Generation of a Nrf2 homozygous knockout human embryonic stem cell line using CRISPR/Cas9

Here, the authors generate a homozygous Nrf2 knockout human embryonic stem cell (hESC) line, H9Nrf2KO-A13, using the CRISPR/ Cas9 genome editing method. The human embryonic stem cell H9 line was grown in Essential 8 Medium on laminin-521 coated plates. Human ESCs were dissociated into single, resuspended in Nucleofection solution and electroporated with 30 μg Cas9 and 40 μg of in vitro-transcribed sgRNA using the Amaxa P3 Primary Cell 4D–Nucleofector Kit (Lonza). After 4 days, cells were replated as single cells at a very low density on laminin 521-coated plates in Essential 8 Medium supplemented with a Rho kinase (ROCK) inhibitor (Stemgent). Individual colonies were picked and expanded. Genomic DNA was then extracted using QuickExtract (Epicentre). The target region was amplified and subjected to paired-end read sequencing using Illumina MiSeq at LAS.