Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds

Jesse G Zalatan; Michael E Lee; Ricardo Almeida; Luke A Gilbert; Evan H Whitehead; Marie La Russa; Jordan C Tsai; Jonathan S Weissman; John E Dueber; Lei S Qi; Wendell A Lim

doi:10.1016/j.cell.2014.11.052

Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds

Cell. 2015 Jan 15;160(1-2):339-50. doi: 10.1016/j.cell.2014.11.052. Epub 2014 Dec 18.

Authors

Affiliations

¹ Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94158, USA.
² Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; Energy Biosciences Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
³ Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, CA 94158, USA.
⁵ Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, CA 94158, USA; Biomedical Sciences Graduate Program, University of California San Francisco, San Francisco, CA 94158, USA.
⁶ Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA.
⁷ Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA. Electronic address: slqi@stanford.edu.
⁸ Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA. Electronic address: wendell.lim@ucsf.edu.

Abstract

Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

CRISPR-Cas Systems*
Gene Expression*
Genetic Techniques*
HEK293 Cells
Humans
Metabolic Engineering
RNA, Guide, CRISPR-Cas Systems / genetics
Saccharomyces cerevisiae / genetics
Streptococcus pyogenes / genetics

Substances

RNA, Guide, CRISPR-Cas Systems

Abstract

Publication types

MeSH terms

Substances

Grants and funding