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.
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Gene Expression Omnibus
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We would like to thank S. Vora, A. Tung, M.K. Cromer, and all the members of the Church and Collins labs for helpful discussions and technical assistance. G.C. acknowledges support from US National Institutes of Health (NIH) National Human Genome Research Institute grant P50 HG005550 and from the Wyss Institute for Biologically Inspired Engineering. In addition, A.C. was funded by National Cancer Institute grant 5T32CA009216-34, R.C. was funded by a Banting postdoctoral fellowship from the Canadian Institutes of Health Research, and J.J.C. was supported by Defense Threat Reduction Agency grant HDTRA1-14-1-0006. B.E.-C. acknowledges funding from the NIH under Ruth L. Kirschstein National Research Service Award F32GM113395 from the NIH General Medical Sciences Division. We would also like to thank J. Lee (Cold Spring Harbor, Cold Spring Harbor, NY), P. Mali (UCSD, La Jolla, CA), and S. Shipman (Harvard Medical School, Boston, MA) for gifting us cell lines.
G.C. has equity in Editas and Caribou Biosciences.
Integrated supplementary information
Data represents the mean + s.e.m. (n = 2 independent transfections). Source data
Data indicate the mean + s.e.m (n = 2 independent transfections). Source data
Supplementary Figure 4 Additional tests of activators on endogenous genes in Hela, U-2 OS, MCF7, N2A, NIH-3T3, and S2R+ cells.
(a) Each human cell line was transfected with the indicated activators and guides. 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). Source data
Samples were tested on both a single gene and a panel of multiplexed genes. Data represents the mean + s.e.m. (n = 2 independent transfections) See Supplementary Note 1 for more explanation on the canonical activator components. For the purposes of this figure, dCas9-10xGCN4 + normal guide + scFV-VP64 represents the canonical Suntag activator and dCas9-VP64 + SAM guide + ms2-p65-hsf1 represents the canonical SAM activator. Source data
dCas9-VP64 denotes the SAM version of VP64. Data represents the mean + s.e.m. (n = 2 independent transfections). See Supplementary Note 1 for more explanation on the canonical activator components. For the purposes of this figure, dCas9-VP64 + SAM guide + ms2-p65-hsf1 represents the canonical SAM activator. Source data
All samples contain dCas9 recruiting MCP-p65-hsf1 via different hairpin designs. All chimeras represent the SAM gRNA with the Scaffold tail appended on it with various parts disabled by either point mutation or deletion. Chimera 1 has the first MS2 extension of the SAM gRNA deleted. Chimera 2 has the second MS2 extension of the SAM gRNA deleted. Chimera 3 has the MS2 loop of the scaffold gRNA disabled via point mutation while Chimera 4 has the F6 loop of the scaffold gRNA disabled via point mutation. For full chimera gRNA tail sequences, refer to the Plasmids section of the supplement. Addition of the Scaffold tail to the end of the SAM gRNA resulted in worse activation than each system alone and there was no method of disabling any part of the hybrid hairpin which led greater activation. Data represents the mean + s.e.m. (n = 2 independent transfections). Source data
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Chavez, A., Tuttle, M., Pruitt, B. et al. Comparison of Cas9 activators in multiple species. Nat Methods 13, 563–567 (2016). https://doi.org/10.1038/nmeth.3871
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