Transit x2

HEK293T cells were seeded at 2×10⁴ cells/well in 96-well plates approximately 20 hours prior to transfection. Each transfection reaction consisted of 1.25 μL of TransIT-X2 (Mirus Bio) with 70 ng of nuclease, 30 ng of sgRNA/crRNA, and 110 ng of anti-CRISPR expression plasmids in a final volume of 20 μL otherwise containing Opti-MEM (Thermo Fisher Scientific). For control conditions containing no Acr plasmid, 110 ng of a pCMV-EGFP plasmid was utilized; for non-targeting sgRNA/crRNA conditions, 30 ng of an empty U6 promoter plasmid was used. For titration experiments, cells were transfected with 70 ng of nuclease, 30 ng sgRNA/crRNA, varying amounts of acr expression and DNA stuffer plasmids totaling 96.5 ng (0.5 ng Acr with 96 ng stuffer; 2.75 ng acr with 93.75 ng stuffer; 16 ng acr with 80.5 ng stuffer), and 1.17 μL of TransIT-X2 (Mirus Bio) in 20 μl Opti-MEM. DNA stuffer plasmids were an orthogonal and incompatible pCAG-MbCas12a expression plasmid. Genomic DNA was harvested approximately 72 hours post-transfection by suspending cells in 100 μL of lysis buffer (20 mM Hepes pH 7.5, 100 mM KCl, 5 mM MgCl₂, 5% glycerol, 25 mM DTT, 0.1% Triton X-100, and 30 ng/μL Proteinase K (NEB)), followed by incubation at 65 °C for 6 minutes and 98 °C for 2 minutes. All experiments were performed with at least 3 independent biological replicates.

The Hut-78 cell line was maintained in RPMI 1640 (Gibco) medium with 10% heat inactivated fetal bovine serum (FBS; Atlanta Biologicals). Hut-78 or primary CD4+ T cells from donors with rs1015164AG genotype were plated at a density of 0.5×10⁶ cells/well in a 12 well plate and transfected with 100nM final concentration of either Control siRNA or gene-targeting (CCR5AS or Raly) siRNA optimized using TransIT-X2® (Mirus Bio LLC) protocol. The cells were incubated for 24 hours in a 37°C CO₂ incubator before determining mRNA expression or the cell-surface expression of CCR5. Hut-78 cells were plated at a density of 0.5×10⁶ cells/well and transfected with 1μg/well of the CCR5 3’UTR reporter constructs in the psichek2 vectors using the optimized TransIT-X2® (Mirus Bio LLC) protocol. Transfected Hut-78 cells were incubated for 24 hours at 37°C in a CO₂ incubator. The cells were lysed, and Firefly and Renilla luciferase activity were measured using the Dual Luciferase Reporter Assay System (Promega). Renilla luciferase activity was normalized relative to Firefly luciferase activity for each transfection. All experiments were performed with six replicates in two independent experiments.