Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology

Abstract

One of the goals of synthetic biology is to build regulatory circuits that control cell behavior, for both basic research purposes and biomedical applications. The ability to build transcriptional regulatory devices depends on the availability of programmable, sequence-specific, and effective synthetic transcription factors (TFs). The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR) system, recently harnessed for transcriptional regulation in various heterologous host cells, offers unprecedented ease in designing synthetic TFs. We review how CRISPR can be used to build synthetic gene circuits and discuss recent advances in CRISPR-mediated gene regulation that offer the potential to build increasingly complex, programmable, and efficient gene circuits in the future.

Towards combinatorial transcriptional engineering

“…As far as activation is concerned, dCas9 fused to VP64 transactivation domain leads to approximately three-fold upregulation of target gene expression in S. cerevisiae (Farzadfard et al, 2013). But the activation efficiencies of the simple dCas9 fusion is lower than that of ZNFs and TALEs in mammalian systems (Jusiak et al, 2016). There is a need to simultaneously recruit multiple trans-activators for significant activation.…”

“…Thus, CRISPR/Cas based TFs make for good bidirectional regulators that may be used to switch on or switch off a gene depending on where they are targeted (Jusiak et al, 2016). Farzadfard et al, (2013) noted that the gRNA mediated targeting of a hybrid TF was extremely sensitive to the target sequence.…”

“…The specificity of CRISPR based sTFs is greater than that of ZNFs, TALEs or even the Cas9 nucleases, as indicated by RNA-Seq studies in mammalian systems Konermann et al, 2015). Jusiak et al, (2016) suggested that this may be due to the high position-specific activity of CRISPR based TFs. The high sensitivity and modularity of the CRISPR/Cas9 based sTFs can be exploited in regulating complex transcriptional networks, where each component of the circuit is controlled by a unique gRNA/sTF combination.…”

“…In the context of CRISPR/Cas9 based systems, this is a major challenge as the gRNAs expressed from inducible RNAP II promoter systems are not retained within the nucleus -a critical requirement for their activity in eukaryotes. The nuclear retention of gRNAs, which is currently achieved through their expression from constitutive RNAP III promoters comes at the cost of a low induction potential of the CRISPR/Cas9 systems (Jusiak et al, 2016). This major drawback has been addressed by either regulating the expression of the dCas9 based TF or through innovative gRNA expression systems, which are discussed in the next section.…”

CRISPR genome editing and its medical applications

“…Regulatory pathways in bacterial and mammalian cells have been developed using transcriptional regulatory systems on the basis of CRISPR-Cas9. The progress made in transcriptional regulation by CRISPR allows multiple genes to be set simultaneously [22,23].…”