Amaxa 4d nucleofector kit

GLI2 and SPP1 knockouts in KP4 cells were generated using the RNP-electroporation method as previously described (Liang et al., 2015 (link)). One million KP4 cells were used per electroporation using the Amaxa 4D Nucleofector kit (V4XC-9064, Lonza). Guide RNA and Cas9 complexes were formed using 160 μM crRNA annealed to 160 μM tracrRNA (Dharmacon) and incubated with 40 μM Cas9 protein (purchased from University of California, Berkeley). Cutting efficiency was assessed 48 hr post-electroporation using PCR and sanger sequencing. GLI2 and SPP1 knockout was confirmed using quantitative RT-PCR, immunoblotting and ELISA after clonal expansion of single cells.
Guide RNA sequences 5’−3’:
GLI2 exon 2 – TTTGGCTTCTTGCTTCTCGGSPP1 exon 2 – GTATGGCACAGGTGATGCCTPCR primer sequences 5’−3’:
GLI2 exon 2 FW – GTGAAGGAGTGAGCGAACATGCGLI2 exon 2 RV – TCTTCGCCCTCCATAAACCCAGSPP1 exon 2 FW – GCAAAATTTCCCTTTCCCTTGCCSPP1 exon 2 RV – ACTGTGCTTGGTACTGGCCTAG

To create a mouse Tlr11 knock-out model in RAW264.7 cell line using CRISPR/Cas9 technology, a GFP expression vector containing two Tlr11-specific gRNAs (gRNA1: CTGGTGAGCCTTACCTTGACTGG and gRNA2: GTACAACCAGTGTCACATCTAGG) was constructed (YKO-RP009-mTlr11). Then the YKO-RP009-mTlr11 plasmids were introduced into RAW264.7-Cas9 cells using Amaxa 4D-Nucleofector kit (Lonza, Cologne, Germany), which could generate a large deletion in exon 2 of Tlr11. Two days post nucleofection, GFP⁺ cells were sorted by flow cytometry and single clones were seeded in 96-well plates. Subsequently, single-cell clones were expanded and genotyped using PCR with primers TLR11-F and TLR11-R. The PCR products of clones with predicted Tlr11-deletion were confirmed via Sanger sequencing. After sequencing, the Tlr11^-/- clones were further verified with qRT-PCR using primers Tlr11-qPCR-N-F and Tlr11-qPCR-N-R, which were designed inside the deletion region of Tlr11.