Plasmid- or Ribonucleoprotein-Mediated CRISPR/Cas Gene Editing in Primary Murine T Cells

Forced expression of the non-coding RNA miR-17∼92 restores activation and function in CD28-deficient CD4+ T cells

“…Protospacer sequences were taken from the Brie library ( Doench et al., 2016 ) and ordered as CRISPR RNAs (crRNAs) from IDT. Cas9 ribonucleoproteins (RNPs) were prepared as described ( Dolz et al., 2021 ). During preparation, 1.44 μL of 100 mg/mL of poly-L-glutamic acid (PGA) solution (Sigma) was added to gRNAs to improve cutting efficiency ( Nguyen et al., 2020 ).…”

TGF-β specifies T FH versus T H 17 cell fates in murine CD4 + T cells through c-Maf

“…Cutting efficiencies were assessed using Sanger sequencing and the tracking indels by decomposition method (http://shinyapps.datacurators.nl/tide/) ( 89 ). Cas9 ribonucleoproteins (RNPs) were prepared according to previously described protocols ( 90 – 92 ). CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA; #1072534) (both IDT) were mixed in equimolar concentrations in a sterile microcentrifuge tube or polymerase chain reaction plate, incubated at 95°C for 5 min, and cooled down at room temperature for 10 min.…”

Proteomics of immune cells from liver tumors reveals immunotherapy targets

“…We followed a protocol that was previously established by Lukas Jeker and colleagues. 47 , 79 FACS-purified T cells (human or mouse) were activated with plate-bound anti-CD3ε (1 μg/mL) and anti-CD28 (0.5 μg/mL) antibodies. After 24h, T cells were electroporated with a pSpCas9(BB)-2A-GFP plasmid (PX458, Addgene #48138) in which gRNAs targeting different genes were inserted by Golden Gate cloning (see Table S7 for gRNA sequences).…”