Extended Data Fig. 2: Optimizing the nuclear localizing signal and comparing to enAsdCas12a.
From: Multiplexed genome regulation in vivo with hyper-efficient Cas12a
a. Schematic to test two different nuclear localization signals. Constructs containing either 2×SV40 or 2×Myc NLS fused with WT dCas12a are co-transfected with Tet crRNA in TRE3-GFP HEK293T reporter cells. b, Representative flow cytometry histogram of BFP intensity, showing threshold for BFP + cells, and subset of ‘low BFP + ’ cells (similar to Fig. 1d). c-d, GFP fluorescence in BFP + (c) or ‘low BFP + ’ cells (d). e-g, GFP fluorescence in BFP + (f) or low BFP + cells (g) to compare WT versus hyperdCas12a (hyp) with 2×Myc NLS, as well as BFP and mCherry average fluorescence in each gated BFP group. h-i, Alignment of the structure of LbCas12a versus AsCas12a proteins (h) and alignment of peptide sequences (i) encompassing mutations harboured by enAsCas12a, a reported enhanced variant of Cas12a from Acidaminococcus with the E174R/S542R/K548R mutations5 corresponding to homologous residues (D156R/G532R/K538R) mutations in LbCas12a. j, Comparison of variants containing mutations of homologous residues in LbCas12a in ‘low BFP + ’ cells. Interestingly, D156R combined with G532R and/or K538R did not achieve activation higher than the single D156R mutant, in contrast to results with homologous residues in AsCas12a5. k, Comparison of hyperdCas12a versus enAsdCas12a with a single crRNA driven by U6 promoter in 1:1 versus 1:0.2 ratio of dCas12:crRNA, in TRE3G-GFP HEK293T cells. l, Comparison of hyperdCas12a versus enAsdCas12a with single crRNA driven by CAG promoter flanked by direct repeats (DR) specific to LbCas12a versus AsCas12a. m, Comparison of hyperdCas12a versus enAsdCas12a with dual crRNAs containing crTet on the second position and non-targeting crLacZ on the first position flanked by As or Lb direct repeats (DR). All transfections in this figure were carried out in TRE3G-GFP HEK293T reporter cells. Bar graph in f, g and k-m shows the mean of n ≥ 3 independent experiments; bar graph in j shows the mean of n ≥ 2 independent experiments; each data point represents value of an independent experiment.
