Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The overall goal of this procedure is to use the CRISPR Cas nine nuclease system to create genomic deletions. First electro purate, the two CRISPR plasmids and A GFP reporter plasmid simultaneously into the cells. Then using fluorescence activated cell sorting, isolate the top 3%of GFP expressing cells.

Next plate the sorted cells at limiting dilution and screen for bio allelic deletion clones using conventional PCR. Ultimately, CRISPR Cas nine can be used to study genes and genetic elements by producing loss of function deletion alleles Compared to frameshift mutations produced by a single guide RNA large deletions generated by the CRISPR Cas nine nuclease system can help ensure loss of function mutations. This approach also allows for easy and cheap screening via a conventional PCR.

We First had the idea for this method when we found screening for small indels created by a single guide RNA to be technically challenging, laborious, and expensive deletion alleles are also particularly informative for the study of non-coating genetic elements. For each CRISPR pair pellet, two times 10 to the six cells grown in suspension. Resuspend the cells in 100 microliters of electroporation solution and then transfer to an electroporation QAT.

Add five micrograms of CRISPR Cas nine construct SG RNAA five micrograms of CRISPR Cas nine. Construct SG a B and 0.5 Micrograms of the GFP expression construct pipette up and down several times gently to mix. Try to avoid producing bubbles using an electroporation system.

Electro purate the cells with 250 volts for five milliseconds in a two millimeter vete with a sterile transfer pipette immediately transfer the solution to one milliliter of prewarm culture media. Incubate the cells at 30 degrees Celsius for 24 to 72 hours. Pass the cells through a sterile 50 micron filter into a fax tube.

Then fax sort the top 3%of GFP positive cells in order to enrich for cells that received high levels of the plasmids. After optimizing limiting dilution conditions for the cells of interest, plate sorted cells into a 96 well plate and a dilution of 30 cells per plate with 100 microliters of cell culture media per well. For the remaining sorted bulk cells freeze half of the cells for future plating.

Plate the other half for screening and primer validation. Incubate these bulk cells at 37 degrees Celsius for three to seven days. Allow the clones to incubate at 37 degrees Celsius for seven to 14 days depending on the doubling time of the cell line.

Used to isolate genomic DNA Resus, suspend the parental and the bulk cell pellets in 50 microliters of DNA extraction solution. Run the sample in a thermocycler to extract genomic DNA. Then measure the DNA concentration.

Now assemble a 20 microliter PCR reaction by combining 10 microliters of two XPCR mix. 0.5 microliters of 10 micromolar forward primer 0.5 microliters of 10 micromolar reverse primer, 50 to 100 nanograms of genomic DNA and water up to 20 microliters. Conduct PCR for the non deletion band and the deletion band in separate reactions.

Next, place the samples in a thermocycler and run using 35 cycles with an A kneeling temperature of 60 degrees Celsius as detailed in the accompanying written protocol. Resolve the samples on a 2%aros gel at 10 volts per centimeter using one XTAE buffer. Then examine the samples for the presence or absence of non deletion and deletion bands.

Consider a strategy to multiplex the deletion and non deletion PCR primer pairs in a single reaction. Alternatively, you may run deletion and non deletion PCR reactions separately. For suspension cells transfer all clones to a single 96 well plate that already contains 50 microliters of cell culture media per well.

Then transfer 50 microliters from each well to a 96 well PCR plate. Alternatively, for adherence cells, trypsin, eyes, clones prior to transfer to a single 96 well plate. Then transfer 50 microliters from each well to a 96 well PCR plate centrifuge the PCR plate at 400 times G for five minutes and remove the supernatant by flicking the PCR plate over a sink.

Now add 50 microliters of DNA extraction solution per well and resuspend the cell pellets after genomic DNA extraction run PCR reactions to detect non deletion and deletion bands from the clones. Resolve PCR products on a 2%aros gel at 10 volts per centimeter. Using one XTAE buffer.

Identify the clones with the desired deletion and amplify the cultures in a larger plate or flask. In the example, shown observe from left to right parental cells, three non deletion clones, three mono allelic deletion clones, and three bi allelic deletion clones. The use of multiple non-overlapping SG r NNA pairs may help to control for off target effects.

Each pair would lead to production of a unique deletion. Breakpoint targeting SG pairs in various locations with respect to the gene can be used to delete the entire gene body to create frameshift indels, even if one or both alleles were not deleted or to allow disruption of a particular isoform. In this experiment.

Regarding the deletion of the entire gene, PIM one two SG RNA pairs were designed cloned into the PX 3 3 0 expression vector and delivered to MEL cells by electroporation. Along with the GFP reporter, the top 3%of GFP positive cells were plated clonally at limiting deletion and screened by PCR for non deletion mono allelic and bio allelic deletion clones for this PIM one deletion. Bio allelic deletion clones were selected for further proliferation after allowing five days for expansion.

Each clone was retested by PCR of genomic DNA to confirm BIC deletion and deletion. Amplicons were subjected to Sanger sequencing to identify the precise deletion. RNA was isolated from BIC deletion clones and analyzed by R-T-Q-P-C-R to confirm loss of PIM one expression.

Once mastered, this technique can be completed in a matter of weeks to identify vitalic deletion clones. After watching this video, you should have a good understanding of how to use the CRISPR CAS nine nuclease system to create genomic deletions by poring CRISPR plasmids sorting GFP bright cells, and screening individual clones by conventional PCR for BioE deletion clones.