Peer review of: A non-invasive far-red light-induced split-Cre recombinase system for controllable genome (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
This paper describes the development of a far red light inducible Cre-lox system in diverse 
mammalian cells and mice. Light is an attractive modulator of Cre activity in mice due to its 
relative non-invasiveness and simplicity, and its super spatial, temporal, and quantitative control 
properties. Far red light is particularly desirable due to its deep tissue penetration and low 
phototoxicity. In previous work, groups have developed UV and blue light inducible Cre-lox 
systems. However those systems have limitations including phototoxicty and poor tissue 
penetration of the controlling light wavelength. Thus, the current work is interesting to a broad 
audience. 
 
To achieve this goal, the authors express a previously developed N-terminal fragment of a split Cre 
recombinase (CreN) fused to the Coh2 domain, which heterodimerizes with the DocS domain. They 
then express a DocS-fusion to the corresponding C-terminal domain of Cre (CreC) under control of 
their previous Bacteriophytochrome-based far red light inducible mammalian promoter system. Far 
red light exposure results in expression of DocS-CreC, which complements CreN-Coh2 in order to 
activate Cre recombinase activity, and thereby excision of DNA residing between two loxP sites. 
The authors demonstrate their new system can be delivered with adeno-associated virus (AAV), 
and can be activated with a far red LED, which should make it widely useful as an in vivo research 
tool. The system outperforms the blue-light inducible alternatives in vitro. Furthermore, it has an 
advantage over the recent near infrared light up conversion nanoparticle methods because it is 
fully genetically encoded and requires no introduction of nanoparticles to the tissue site of interest 
in the animal. 
 
Overall, the work is well done, the results clear, and the paper well written. I believe it is suitable 
for publication in Nature Communications if the authors address several minor issues. 
 
*The 4 plasmid design that the authors use to optimize their system is described in a way that is 
very difficult to understand (lines 120-124). Rather than relying upon the unwieldy names of each 
of these plasmids, the authors should describe the important components of each of the plasmids 
and the role of each plasmid in the overall design. I have a similar comment for lines 175-176, 
where they combine the plasmids for in vivo work. 
 
*There are a number of undefined abbreviations (e.g. pA, STOP, GOI, 3*whiG) in Figure 1. Due to 
the very large number of elements in these figures, the authors should define everything in the 
figure legend. 
 
*Why is EGFP fluorescence reported in the unusual and non-intuitive units of “Weighted EGFP” in 
Figure 2a as opposed to standard units like EGFP fluorescence per cell? What weighted EGFP is, 
why it is used, and how it is calculated should be clearly described in the legend. 
 
*It is not immediately clear that Cre activity induces TdTomato expression in livers of the 
Gt(ROSA)…mouse strain. This lack of clarity makes the results of light exposure difficult to 
interpret at first. The authors should explain the design of this mouse strain, and the rationale for 
the experiment prior to discussing their experiment and results. 
 
*The data in Figure 4, where the authors are injected the plasmids into the mice, are weak relative 
to the AAV-delivery data in Figure 5. It seems that researchers will be interested in the AAV 
delivery mode, as it is very common, and not the free plasmid delivery mode. I think Fig. 4 is a 
distraction and should be moved to the supplemental materials. 
 
*There is a red light activated AAV that has been used for optogenetic activation of gene 
expression in vitro (Gomez et al. ACS Nano 2016). This paper is relevant prior work and should be 

cited. 
 
*The authors should move descriptions of how their system works from the Discussion to the 
Introduction. The Discussion should focus on the benefits and limitations of the system, future 
work that can be done to better understand how well it works (e.g. single cell-level, different 
tissues, organs, etc.), improve upon it, and future applications of the system. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In this report, the authors describe a far-red light-induced split Cre (FISC) system. Far-red light 
(FRL) stimulates the system allowing for expression and reconstitution of a split Cre recombinase 
under the control of a previously described FRL inducible system. The transcriptional system works 
in response to FRL through generation of c-di-GMP through a bacterial phytochrome Bph and 
subsequent activation of a c-di-GMP responsive transcriptional activator (P65-VP64-BldD). Activity 
of a split-Cre (split along previously identified residues and reconstituted through the Coh2-DocS 
interaction) is controlled by putting the CreC component under the FRL transcriptional system 
promoter (PFRLx). The authors optimize many aspects of the system (PFRL promoters, linkers, lox 
sites) in HEK cells to allow for low background (dark) recombination and low phototoxicity, with 
significant recombination in FRL. Red-light is able to penetrate the organs of mice, allowing for 
Cre-recombinase mediated DNA recombination in the livers of BALB/c wild-type mice and in a CRE-
tdTomato reporter mouse model. They can split their system between several vectors, allowing for 
packaging and delivery with adeno-associated virus (AAV). 
 
Site-specific DNA recombination using Cre recombinase (which catalyzes homologous DNA 
recombination between specific 34-bp loxP sites) is a commonly used genome engineering 
technique. Certainly, an FRL light controlled Cre is a very useful alternative to chemically inducible 
systems (which lack spatial resolution) and blue/UV-light inducible systems (which lack depth 
penetration and cause phototoxicity). Overall this article is well written and the data well 
presented. I have a few comments that should be addressed before publication: 
 
Major comments: 
• The results in Supplemental Figure 1 are confusing to me. Unless I am reading the figure 
incorrectly, it shows that background activity of the split-Cre is actually higher in the dark than in 
the light. How is this possible? I have a similar question about Supplemental Figure 2a, b. How is it 
that the activity is consistently higher in the dark, across multiple transfection conditions, for 
PFRLAa and PFRLc? 
• I don’t have a perfect suggestion for how to fix this, but in places the paper is hard to follow 
simply because of the number of multi-component labels to follow (e.g. lines 121-124). I don’t 
know if there is a way to shorten the “code name” for some of the components, e.g. just use 
CreC? Again, I don’t have a perfect suggestion on how to fix this but there were a lot of construct 
names specific to this paper that at times became a bit unwieldy. 
• Is background induction at all a function of time? For example, in Figure 2d we see that the SEAP 
production increases and the fold-change, but not the background induction. The data in Figure 1e 
(and Supplemental Figures 1,2) is taken after a specific amount of time so I don’t have a sense for 
how it varies with time or if it continues increasing (which I would expect). 
• What isn’t clear to me, but should be known from experiments like the experiment depicted in 
Figure 2a, is what the fraction of cells completing recombination is. This seems like a relevant 
piece of information unless the heterogeneity of recombination is not important in downstream 
applications. Figure 2f does make it appear that there is some heterogeneity, though whether this 
is due to light leakage from the mask edges isn’t obvious. It is also interesting to ask how 
recombination varies as a function of light intensity. Is it bimodal? I.e. at intermediate light 
intensities does some cells switch while others do not? Or left in intermediate light long enough 
would all cells switch, and so it is really the time to switching that is relevant? 

• What is also not clear to me is how long the light needs to be on for maximum recombination. In 
Figure 2d SEAP production is measured, but I assume this just keeps increasing whether or not 
recombination continues happening. It might be interesting to ask how different light durations 
affect SEAP production (i.e. at what point do you saturate the efficacy of the system with light?) 
• Line 174—You mention optimizing the distance between the two split Cre sequences, but Supp 
Figure 8 shows only two constructs. Did you do extensive optimization, and did you find any 
trends? Or was trying one other spacing enough to dramatically improve performance? Looking 
through Supp Table 3 I don’t see those intermediate plasmids? 
• Can you explain why your results for the PA-Cre differ from the results of Kawano, et al? They 
showed more induction with the PA-Cre system in vivo. 
• Should I take Figure 3C to mean there is substantial background in the mice? I don’t know what 
the background measurement is in mice with a luciferase reporter plasmid for example. 
• Line 245, 246---How do you conclude that the AAV is more efficient than other delivery 
methods? What delivery methods are you directly comparing to and can you make a clear 
comparison figure? 
• Please compare/contrast your method to methods like Kinjo, et al 2019 (Nature Methods) which 
allow for two-photon (2P) excitation by developing a FRET transfer from BFP to CRY2. 
Minor comments: 
• Line 73—“of UCNPs” 
• Line 101—FRTA acronym introduced without explanation. I had to go to Shao, et al to know that 
it refers to synthetic mammalian transactivators. 
• Is there a justification/explanation for why only inducing expression of one of the split-Cre 
components is preferable (particularly in terms of background) to inducing both of them? This 
doesn’t need to be tested experimentally but could be minimally discussed as part of an 
explanation for the design or in the discussion. 
• In a similar vein, is there any intuition for why the different linkers were tested and why they 
worked more or less well? 
• The explanation of the promoter construction (mostly given in Figure 1c without a detailed 
caption) could be expanded in the main text to explain the promoter structure. 
• Can you discuss why there is such a range of efficiency between cell lines, and does this mean 
tissue specific/cell line optimization would be necessary for this technology to be adopted by other 
labs? 
• Supplemental Figure 6—please double check that the 72h FISC points are not identical—the 
three points look identical to my eyes between the dark and illuminated samples, but it could just 
be my eyesight. 
• Line 195—I think there is an “it” missing before “superior” 
• Please clarify in the methods what the epi-fluorescence units are (counts/total counts) and how 
they were measured. 
• It is unfortunately that the spatial resolution of light is not employed in this paper in vivo. That 
would be exciting. 
• Line 272—I think potentially two sentences were accidentally concatenated here. The sentence 
as is doesn’t make sense.