Multi‐input CRISPR/Cas genetic circuits that interface host regulatory networks

Molecular Systems Biology

Alec AK Nielsen¹ 

[1] Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

关键词: CRISPR;    genetic compiler;    synthetic biology;    TALE;    TetR homologue;   

DOI  :  10.15252/msb.20145735

来源: Wiley

【 摘 要 】

Abstract

Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and designed sgRNAs to build transcriptional logic gates and connect them to perform computation in living cells. We constructed a set of NOT gates by designing five synthetic Escherichia coli σ₇₀ promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These sgRNAs exhibit high on-target repression (56- to 440-fold) and negligible off-target interactions (< 1.3-fold). These gates were connected to build larger circuits, including the Boolean-complete NOR gate and a 3-gate circuit consisting of four layered sgRNAs. The synthetic circuits were connected to the native E. coli regulatory network by designing output sgRNAs to target an E. coli transcription factor (malT). This converts the output of a synthetic circuit to a switch in cellular phenotype (sugar utilization, chemotaxis, phage resistance).

Synopsis

Genetic gates are assembled based on dCas9 and engineered small guide RNAs (sgRNAs) that drive Cas9 to target promoters. These transcriptional gates are linked to build larger genetic circuits that are connected to the natural regulatory network of the cell.

• Synthetic promoters and cognate sgRNAs exhibit large dynamic range and negligible crosstalk.
• sgRNA-based NOT gate response functions are non-cooperative and quantitatively different from those based on transcriptional repressors.
• Multi-input logic gates are constructed by layering orthogonal sgRNAs.
• Host regulatory networks can be interfaced through the design of sgRNA circuit outputs targeted to native transcription factors.

【 授权许可】

CC BY   
© 2014 The Authors. Published under the terms of the CC BY 4.0 license

Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.