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Multistage Promoter Design Guideline

*The definition of symbols and abbreviation is the same as our TP model

In order to employ multi-stage promoter (mentioned as MP in the following steps), we must know how to deign one, and to predict the output strength of every-stage of it. Therefore we propose the following procedure for the users to design their multi-stage promoters.

1.Determine the required output levels.

2.Design the “base” of the MP.

1) Employ our TP model to design a tandem promoter system that have the potentials to produce the required output level. To avoid unpredictable interaction among sub-promoters, we recommend spacer sequences of ~20bp between each sub-promoters. ( Check our design)

2)Notice that dCas9 can not completely repress a sub-promoter, usually it can repress 90-99% of the output of it targeting promoter (depends on the concentration of dCas9 and gRNA). In our experiments, dCas9 achieve 90% repression. In the data of [1], dCas9 achieve 99% repression.

Figure 1. dCas9 regulation data of our experiments.
Complete repressed strength = Total strength * 0.1 Partial repressed strength = (Total strength - Single strength) * 0.1+ Single strength

Figure 2. dCas9 regulation data from [1]

3.Design the corresponding gRNA for the dCas9 regulation.

1)Employ well studied promoter to express gRNA, as both extension and truncation of the 5’ terminal will undermine the guiding ability of gRNA [1].

2)Choose gRNA targeting site carefully. Make sure the dCas9 inhibit and only inhibit the target sub-promoter. dCas9 will affect the expression of promoters within 20bp away from its targeting site. Our design can serve as a reference.

3)Choose gRNA targeting site carefully. Insure there are no potential off-target site in the genome. The best way to achieve this is choose a target sequence that has at least 4bp difference with its most similar sequence in the genome. If this cannot be guaranteed, our Cas9Off model can provide some help to find the relative better site with lesser off target possibility.

4. Transfer the system into the chosen host.

the required out levels are Normalized Strength 0.6, 0.3 and 0.06.
Then we can employ the TP model and find that a tandem promoter consists of two sub-promoter with Normalized Strength 0.3 will satisfy the needs.
1) The total output will be Normalized Strength 1-(1-0.3*2^0.4)^2=0.63
2) The single promoter output will be Normalized Strength 0.3, Thus the regulated partial output of the TP will be 0.3+(0.63-0.3)*0.1=0.33
3) The repressed output will be Normalized Strength 0.63*0.1=0.06
Then we can design two gRNA to target the 5’ sub-promoter and the 3’ sub-promoter respectively. This will result in dCas9 repression of only the 5’ sub-promoter and the whole TP depends on which gRNA is expressed.

1. Qi, Lei S., et al. "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression." Cell 152.5 (2013): 1173-1183.