Geneart crispr grna design tool

DNA oligonucleotides used for gRNA targeting were designed with the GeneArt CRISPR gRNA Design Tool (Thermo Fisher Scientific; Invitrogen Life Technologies, Carlsbad, CA, USA). To examine the cleavage efficiency of gRNA, a series of gRNAs flanking the target site was designed and synthesized. Each individual gRNA was combined with Cas9 nuclease (Invitrogen Life Technologies, Carlsbad, CA, USA) to form Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs). The Cas9 RNPs were then used to transfect the iPSCs, a task for which the Neon Transfection System (Invitrogen Life Technologies, Carlsbad, CA, USA) was used. Genomic editing efficiency was then evaluated through T7 Endonuclease I (T7E1) assay 48 h after transfection. The gRNAs that had both the highest cleavage efficiencies and closest proximity to the target site were selected for the subsequent genome editing. For precise genome editing, the Cas9 RNPs and repair template (ssODN from IDT, Coralville, IA, USA) were coelectroporated into the iPSCs. The transfected iPSCs were then clonally expanded to derive isogenic cell lines. The single-nucleotide substitution was screened using a TaqMan SNP Genotyping Assay (Applied Biosystems, Forster City, CA, USA) and confirmed through Sanger sequencing.

To generate keratinocytes that are deficient for IL-20R1, we transfected HaCaT cells with three different CRISPR/Cas9 ribonucleoproteins (RNPs) targeting the IL20R1 gene using the GeneArt CRISPR gRNA Design Tool from Thermo Fisher Scientific. Synthesis of three different guide RNAs (gRNA) was performed using probes designed by the Invitrogen™ TrueDesign™ Genome Editor as following: IL20R1-gRNA-C1 5′-AGACACGTTATACTTCAGAT-3′ PAM TGG targeting exon 4, IL20R1-gRNA-C2 5′-CACTCTTTACTGCGTACACG-3′ PAM TGG targeting exon 5, and IL20R1-gRNA-C3 5′-CGTGTGGTTGGTCACACACT-3’ PAM GGG targeting exon 5. The three gRNA were synthesized in vitro using the GeneArt Precision gRNA Synthesis Kit (Thermo Fisher) and were purified using the gRNA Clean Up Kit. Transfection of cells with CRISPR/Cas9 RNP complexes were performed by mixing 1 μg of TrueCut™ Cas9 Protein v2 (Thermo Fisher) with 240 ng of gRNA in 1.5 μl of Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (Thermo Fisher) for 10 min at room temperature. As negative control, a non-targeting TrueGuide™ sgRNA Negative Control was used. RNPs were then added to 5.10⁴ HaCaT cells in a 24-well plate for 2 days before knockdown evaluation by western blot. Cells were further cultured by limiting dilution to generate IL20R1^−/− HaCaT clones.

DNA oligonucleotides used for gRNA synthesis were designed with the GeneArt CRISPR gRNA Design Tool available at (www.thermofisher.com, accessed on 9 June 2017). The two gRNAs were then synthesized using the GeneArt Precision gRNA Synthesis Kit (Cat no. A29377, Thermofischer, Waltham, MA, USA) according to the manufacture’s protocol. We estimated the concentration of gRNA by using the Qubit RNA BR Assay Kit (cat. no. Q10210). Each gRNA was combined with GeneArt Platinum Cas9 Nuclease (cat. no. B25640, Thermofischer, Waltham, MA, USA) to form the Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs), as seen in Figure S3 and Table S2.