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.

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Figure 1: Initial tests of all second-generation activators on endogenous genes in HEK293T cells.
Figure 2: Activation of endogenous genes in HEK293T cells.
Figure 3: Evaluation of activator specificity by RNA sequencing.
Figure 4: Activation of endogenous genes in alternative human, mouse, and fly cell lines and effects of multiple guides.

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Acknowledgements

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.

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Contributions

A.C. and M.T. conceived of the study. A.C., M.T., B.W.P., and R.C. designed and performed experiments. S.J.H., R.J.C., and J.B. performed experiments. B.E.-C., B.E.H., and N.P. designed and performed all experiments in Drosophila melanogaster. D.T.-O. and E.J.K.K. performed RNA-seq experiments and analyzed data. J.J.C. and G.C. supervised the study. A.C. and M.T. wrote the manuscript with support from all authors.

Corresponding authors

Correspondence to Alejandro Chavez or George Church.

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Competing interests

G.C. has equity in Editas and Caribou Biosciences.

Integrated supplementary information

Supplementary Figure 1 Additional tests of activators on endogenous genes in HEK293T cells.

Data represents the mean + s.e.m. (n = 2 independent transfections). Source data

Supplementary Figure 2 Effect of Baseline Level of Expression on Activation.

Data represents the mean activation from Fig. 2b and Supplementary Fig. 1. Baseline expression data calculated from the Puc19 control sample for each experiment. Source data

Supplementary Figure 3 Multiplexed activation of two sets of three endogenous human genes.

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

Supplementary Figure 5 Combinations of different activator components.

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

Supplementary Figure 6 Recruitment of different activation domains to SAM and Suntag.

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

Supplementary Figure 7 SAM and Scaffold gRNA chimeras.

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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Supplementary Figures 1–7, Supplementary Tables 1 and 2, and Supplementary Notes 1 and 2 (PDF 1029 kb)

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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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