Peer review of: Multistable and dynamic CRISPRi-based synthetic circuits (2019)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Manuscript by Santos-Moreno and Schaerli describes application of dCas9 to design diverse 
transcriptional regulatory circuits in E.coli. This includes several “classical” circuits such as a 
bistable switch, IFF loop and an oscillator. Results in terms of the performance of dCas9/sgRNA 
instead of natural and designed DNA binding proteins used previously are quite impressive, 
although it is not clear how some of those designs could function as they apparently violate some 
of the design principles. For publication in NatCommun this should be elucidated. 
I have the following specific comments: 
1. Repression by DCas9 downstream from the promotor has been shown before. However this 
depends strongly on the distance from the promotor, while the results in this MS show very similar 
repression by different sgRNAs. Sequences of all annotated DNA constructs should be provided in 
the supplement in order to enable others to check the design and replicate the study, for this 
segment but also for all presented results. It would also be interesting to identify the specific 
context of the csy4 cleavage sequences. Are all cleavage sites really necessary ? 
2. Demonstration of hysteresis is not sufficient to claim functional bistable switch. There are many 
potential reasons for the occurrence of hysteresis – from the reporter protein degradation rate, 
remaining inducer in the medium etc. Authors should demonstrate the time course of the two 
states and that the state remains stable even when the inducer has been removed. 
3. Also as shown by theoretical analysis and experiments using monomeric DNA binding domains, 
nonlinear response, predominantly provided by cooperativity is required for the bistability. None of 
this seems to be present in the dCas9/sgRNA. If this system indeed exhibits a bistability authors 
should provide an explanation, although they argue that this is beyond the scope of this work. 
4. Performance of the bandpass filter by an IFFL looks very nice. What is the advantage of a 
double stripe phenotype if they occur at exactly the same concentration range. Demonstration of a 
double stripe phenotype would have been much more exciting by a noncoincident inducer 
responsiveness or responsiveness to two different inducers. 
5. As authors also comment synchronization of cells by CRISPRilator is completely unexpected as 
there do not seem to be any soluble mediator that would transfer the information between cells. In 
the absence of a plausible and verifiable mechanism we cannot exclude the possibility of some 
experimental setup bias, particularly since the oscillations do not seem to dampen as in almost all 
other oscillators and they start without adding an inducer. Controls should have been performed 
e.g. by omitting some components of the oscillator. Does the frequency depend on the flow rate as 
in case of soluble mediator driven quorum-based oscillations (Danino, Nature 2010)? 
 
 
 
Reviewer #2: 
Remarks to the Author: 
This paper demonstrates that some of the standard example circuits in synthetic biology can be 
constructed in a very clean and straightforward way with CRISPRi components. CRISPRi is 
desirable compared to protein based regulation because it is much easier to program, or re-wire, 
circuits. Thus, using essentially the same types of parts, the authors build a not gate, a bistable 
switch, an IFFL, and a repressilator. They authors employ a number of good engineering 
techniques, such as putting the entire circuit on one plasmid and using orthogonal degradation 
tags, to get what look like very nice behaviors from circuits that are often quite difficult to tune. 
One of the main assumptions the authors test, and disprove, is that CRISPRi is might not exhibit 
sharp enough deactivation (sometimes called cooperatively) to make these circuits work. 
 
It is been 19 years since the original bistable switch and repressilator were published. The paper 
shows we have simultaneously come a long way with building predictable circuits, while also 
showing that we have not come that far. We are still tuning circuits with three components without 
applying them to real world applications. 

 
In terms of impact, this paper clearly shows that these circuits can be more easily engineered than 
they were 19 years ago. However, it does not provide much insight as to why other than relying 
on what seem like better informed, and more rational choices about some specifics and some 
unsupported (but believable) claims about their circuits incurring lower metabolic load. There is no 
mathematical modeling, for example, showing a robustness analysis of the old architecture versus 
the new (this is just an example of something that I would qualify as a deeper analysis). 
 
Some specific comments: 
 
Measuring hysteresis using cells pre-cultured in one of the inducers, is an interesting way of 
characterizing the switches. However, other papers presenting bistable switches typically should 
show a time series [inducer one, no inducer, inducer two, no inducer]. So the data here are hard 
to compare. 
 
As for the oscillator: The say it rivals [40], in which the original repressilator was optimized to 
function much more robustly. It would be nice to see if the circuit in this paper works in liquid 
culture as in the corresponding one in [40] does. Otherwise it is hard to evaluate their claims. 
 

Multistable and dynamic CRISPRi-based synthetic circuits 

 

Javier Santos-Moreno, Eve Tasiudi, Jörg Stelling and Yolanda Schaerli