Our team is dedicated to developing induced pluripotent stem cell, iPSC therapies, for severe, currently incurable skin diseases such as recessive dystrophic epidermolysis bullosa. We aim to determine if precise gene editing coupled with iPSC's differentiation into skin cells can provide a viable treatment option for these conditions. Gene editing in somatic cells and iPSCs using CRISPR-Cas9 technology is transforming regenerative medicine by enabling personalized therapies for various diseases, including genetic skin disorders.
Current challenges in genetic engineering include off-target effects of gene editors. The therapeutic requires gene editors that precisely correct the gene of interest without altering any other locations in the genome. Our challenge is to demonstrate that gene editors do not introduce unintended edits beyond the target gene.
The CIRCLE-seq protocol enables the identification of bonafide potential off-target sites that other similar techniques might miss. A comprehensive analysis of these off-target sites provides valuable insight into the activity of genome editors, enhancing the safety of gene therapies, including our IPSC-based therapy for skin diseases. After growing induced pluripotent stem cells for five days, collect the cells by centrifugation and resuspend them in 10 milliliters of PBS.
Mix six microliters of cell suspension in six microliters of trypan blue for cell counting. After counting, aliquot two times 10 to the power of seven cells per tube. Centrifuge the cells at 300 G for three minutes at 25 degrees Celsius and discard the supernatant.
Prepare the focused ultrasonicator by homing the control arm. Fill the reservoir with purified, deionized water. On the control station laptop, access waterworks and click fill to start filling the system.
Adjust the temperature to 4.5 degrees Celsius. Transfer 25 micrograms of genomic DNA isolated from induced pluripotent stem cells into a microtube. Fill the tube to a total volume of 130 microliters with TE buffer.
Set the conditions to shear the DNA to an average length of 300 base pairs, duration of 10 seconds, peak power of 70, duty factor percent to 20, and cycles burst to 50. Divide the sheared genomic DNA into two portions of 65 microliters each. Purify the samples using 1.8 times the volume of XP beads, followed by magnetic rack separation.
Transfer the supernatant to a new PCR plate and measure the DNA quantity using a spectrophotometer. Finally, run one microliter of the alluded shear genomic DNA on a tape station. To begin, resuspend oSQT1288 and the hairpin adapter to a final concentration of 100 micromolar in TE buffer.
For adapter annealing, mix 40 microliters of oSQT1288, 10 microliters of 10X STE, and 50 microliters of nuclease-free water to reach a total volume of 100 microliters. After adapter annealing, mix 10 microliters of 5X ligation buffer, five microliters of DNA ligase, and five microliters of annealed hairpin adapter, Pipette 20 microliters of the adapter ligation master mix into each eluted DNA specimen containing beads. Place the samples in a thermocycler at 20 degrees Celsius for one hour, then hold at four degrees Celsius indefinitely.
Next, transfer 50 microliters of PEG/NaCl spray solution to the adapter-ligated DNA. Purify the samples using XP beads and magnetic rack separation. Elute the samples with 30 microliters of TE buffer pH eight and decant the supernatants into a new semi-skirted PCR plate.
Combine the samples and quantify the DNA using the dsDNA BR assay. To prepare the circularization master mix, combine eight microliters of nuclease-free water, 10 microliters of 10X TD4 DNA ligase buffer, and two microliters of T4 DNA ligase for a total volume of 20 microliters. Pipette 20 microliters of the circularization master mix to 80 microliters of 500 nanograms of DNA treated with user PNK.
Incubate the sample in a thermocycler at 16 degrees Celsius for 16 hours. The next day, add 100 microliters of XP beads to the circularized DNA and purify the samples, as demonstrated previously. Elute the sample with 38 microliters of TE buffer and decant the supernatant into a new semi-skirted PCR plate.
To cleave purified circularized genomic DNA, prepare in vitro cleavage master mix by combining five microliters of 10X Cas9 buffer, 4.5 microliters of Streptococcus pyogenes Cas9, and 1.5 microliters of genomic RNA for a total volume of 11 microliters. Incubate the cleavage master mix at room temperature for 10 minutes. to form the Cas9 gRNA RNP complexes.
Dilute 125 nanograms of plasmid-safe DNase-treated genomic DNA to a final volume of 39 microliters in nuclease-free water. Then, add 11 microliters of the cleavage master mix to the 39 microliters of DNA for a total volume of 50 microliters. Incubate the mixture in a thermocycler for one hour at 37 degrees Celsius and maintain it at four degrees Celsius indefinitely.
After incubation, add 50 microliters of XP beads to the in vitro cleaved DNA and purify the DNA on a magnetic rack. Elute the purified DNA in 42 microliters of TE buffer. For the analysis of next-generation sequencing data, install Python version 2.7, Burrows-Wheeler Aligner, and SAMtools.
Download the reference genome from the given website. Define the reference genome FASTA file, the output directory for the analysis, and the paths to the BWA and SAMtools commands. Specify the target sequences and the paths to the demultiplexed FASTQ files for both the nuclease-cleaved and control samples.
Execute the standard reference-based analysis using the following command. When executing the full pipeline, locate the output results of each step in a distinct output folder designated for that specific step.
