For knock-in of mRuby411 into the HIST2H2BE locus, we ordered 200-nt HDR templates in single-stranded DNA (5′-gcccggcgagctggccaagcacgccgtgtccgagggcaccaaggcggtcaccaagtacaccagctccaagGGTGGCGGCGAAACCTACGTAGTGCAAAGAGAAGTGGCAGTTGCCAAATACAGCAACtgagtccctgccgggacctggcgctcgctcgctcgagtcgccggctgcttgactccaaaggctcttttcagag-3′, Integrated DNA Technologies). Cas9 protein was expressed in E. coli and purified by the Kipreos laboratory at UGA as described previously11 (link). sgRNA and Cas9/sgRNA ribonucleoprotein complexes were prepared as described before12 (link). After the treatment of HEK293 FT cells with nocodazole (200 ng/mL, Sigma-Aldrich) for 16 h, we performed electroporation on an Amaxa Nucleofector 2b device with Nucleofector Solution V reagents (Lonza).
nocodazole-treated cells were resuspended at a concentration of 1 × 104 cells/μL in 100 μL of Nucleofector Solution V. We added cells to the RNP/donor template mixture (50 μL), electroporated using the Q-001 program, and quickly transferred to 12-well plates with pre-warmed media. Electroporated cells were cultured for 2–5 days and transfected with mRuby41-10 plasmid.
nocodazole-treated cells were resuspended at a concentration of 1 × 104 cells/μL in 100 μL of Nucleofector Solution V. We added cells to the RNP/donor template mixture (50 μL), electroporated using the Q-001 program, and quickly transferred to 12-well plates with pre-warmed media. Electroporated cells were cultured for 2–5 days and transfected with mRuby41-10 plasmid.
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