Multiplex CRISPR-Cas9

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.

A one-step solution for multiplex gene knockout

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.

Experimental considerations

• A Cas9 stably expressing cell line will increase the efficiency of multiplexed editing, but may not be desired for cell line generation. Consider using either Cas9 mRNA or Cas9 protein to create a DNA-free system for cell line generation if your cells can be transfected or electroporated.
• The use of Edit-R Fluorescent Cas9 mRNA (Cat #CAS11860 or #CAS11859) and flow-sorting the cells prior to single cell isolation will enrich transfected cells and reduce the number of clonal isolates to characterize.
• Confirm population editing with a mismatch detection assay prior to clonal cell line generation to ensure that all targeted genes were edited at a population level.
• Functional knockout of your genes of interest should be confirmed by western blot or a functional assay.
• Many established cell lines are highly polyploid and will have more than the standard two copies of a specific gene. Although most single-gene knockdown studies with crRNA will show editing for all copies of a loci, targeting multiple sites increases the chances of some alleles remaining unedited.

Example of multiplex application

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.

• Cells: HEK293T and U2OS, plated at 20,000 and 10,000 cells/well, respectively, on the day prior to transfection
• Gene targets: AXL, DNMT3B, PPIB
• Cas9 nuclease source: stably expressed (mixed population), mRNA (200 ng, Cat #CAS11195), or protein (25 nM, Cat #CAS11201)
• crRNA: Edit-R crRNA (AXL, Cat# CM-003104-01-05, DNMT3B, Cat# U-007010-01-05, PPIB, Cat# U-007000-01-05), complexed 1:1 with tracrRNA (Cat #U-002005-05), used at 25 nM each (Cas9 stable cells, Cas9 mRNA) or 50 nM total RNP (Cas9 protein)
• Transfection reagent: DharmaFECT Duo (0.1 μL/well, Cat # T-2010-01)
• Cells were isolated for clonal cell line generation 72 hours post-transfection (dilution method)
• Each clonal cell line was sequenced for each individual edited gene using Sanger sequencing. Editing of genes was determined by deconvolution of the sequencing using the CRISP-ID tool.

Successful multiplex knockdown

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.

References

1. Dehairs, J. et al. CRISP-ID: decoding CRISPR mediated indels by Sanger sequencing Sci. Rep. 6, 28973; doi: 10.1038/srep28973 (2016).