Extended Data Fig. 2: Optimizing inducible crRNAs for lentiviral transduction.
From: Scalable biological signal recording in mammalian cells using Cas12a base editors
a, Left, designs tested for transferring the inducible guide cassette into a lentiviral backbone. The inducible construct was placed in reverse orientation, to prevent the polyA from disrupting lentiviral packaging. Right, quantification of reporter GFP fluorescence in HEK cells transiently transfected with inducible guide, mutant base editor, and reporter, as well as additional rtTA for design 2. Data shown for 2 independent replicates, 3 days post-transfection and stimulation with dox. Design 1 was selected moving forward, as despite slightly worse performance than design 2 it had benefit of including constitutive open reading frame for selection. b, Left, schematic showing incorporation of additional repeat-spacer cassette to increase amount of guide available per mRNA transcript. Right, GFP fluorescence in GFP* HEK cells stably transduced with dox-inducible guide, 3 days post-transfection with base editor and stimulation with dox. Data shown for 3 independent replicates. c, Left, representative histogram of GFP* HEK cells stably transduced with NFKB-crRNA, 3 days post-transfection with base editor and stimulation with TNFα. Right, quantification of mean fluorescence and percent GFP + for 3 independent replicates. d, Quantification of A→G mutations in stably transduced NFKB-crRNA cells at the targeted stop codon in the GFP* locus as measured by MiSeq. Data are shown for 3 independent replicates.
