Highly-efficient Cas9-mediated transcriptional programming

Alejandro Chavez; Jonathan Scheiman; Suhani Vora; Benjamin W Pruitt; Marcelle Tuttle; Eswar Iyer; Samira Kiani; Christopher D Guzman; Daniel J. Wiegand; Dimtry Ter-Ovanesyan; Jonathan L Braff; Noah Davidsohn; Ron Weiss; John Aach; Collins J Collins; George M Church

doi:10.1101/012880

bioRxiv · 10.1101/012880

Highly-efficient Cas9-mediated transcriptional programming

Alejandro Chavez·Jonathan Scheiman·Suhani Vora·Benjamin W Pruitt·Marcelle Tuttle·Eswar Iyer·Samira Kiani·Christopher D Guzman·Daniel J. Wiegand·Dimtry Ter-Ovanesyan·Jonathan L Braff·Noah Davidsohn·Ron Weiss·John Aach·Collins J Collins·George M Church

View original source ↗Export metadata ↓

Abstract

The RNA-guided bacterial nuclease Cas9 can be reengineered as a programmable transcription factor by a series of changes to the Cas9 protein in addition to the fusion of a transcriptional activation domain (AD)1-5. However, the modest levels of gene activation achieved by current Cas9 activators have limited their potential applications. Here we describe the development of an improved transcriptional regulator through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to Cas9. We demonstrate its utility in activating expression of endogenous coding and non-coding genes, targeting several genes simultaneously and stimulating neuronal differentiation of induced pluripotent stem cells (iPSCs).

Source connections

Published article ↗

Explore related subjects

Synthetic Biology Preprint

Keep this discovery

BibTeX RIS

Alejandro Chavez, Jonathan Scheiman, Suhani Vora, Benjamin W Pruitt, Marcelle Tuttle, Eswar Iyer, Samira Kiani, Christopher D Guzman, Daniel J. Wiegand, Dimtry Ter-Ovanesyan, Jonathan L Braff, Noah Davidsohn, Ron Weiss, John Aach, Collins J Collins, George M Church. 2014-12-20. Highly-efficient Cas9-mediated transcriptional programming. https://doi.org/10.1101/012880

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2016

Programming mRNA decay to modulate synthetic circuit resource allocation

Synthetic biomolecular networks embedded in host-cells compete with cellular processes for limited intracellular resources. Resource utilization is a major variable that impacts synthetic circuit behavior. Here we show that intracellular resources could be diverted from cellular operations to a synthetic circuit by programming global mRNA decay using the sequence-dependent endoribonuclease MazF. Synthetic circuit genes were protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and the metabolic flux of a high-value metabolite gluconate were significantly enhanced using this genome-scale control strategy. Proteomics measurements discovered a key host translation factor in need of protection to optimize the resource redistribution activity. A dynamic computational model demonstrated that the MazF mRNA-decay feedback loop achieved proportional control of MazF levels in an optimal operating regime. RNA-seq time-series measurements of MazF-induced cells elucidated the dynamic shifts in the transcript abundance and discovered regulatory design elements that could be used to expand the control mechanisms of the MazF resource allocator. Together, these results demonstrated that manipulation of resource allocation is a tunable parameter that can be used to redirect resources away from cellular processes to synthetic circuits to enhance target functions.

Synthetic Biology↗

bioRxiv2016

Engineering species-like barriers to sexual reproduction

We introduce a novel approach to engineer a genetic barrier to sexual reproduction between otherwise compatible populations. Programmable transcription factors drive lethal gene expression in hybrid offspring following undesired mating events. As a proof of concept, we target the ACT1 promoter of the model organism Saccharomyces cerevisiae using a dCas9-based transcriptional activator. Lethal over-expression of actin results from mating this engineered strain with a strain containing the wild-type ACT1 promoter.

Synthetic Biology↗

bioRxiv2016

Biosynthesis of the Antibiotic Nonribosomal Peptide Penicillin in Baker’s Yeast

Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. By taking genes from a filamentous fungus and directing their efficient expression and subcellular localisation, we here engineer the bakers yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide. Using synthetic biology tools combined with long-read DNA sequencing, we optimise productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

Synthetic Biology↗