Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

This protocol helps you to identify mutations that occur during the repair of a double-strand break induced by single-guide RNA and CAS9. The main advantage of this technique is that it is a cost-effective and robust way to detect mutations at a specific genomic locus. We use human keratinocyte as an example, but this protocol can be adapted to any transfectable cell lines.

Demonstrating this protocol will be Taylor Bugbee, an undergraduate assistant from our laboratory. To begin, culture HFK LXSN and HFK 8E6 cells in 10-centimeter plates at 37 degrees Celsius with 5%carbon dioxide in a jacket incubator containing keratinocyte culture media with human keratinocyte growth supplement and 1%penicillin streptomycin. Replace the culture media with three milliliters of trypsin-EDTA and incubate at 37 degrees Celsius for three minutes.

Then, neutralize the trypsin with an equal volume of FBS-supplemented media. Now, transfer the cells to a 15-milliliter centrifuge tube and centrifuge at 300 times G for five minutes. Re-suspend cells with 10 milliliters of keratinocyte culture media with human keratinocyte growth supplement and determine the concentration of cells with a hemocytometer.

Afterward, seed two plates each for HFK LXSN and HFK 8E6 cells in four milliliters of keratinocyte culture media with human keratinocyte growth supplement and 1%penicillin streptomycin. Following seeding, incubate the cells at 37 degrees Celsius and 5%carbon dioxide in a jacket incubator. On the day of transfection, replace media with three milliliters of antibiotic-free supplemented media and incubate for two hours at 37 degrees Celsius in a jacket incubator.

To transfect the cells, warm transfection reagents to room temperature and pipette them gently before using. Place an appropriate amount of transfection buffer in two sterile 1.5-milliliter centrifuge tubes for each cell line and label them as tube one and mock transfection, or tube two. Then, add two micrograms of plasmid DNA-expressing, CAS9, single-guide, RNA-targeting human CD4 to tube one and pipette gently to mix completely.

Add an equal volume of sterile water to tube two. Add an appropriate amount of the transfection reagent to tube one with DNA mixture and the mock transfection or tube two. Using a pipette, gently mix them completely and incubate at room temperature for 15 to 30 minutes to form the complexes.

Add the transfection mixture drop-wise to the plate and gently shake the culture plate for one minute to evenly distribute the transfection mixture. To harvest the cells by trypsinization, replace culture media with one milliliter of trypsin EDTA and incubate at 37 degrees Celsius for three minutes. Then, neutralize trypsin with an equal volume of FBS-supplemented media.

For each plate of cells, transfer the cell suspension to two microcentrifuge tubes with equal aliquots and centrifuge at 300 times G for five minutes. After centrifugation, re-suspend the cell pellet from one tube in one milliliter of PBS for sequencing, and in another tube, with ice cold PBS for immunoblot. Immunoblot assay was performed to compare the CAS9 expression in untransfected and transected HFK cells.

The results show that untransfected and transected HFK cells expressed a similar amount of CAS9, indicating that transfection efficiency is similar between two cell lines. Immunofluorescence microscopy images of phosphorylated histone H2AX S139 in SgRNA/CAS9 transfected cells and untransfected control were obtained. The results indicated that two double-strand breaks were induced by CAS9 SgRNA.

Genomic variations were grouped by types of mutational events in HFK LXSN and HFK 8E6, where each group of genomic variations and the total number of variations were compared between HFK LXSN and HFK 8E6. The results showed that 8E6 increases genomic variations within 200 kilobase around the CAS9-induced double-strand breaks, indicating that HPV 8E6 deregulates double-strand break repair and increases genomic instability. The purpose of immunoblot is to measure transfection efficiency, which should be considered during data analysis.

In addition to transfection efficiency, immunofluorescence microscopy could be used to confirm double-strand break induction using phospho-H2AX as a marker. We use the beta HPV 8E6 as an example. This technique can be used to detect changes in mutation frequency or type caused by other reagents such as small molecule inhibitors.