Genomic DNA purified from DRG and spinal cord tissues using Quick-gDNA MiniPrep Kit (Cat# 11–317AC; Genesee, San Diego, CA). PCR was performed using 5X HOT FIREPol Evagreen (Cat# 08–24-00020; Solis Biodyne, Tartu, Estonia) according to the manufacturer’s instructions. Specific primer sequences used are given in Supplementary Figure 1 (available online at http://links.lww.com/PAIN/A446 ). PCR efficiency was tested by running the samples on a 1% agarose gel containing SYBR Safe DNA Gel Stain (Cat# S33102; Thermo Fisher Scientific). Samples were sent for Sanger sequencing and obtained sequences were analyzed by alignment with the target regions within the Rattus norvegicus genome. As a control for off-target effects of our CRISPR/Cas9 editing strategy, we sequenced 11 potential off-target binding sites predicted using COSMID (crispr.bme.gatech. edu).10 (link) We sequenced 11 genomic regions, in 16 tissues, DRG (L4, L5, and L6) and dorsal horn of the lumbar region of the spinal cord (n = 4 controls and n = 4 CRISPR Nf1 per tissue). We found only 1 occurrence of an off-target alteration among the 176 sites screened (Supplementary Figure 1 , available online at http://links.lww.com/PAIN/A446 ); this occurred in a noncoding region of the DNA.
Protocol detail
CRISPR/Cas9 Off-Target Screening in Rat Tissues
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Agarose
Binding sites
Cosmid
Crispr
Dorsal horn of spinal cord
Genomic
Lumbar region
Pain
Primer
Rattus norvegicus
Spinal cord
Stain
Tissue
Corresponding Organization :
Other organizations : University of Arizona, Korea Institute of Brain Science, Korea Institute of Science and Technology, Korea University of Science and Technology, Mayo Clinic in Florida
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Variable analysis
independent variables
- CRISPR/Cas9 editing strategy
- Occurrence of off-target alterations in the 176 sites screened
- Genomic DNA purified from DRG and spinal cord tissues using Quick-gDNA MiniPrep Kit
- PCR performed using 5X HOT FIREPol Evagreen according to the manufacturer's instructions
- Specific primer sequences used (see Supplementary Figure 1)
- PCR efficiency tested by running the samples on a 1% agarose gel containing SYBR Safe DNA Gel Stain
- Obtained sequences analyzed by alignment with the target regions within the Rattus norvegicus genome
- 11 potential off-target binding sites predicted using COSMID and sequenced in 16 tissues (DRG L4, L5, L6 and dorsal horn of the lumbar region of the spinal cord, with 4 controls and 4 CRISPR Nf1 per tissue)
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