Spcas9

Two gRNA sequences were designed to flank each side of B2_Mm2.Dicer1 in order to delete the element and generate knockout J774A.1 cells via SpCas9 (Integrated DNA Technologies #1081060). All gRNA sequences were also verified to uniquely target the locus of interest using the UCSC BLAT tool (Kent, 2002 (link)) against the mm10 genome assembly.

pCMV-GFP (plasmid DNA; 3705 bp) was purchased from Altogen Biosystems (Las Vegas, NV, USA). Dasher GFP mRNA (1 kb) was purchased from Aldevron. SpCas9 and sgRNA targeting the TRAC or TRBC locus (TRAC: 5′-AGAGUCUCUCAGCUGGUACA-3′; TRBC: 5′-GGAGAATGACGAGTGGACCC-3′) were purchased from Integrated DNA Technologies (Coralville, IA, USA). Ribonucleoprotein (RNP) complexes were formed by mixing equimolar quantities of SpCas9 and each sgRNA and incubating for 10 min at room temperature before addition to cell suspensions in electroporation buffer.

For the generation of CD147 knockout Jurkat cells, the sgRNA targeting CD147 molecule^{34 (link)} was generated using the GeneArt Precision gRNA Synthesis kit (Invitrogen, A29377). The RNP complex was prepared by mixing 150 μg/mL of SpCas9 (Integrated DNA Technologies) and 90 μg/mL of sgRNA at a 1:3 molar ratio, then incubated for 10 min at room temperature. 1 × 10⁶ Jurkat cells were nucleofected with RNP using the Cell Line Nucleofector Solution V (Lonza, VCA-1003) and the program X-005. On day 11, the edited cell pool (CD147 knockout Jurkat cells) was used to analyze the specificity of HuScFvM61B9 by immunofluorescence analysis using the protocol described above except 40 μg/ml concentration was used. Untransfected Jurkat cells were used as a positive control.