Abstract
Controlling gene expression with sophisticated logic gates has been and remains one of the central aims of synthetic biology. However, conventional implementations of biocomputers use central processing units (CPUs) assembled from multiple protein-based gene switches, limiting the programming flexibility and complexity that can be achieved within single cells. Here, we introduce a CRISPR/Cas9-based core processor that enables different sets of user-defined guide RNA inputs to program a single transcriptional regulator (dCas9-KRAB) to perform a wide range of bitwise computations, from simple Boolean logic gates to arithmetic operations such as the half adder. Furthermore, we built a dual-core CPU combining two orthogonal core processors in a single cell. In principle, human cells integrating multiple orthogonal CRISPR/Cas9-based core processors could offer enormous computational capacity.
Highlights
Controlling gene expression with sophisticated logic gates has been and remains one of the central aims of synthetic biology
The OFF system consisted of three core components, namely dCas9-KRAB, an input guide RNAs (gRNAs), and a reporter construct with binding sites for igRNA between the human cytomegalovirus immediate-early promoter and the transcription start site (TSS)
The ON system consisted of four constructs: dCas9-KRAB, an igRNA, a regulatory gRNA with binding sites for the igRNA between the human U6 (hU6) promoter and the TSS of the rgRNA, and a reporter construct with binding sites for the rgRNA
Summary
Observed correct circuit performance after 24 h, the performance was improved after 48 h (SI Appendix, Fig. S7). Compared with synthetic DNA-binding domains, the generation of gRNA is both cost-effective and userfriendly, and future users can modify and extend the existing circuits In this context, the tRNA-expression system is essential to enable us to build up gene circuits by adding multiple layers of regulation by regulatory gRNAs. For consistency, we equipped all gRNA constructs with the tRNA-processing system, as the compatible structure facilitates the modularity of circuit design. As gRNA is itself used as an input signal, the system does not require additional induction Another advantage of CRISPR-mediated transcriptional regulation is the orthogonality of custom-designed gRNAs and their corresponding promoters [9]: Each gRNA-promoter set represents a basic unit for building up circuits, and these units can be combined to generate multiple layers of regulation for transcriptional control. The cell may be loaded with custom-programmed circuits, enabling the performance of various functions, as a “biocomputing core.” Each single cell can be considered a single-bit
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