncRNAs we find will give us new insights into the molecular pathways controlling oncogenesis, and hopefully provide new targets for therapy.
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.
CRISPETa is a webserver (standalone version also available) for paired sgRNA design, such as required by DECKO. A flexible tool to design optimal pairs of sgRNAs for deletion of desired genomic regions. CRISPETa can be run on any number of targets – from one to thousands. At present, designs can be performed for 5 genomes: human, mouse, zebrafish, fruitfly and worm
Access it here: http://crispeta.crg.eu/