Comparison of Cas9 activators in multiple species

Abstract

Several programmable transcription factors exist based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems, and we assess the role of cooperativity in maximizing gene expression.

Figure 1. Initial tests of all second-generation activators on endogenous genes in HEK293T cells

(a)dCas9-VP64 and dCas9-VPR both work via activation domains fused to the C-terminus of Cas9. SAM uses dCas9-VP64, but recruits more activation domains to the gRNA. Scaffold recruits multiple copies of VP64 to the gRNA. Suntag uses single chain antibodies to recruit multiple copies of VP64 to the peptide tail. P300 uses the catalytic core of the epigenetic modifier fused to dCas9 to modify the chromatin around the promoter to drive transcription. VP160 is the direct fusion of 10 repeats of VP16 protein to dCas9 instead of the usual four that makes up VP64. VP64-dCas9-BFP-VP64 drives transcription via the fusion of VP64 to both the N and C-termini of Cas9 (b)Data indicate the mean + s.e.m (n = 2 independent transfections).

Figure 2. Activation of endogenous genes in HEK 293T cells

(a) RNA expression analysis on 6 endogenous human genes. Data indicate the mean + s.e.m (n = 2 independent transfections). (b) Multiplexed activation of six endogenous human genes. Data indicate the mean + s.e.m (n = 2 independent transfections).

Figure 3. Evaluation of activator specificity by RNA sequencing

(a) Gene expression levels (log₂TPM, Transcripts Per Million) in cells transfected with the indicated activator targeting HBG1 (y axis) vs. expression in cells transfected with guide RNA only (x axis). R indicates Pearson's correlation coefficient, calculated for log-transformed values on all genes except HBG1. Genes with 0 TPM in both replicates of either the activator or control were excluded before log transformation. Average of two biological replicates shown. (b) Histograms showing the distribution of fold-changes in gene expression (activator/guide control). Genes were filtered to include only those with TPM > 0.1. Bottom panel is zoomed in on the range 2-400. In both panels arrows indicate fold-change of HBG1 for the indicated activator. Average of two biological replicates shown.

Figure 4. Activation of endogenous genes in alternative human, mouse and fly cell lines and effects of multiple guides

(a) Activation of endogenous genes in Hela, U-2 OS and MCF7 cells. Data indicate the mean + s.e.m (n = 2 independent transfections). (b) Activation of endogenous genes in mouse and fly. Data indicate the mean + s.e.m (n = 2 independent transfections) (c) Samples were transfected with the indicated guide or mixtures of guides. Theoretical sum indicates sum of the relative RNA expression for each activator of the individual gRNAs. Data indicate mean +S.E.M. (n = 2 independent transfections)