Edit multiple genes in a single reaction with synthetic crRNA
The application of CRISPR-Cas9 components to achieve targeted gene knockout is quickly becoming a standard practice, but what about knockout of more than one gene? Researchers who wish to create an engineered cell line, potentially to mimic a disease state, will often require knockout of two or more loci. However, the time and expense of doing serial edits and clonal cell line characterizations is onerous, and the transfection efficiency of multiple sgRNA expression plasmids is very low. While it is possible to engineer a single expression plasmid with multiple guide RNA constructs, there is potential for recombination of the plasmid, and a new one must be generated for every combination of genes.
The ease and efficiency of synthetic CRISPR RNA (crRNA) transfection makes it an ideal system to knock down one, two, or three target genes in a single reaction. Edit-R™ crRNAs are predesigned against all human, mouse, and rat genes, so they arrive within a few days, ready to use, and there is no time lost in engineering custom plasmids! Edit-R crRNA are compatible with co-transfection of Cas9 mRNA or protein, or can be delivered into Cas9-expressing cells to achieve functional target gene knockout.
Here we demonstrate clonal cell line results of a three-gene multiplex knockout experiment using Edit-R synthetic crRNA with three different Cas9 nuclease sources: mRNA (co-transfection), protein (RNP transfection), and stable expression from lentiviral transduction.
Multiple genes can be edited in a single cell with a single transfection using crRNAs targeting each individual gene, and is effective with various Cas9 sources. In our delivery of Cas9 protein combined with three different crRNAs, all guide RNAs were complexed at the same time to make the RNPs. There may be improved efficiency if each crRNA is complexed separately with Cas9 protein and then combined prior to transfection. U2OS cells were sensitive to growing in single-cell isolation, so fewer colonies were isolated than for HEK293T. The stable expression of Cas9 improved the overall efficiency of knockout in both cell lines tested, and showed exceptional success in U2OS cells, where there are up to four copies of a single gene.
Authors: Louise Baskin, Senior Product Manager, and Eldon Chou, Associate Scientist II
Guaranteed to edit your target! Algorithm-optimized crRNA for genome-wide coverage of human, mouse, or rat genes. Modifications for nuclease resistance improve DNA-free editing. Simply search for your gene!
Purified Cas9 protein ready to use for DNA-free nuclease expression.
Purified Cas9 mRNA for transient Cas9 Nuclease expression. Fluorescent options available for sorting, enrichment and visualization of delivery.
Chemically synthesized trans-activating CRISPR RNA required for use with synthetic crRNA for fast and easy gene editing.
Dehairs, J. et al. CRISP-ID: decoding CRISPR mediated indels by Sanger sequencing Sci. Rep. 6, 28973; doi: 10.1038/srep28973 (2016).