Inhibition of the DNA damage response increases detection sensitivity for mapping CRISPR off-target sites in vivo. NBThighlight
). In short, a femtosecond laser beam with a repetition rate of 80 MHz from a Ti:Sapphire laser head is split into two parts: one part produces an excitation beam coupled into a photonic crystal fiber for wide-spectrum light generation. The beam is further filtered by a frequency-tunable acoustic optical filter for multi-color excitation. The other part of the laser pulse is temporally stretched to ~300 ps , collimated and expanded, and wave-front modulated with a vortex phase plate .
For WTC-11 iPSCs, cells were dissociated from the plate using accutase . Electroporation was performed using the Lonza P3 Primary Cell 4D-Nucleofector X Kit L using code CA-137, on 10 million cells, and using 65 µl of the P3 solution mixture with EP enhancer per electroporation cuvette .
For adherent cells, approximately 10 million cells were gently rinsed with room temperature PBS, washed off the plate using 10 ml of DMEM with assistance from pipette squirts and a cell scraper, then transferred to a 15-ml Falcon tube. For suspension cells, approximately 10 million cells were transferred to a 15-ml Falcon tube, spun down at 200for 1 min, decanted, then resuspended with 10 ml of DMEM.
Beads pre-loaded with antibodies were prepared before cell collection. First, 50 µl of Protein A beads were used per immunoprecipitation and transferred to a 2-ml Eppendorf tube on a magnetic stand. Beads were washed twice with blocking buffer BB , then resuspended in 100 µl of BB per immunoprecipitation. Then, 4 µl of MRE11 antibody per immunoprecipitation was added and placed on a rotator for 1–2 h.
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