Trucut v2 cas9 protein

sgRNA targeting the first exon of gene encoding GM-CSF was designed and synthesized according to the protocol described in the kit (Thermo Fisher). Primary T cells from a healthy donor (purchased from vendor PPA research) were activated with anti-CD3&CD28 dynabeads (Thermo Fisher) and then electroporated with RNP complex of the sgRNA candidates and Trucut V2 Cas9 protein (Thermo Fisher). Afterward, T cells were analyzed by intracellular staining of cytokines (LSR II, BD) to assess the efficiency of gene editing. The sgRNA targeted sequences before PAM motif are listed as:
1 GCTGCAGAGCCTGCTGCTCT; 2 GGAGCATGTGAATGCCATCC;
3 GCATGTGAATGCCATCCAGG; 4 GAGACGCCGGGCCTCCTGGA;
5 GATGGCATTCACATGCTCCC; 6 GCTCCCAGGGCTGCGTGCTG;
7 GCGTGCTGGGGCTGGGCGAG; 8 GCTGGGGCTGGGCGAGCGGG.

Peripheral blood was collected from the enrolled patient and processed with Ficoll (GE Healthcare) gradient centrifugation to isolate PBMC. T cells were purified from PBMC with the pan T isolation kit (Miltenyi). Purified T cells were activated with anti-CD3&CD28 dynabeads (Thermo Fisher) and transduced with 3rd generation lentivector encoding anti-CD19 or anti-BCMA CAR co-expressing IL6/IL1 blockers (scFv derived from Sirukumab and IL1RA), followed by electroporation with RNP complex of the sgRNA (targeting GM-CSF and TRBC) and Trucut V2 Cas9 protein (Thermo Fisher). T cells were further ex vivo expanded and analyzed for CAR expression and gene editing efficiency (Novocyte, Agilent). The CART cells were also tested for sterility and in vitro killing of leukemia cells Nalm6 (ATCC) expressing GFP or RPMI-8226 (ATCC) cells (Novocyte, Agilent).