Team:Peking/Demonstrate
Results and Demonstration
Sequence-Specific Binding of dCas9 to DNA is Validated by Classical CRISPR-interference Method
To investigate CRISPR-dCas9 binding specificity and affinity with DNA, we started with arabinose-induced expression of dCas9 targeted to mRFP coding region, with assistance of constantly-expressed single guide RNA that is complementary to the corresponding sequence (Figure 1A). Expression level of dCas9 induced by arabinose is tested by fluorescence of fused dCas9-GFP (Figure 1B). Since normal mRNA elongation is interrupted by occurrence of dCas9, fluorescence is greatly decreased as the arabinose concentration increases (Figure 1C), comparing to a single guide RNA that has no binding specificity to DNA. This proves the basic concept that a dCas9 protein is able to bind to DNA with sequence specificity and interferes with the physiological process. All plasmids we use to interfere with DNA replication subsequently are derived from this.
Figure 1. Sequence-specific binding of CRISPR-dCas9 to DNA is validated by classical CRISPR-interference method. (A) CRISPR-interference inhibits mRFP expression by interrupt the elongation of mRNA. (B) Arabinose-induced dCas9 expression level is tested by measuring fluorescent intensity of fusion protein dCas9-sfGFP in cytometer. (C) CRISPR-interference causes a rapid decrease in mRFP as the arabinose concentration increases. Target sequence of CRISPRi, NT1, is located on the coding region of mRFP/. The control group, of which the sgRNA is composed of a Poly-adenine that has no binding affinity to DNA strand.
An sgRNA Library Allows for a Rapid Scan for Essential DNA Boxes in vivo
In-vitro methods for identifying protein-nucleic-acid interactions are always controversial for significant distinctions between their experimental conditions and real in-vivo physiological environment. However, classical in-vivo method to search for essential DNA boxes involved in biological process is mainly by site-directed random mutagenesis, which is almost impossible for rapid and high-throughput scanning. Based on CRISPR-interference method for transcription inhibition, we develop a novel approach for prokaryotic genome replication interference (CRISPRri). Hence, a 20-bp sgRNA is designed to be complementary to OriC, the genome replication origin (Figure 2A). Instead of site-directed mutations one by one, CRISPRri allows for 20-bp scan each time. Although CRISPRri requires a PAM ("NGG") sequence to execute its function, we found a high occurrence frequency of PAM in the region of replication origin and all available sgRNAs can cover 76.2% (221 out of 290) of OriC.
Seven different targeting sites for dCas9 is designed to test the effect on cell growth. Binding box nomenclature used here is the conventional name of binding box (e.g. "M") followed by a "+" or "-", which stands for the direction of sgRNA from 5' to 3'. For instance, M box with its bottom strand bound by dCas9 is thus called M+ box. TOP 10 strain is used as the chassis organism.
Figure 2. Designed targeted box of CRISPRri and observation methods. (A) Functional DNA boxes located on the genome replication origin. The diagram includes high-affinity DnaA binding boxes (R1, R2 and R4), IHF binding site and region for DNA unwinding. Low-DnaA-binding-affinity boxes, R3 and M, are not shown here. Among these boxes, utilized in the experiment are R1, R3, M, IHF binding box and a target box located at the unwinding site (MR13). Another target box, which is located at the linker sequence between M and R2, is also designed. Control group is poly-adenine.This figure is adapted from the review of Wolanski, M. et al.[1] (B) Structure of microfluidic chip used for microscopic imaging. There are 4 main channels on one chip and thus 4 groups of bacteria can be tested each time. Small chambers are arranged along the both sides of main channel, which serves as a microscopic view. 8 view fields are taken for each group, which are at the side of the same main channel. Structural parameters are shown on the left while a real microscopic image of a main channel is shown on the right.This figure is adapted from the review of Wang, Y. et al.[2]
To precisely record the bacteria growth under stable conditions, a microfluidic chip is developed to adapt to observed features of bacteria (Figure 2B). All repeat groups are under flow of the same culture to ensure that the experiment results will not be affected by irrelevant external conditions. We have pointed out that interference of genome replication initiation would result in longer cell cycle and cell number doubling time. Here we take a 90 um * 90 um microscopic view each repeat group for cell counting every half an hour. It turns out that CRISPRri targeted to different boxes on OriC results in variant levels of cell doubling time extension(Figure 3 GIF), even though intervals between these boxes are only tens of base pairs. This is consistent with our expectations based on literatures, that functions and essence of different DNA boxes on the OriC and their contributions to genome replication vary a lot. Combined with known mechanism in DNA replication initiation, it is found out that our results accord with the DnaA binding affinity reported previously. High DnaA affinity boxes, like R1 and R3, were shown to have severe inhibition effect when targeted by dCas9. For typical low affinity box, like M box, the effect of CRISPRri is much milder. The only exception is R4, which was reported to be a high-affinity box but shows slight effect on cell growth..
Other boxes that are not considered to be DnaA binding boxes, like MR13 and IHF—they are, in fact, binding boxes for proteins other than DnaA—have mild effects. Moreover, we found another box which shows no inhibition ability on cell growth and the box happens to locate at the linker sequence of two DnaA binding boxes (M and R2) and is never reported to have specific biological functions before. These phenomena conform to our knowledge about DNA replication initiation that cooperative binding of DnaA is the rate-determining step.
Figure 3. Microscopic GIFs of bacteria transformed with CRISPRri system targeted to different boxes from microfluidic system. Transformed Top 10 strain is transferred to M9 medium in the ratio of 1:10 after overnight cultivation in LB medium. About 2 hours after transferring, bacteria in its log phase is precipitated by 5000-rpm centrifuging for 4 min and is re-suspended by M9 medium arabinose. Re-suspended bacteria are injected into the chip and observed and recorded continuously for 10 hours, under constant flow of 1 mL/16 hours.
Development, Characterization and Optimization of CRISPR-Based DNA Replication Interference (CRISPRri)
To develop a programmable and highly-adjustable method to realize precise control of DNA replication, we finely tuned the CRISPRri on multiple aspects , including plasmid copy number, inducer, targeted boxes and other extension for wider and smarter use of the system(Figure 4A). Multiple methods are developed to characterize and measure the system in detail and completeness. Cell number doubling time, nucleo-cytoplasmic ratio, morphology and irrelated protein productivity are seen as the outputs of the system and are all well described and tuned. These four parameters of E. coli are taken into consideration because they stand for different features of evaluating the cell state. A rounded characterization system, including multiple measurement methods, is well developed as a full-scale quantitative description of E. coli general states..
We picked up M+ box as the target site of dCas9 due to its relatively milder effect on cell growth, which avoids over-inhibition under low inducer concentration. Another important reason for choosing M+ box instead of other mild boxes like IHF box, is that its relation with DnaA binding shows a clear picture on how DNA replication initiation is delayed and might have higher predictability. For a same reason, we chose the vector with medium copy number to carry the dCas9 gene and sgRNA. We found an arabinose-dose-dependent increase in cell number doubling time with a considerable dynamic range (Figure 4B). This realizes preliminary adjustment of bacteria division time.
Figure 4. Overview of multi-input CRISPRri system and inducer dose adjustment. (A) A general picture of multi-input CRISPRri system.This figure is adapted from the review of Mott, M. L. et al.[3] (B) Cell number change of bacteria with CRISPRri targeted to M+ box under induction of different arabinose concentration. Cell counting is finished in hemocytometer every 2 hours.
It has been pointed out that longer cell cycle is mainly caused by a longer time to initiate the DNA replication. Since that there is still normal biochemical synthesis and metabolic reactions occurring in the cell, temporary blocking of genome replication would result in a bigger mass per cell unit. Nucleic acid staining enables us to observe the distributions of nucleoids in single cell under laser scanning confocal microscope. As before, we use poly-adenine as the sgRNA control group. We found a decrease in average nucleo-cytoplasmic ratio when treated with CRISPRri targeted to OriC (Figure 5).
Figure 5. Nucleoid staining followed by imaging under laser scanning confocal microscope.The top row of images is PolyA group and the bottom row is R1+ group. DAPI is used to stain the nucleoids in E. coli with a working concentration of 10 μg/mL. About a minute after mixing bacteria with DAPI, the medium is replaced by PBS through precipitation-resuspension process. After washing for three times, the bacteria are available for microscopic imaging. Z-axis scanning for 2 μm with 0.2 μm each step overcomes the imaging difficulty caused by rise and fall along the long cell body. Nucleo-cytoplasmic ratio is calculated by the total number of nucleoids being divided by cell length. Sample number N1 = 6 for R1+ group and N2 = 24 for control group. Different sampling number coincide with different cell density in solution for each group.
In order to extend this system to other boxes which are shown to have over-inhibition on cell growth and small dynamic range, we improve the performance of the system by weakening its effect by adding a degradation signal peptide ssrA to dCas9. This largely accelerates the degradation rate of dCas9 and thus weaken its effect. Again, the CRISPRi system provides solid evidence for retention of dCas9 binding ability and degradation-promoting effect of ssrA. As a matter of fact, CRISPRi system with sgRNA targeted to mRFP coding region shows a gentler decrease in fluorescence when dCas9 is fused with ssrA tag, while non-binding dCas9 with or without ssrA has no influence on mRFP expression (Figure 6A). We tested the improved CRISPRri-ssrA system with target site to boxes which are shown to have excessive inhibition on cell growth, and found that the degradation tag make inhibition effect much milder, which allows for a wider adjusting range (Figure 6B GIF).
Figure 6. Characterization of CRISPRri-ssrA system. (A) Comparison between dCas9 and dCas9-ssrA system by expression level and CRISPRi effect on mRFP fluorescence. (B) Comparison of effect on cell growth between CRISPRri and CRISPRri-ssrA, both targeted to R1+ box. (C) Reversibility of CRISPRri-ssrA system targeted to R1+ box. Hollow arrows stand for removal of arabinose while solid black arrows stand for addition of arabinose.
Traditional methods to inhibit bacterial growth, including antibiotics, self-killing switches and endogenous expression of toxic proteins, all cannot enable affected bacteria to restore its normal functionality and morphology. We examined the reversibility of CRISPRri-ssrA system by repeated addition and elimination of the inducer. After we treat bacteria with high inducer concentration and cultivate for 8 hours, we dilute the bacteria solution and transfer a portion of it into non-inducer medium, and again cultivate for 8 hours. The morphology of almost all cells in the non-inducer medium restore to normal (Figure 6C). This supports good reversibility of the CRISPRri system, and also implies that our bacteria are kept viable (that is, they are still able to form a colony) throughout the process. To further validate this conclusion, again bacteria are transferred into high-inducer-concentration medium and the growth was found to be inhibited. This process can be repeated for over three times. Although we notice the reduction in effect of the system as the times of repetition increases, it is thought be attributed to instability of plasmid-based expression and might be overcome if CRISPRri system is knocked in to the genome.
CRISPRri Enhances Cell Adhesiveness to Surface Through a Harmless Lengthening in Morphology
Measuring the 600-nm light absorbance is a traditional approach for rapid and real-time measurement of bacterial concentration. We found no remarkable decrease in OD600 for bacteria solution treated with CRISPRri (Figure 7A), which is attributed to decrease in cell number but lengthening in cell morphology. As a proof of this concept, we calculated the total covering area of bacteria in microfluidic system, and found the same results (Figure 7B). CRISPRri-implanted E. coli is able to reach a length of 100μm on average and over 500μm at most.
Figure 7. Biomass of CRISPRri-implanted bacteria (A) Time-course OD600 of bacteria with CRISPRri targeted to M+ box under different arabinose concentration, tested by microplate reader. (B) Normalized covering area of M+ group bacteria in a microfluidic chip chamber, under flow of M9 medium with different arabinose concentrations. Covering area at the beginning is treated as 1. 8 chambers is measured for each group.
There are numerous ways of turning E. coli morphology into abnormality, and many of them are harmful to cell. E. coli under high stress, like antibiotics environment, is often observed to have abnormal morphology. To distinguish CRISPRri effect on replication initiation from potential dCas9 protein toxicity from the system, we stained the nucleoids of cell and compared the CRISPRri-implanted long cells with cells in abnormal morphology in control group (very rare, but can be found) (Figure 8). An apparent distinction in nucleo-cytoplasmic ratio can be observed, which suggests that morphology change is not from dCas9 toxicity.
Figure 8. Comparison of long cells between R1+ group and control group. Most of cells in control groups are in normal length and shape and cells in abnormal morphology is rare but can be found. Stained nucleoids are marked by number in each cell.
Moreover, lengthening of the cell seems to be a persistent process as many of them keep growing even at the endpoint of our recording (Figure 9). The concept of harmless lengthening is further proved by irrelative protein productivity, which will be stated in next session.
Figure 9. Continuous lengthening of a cell with no division. The cell is transformed with CRISPRri system targeted to R1+ box.
Meanwhile, we found morphology of bacteria to be an adjustable and dose-dependent outputs of the system (Figure 10). In other word, a given input including sgRNA sequence and arabinose would result in a predictable morphology of the cell, which is almost impossible to achieve in traditional gene-knockout method.
Figure 10. Morphology change can be tested by flow cytometry. Elongation of cell body can be observed through increase of SSC-A. Slight SSC increase in PolyA group might be attributed to cell stress led by overexpression of dCas9. R1+ group shows a remarkable increase in SSC-A as the arabinose concentration increases. However, flow cytometry is argued to be indirect test of cell morphology change. SSC change is not absolutely positively correlated to cell shape elongation and flow cytometer actually has a upper limit for cell size which is around 150 μm. Therefore, flow cytometry is a qualitative but high-throughput characterization of cell morphology.
One of the biggest advantages for long-shape-type cell is that its membrane area per cell increases. This allows a stronger interaction between a cell and other interface, like surface of human intestinal tract or genital tract. Longer bacteria might be more stable and easy-to-plant compared to normal-sized cell. As a simplified model, we test the adhesiveness of E. coli with a uniquely designed microfluidic chip (Figure 11A). Flow velocity is adjustable by the injection pump. Long cells and normal cells are mixed in the same culture and are placed into the chip. We found ratio of long cells to normal cells greatly increases as the flow gets faster (Figure 11B).
Figure 11. Measurement of cell adhesiveness. (A) A uniquely designed microfluidic chip as a platform to measure cell adhesiveness. The preparation workflow is as follows: (a) Use a puncher to make two holes at proper locations, according to the structural parameters shown here. (b) Stick a double-sided adhesive tape to fully cover the area of cover glass and the tape covering the view field is removed (the dotted box). (c) Add the cover glass onto the glass slide. (d) Fasten the slide with two clamps and heat the slide in 200 degree Celsius in vacuum. (e) The chip is available after natural cooling. (B) A simple experiment is designed to test the cell adhesiveness. E. coli transformed with CRISPRri system targeted to M+ box and induced by 0.50% arabinose M9 medium. Then the elongated M+ group cells is mixed with normal-sized PolyA group to obtain a long and short cell mixture. The mixed bacterial solution is injected into the chip and stand for 20 min to ensure the sedimentation of most cells. After that, the injection pump constantly provides the liquid buffer with a settled flow velocity. The initial velocity is 1 mL/4 hrs, and is slowly developed by 2-fold. Microscope would record the process until the flow velocity reaches the maximum of the injection pump. We defined a cell to be long if it takes more than 80 pixels in a microscopic graph. We counted the ratio of long cells to normal cells every time we develop the flow velocity. Initial ratio is normalized to 1.
Irrelative Protein Productivity can be Largely Enhanced in CRISPRri System
As we have mentioned before, CRISPRri system inhibits the cell growth but not in the way of reducing its total biomass (Figure 7A and 7B). This typical feature means its potential industrial application will not be limited. Furthermore, since cell energy consumption in DNA replication is reduced but nutrition uptake rate might be a constant, production of proteins that has no direct relation with DNA replication would increase. This was proved by measuring the production of GFP per cell mass, which is calculated by GFP fluorescence divided by OD600. We found more than three-fold increase in GFP productivity, and a dose-dependent phenomenon suggests it is caused by CRISPRri system (Figure 12).
Figure12.GFP production per cell mass unit. CRISPRri system targeted to M+ is co-transformed with a constantly-expressed GFP plasmid. Fluorescent intensity and OD600 are both measured by microplate reader. The transformed strain is transferred to M9 medium after overnight cultivation. Three hours after transferring, the bacterial solution is diluted into medium with different arabinose concentration. The ratio of FI to OD600 shown here is at the timepoint of 6 hours.
Figure13.Indigo production of E. coli with or without CRISPRri.(A) Schematic diagram of indigo synthesis process. (B) Photo of indigo production in M+ group and polyA group. (C) Quantitative results of samples shown in Figure 13B.
Based on the up-regulation of GFP expression, we suppose that the introduction of exogenous metabolic synthetase into cells can get higher yields theoretically. We chose the indigo synthesis pathway as a demonstration. This pathway is very simple, which just contains only one enzyme, FMO. At the same time, because of its blue color, indigo can be detected very easily. The synthesis pathway of indigo is shown in Figure 13A.
Through collaboration, we obtained the DNA sequences expressing FMO from SCU-China. The FMO and CRISPRri systems were co-transformed into E. coli, and the bacteria were cultured under conditions with addition of substrate and different concentrations of inducer.
Comparing with the control group (polyA), the strain whose DNA replication initiation is inhibited by the CRISPRri system (M+) could still synthesize indigo under the treatment of high concentration inducer (Figure 13B). We tested the light absorbance indigo at 620 nm by spectrophotometer. The quantitative results showed that when the arabinose concentration was 0.05%, the yield of indigo in the M+ group was relatively high, indicating that the bacteria can put more material and energy into the production of indigo. When the concentration of arabinose rises again, the total indigo production will decline (Figure 13C). We think it may be related to a drop in the total number of bacteria. In general, inhibition of DNA replication initiation under certain conditions can increase the production of exogenous enzymatic reaction system.
Quorum Sensing CRISPRri System Enables Spatial-level Growth Control and Ultrasensitive Autoregulation of Growth
We have revealed that multi-input CRISPRri system with rational modification can realize high-adjustable control of cell growth, morphology and protein productivity. However, fully-artificial interference system might not be feasible enough for medical use, as the state of bacteria cannot be supervised all the time to be adjusted immediately. In our expectation, we hope that our system has the ability to adjust in situ. When applied in therapeutic scenario, lengthening in morphology and enhancement of drug production should initiate when cell density reaches a certain threshold, namely, when a large number of cells gather and colonize near the tumor.
Hence, based on the well-characterized growth control system, we constructed a quorum sensing CRISPRri system (qs-CRISPRri) to realize smart regulation of bacterial overall states. A classical quorum sensing system based on cell secretion of AHL synthesized by LuxI, which in turn activates the transcription factor LuxR, is combined with CRISPRri system to realize cell state control which itself senses the population density (Figure 14).
Figure 14.Synthetic gene circuit of quorum sensing CRISPRri system.
Instead of combination with CRISPRri, we started with GFP to test the performance of quorum sensing system. A complete quorum sensing system is transformed into the cell, in which LuxI and LuxR is endogenously expressed while GFP expression is activated by LuxR. A positive correlation between GFP production per cell mass (FI/OD600) and population density (OD600) is remarkable (Figure 15).
Figure 15. Relation between GFP production and cell density of bacteria transformed with quorum sensing GFP system.
A time-scale quorum sensing system can be transformed to spatial-level through a donor/receiver system (Figure 16). The donor cells, which merely express and release AHL, would activate the GFP expression of receiver cells through AHL diffusion. This is validated on the agar plate, by dropping the donor cells in the center and receiver cells around them with different distances. We found a progressive decrease in fluorescence as the receiver cells locate farther from central AHL donor.
Figure 16.The donor-receiver split quorum sensing system of CRISPRri system. Upper figure is the gene circuit of donor and receiver cells of qs-CRISPRri system. For the figure below, a donor-receiver split quorum sensing GFP system enables fluorescent intensity control on spatial scale. Left figure is the sketch map of how donor and receiver bacteria is dropped onto the agar plate. Right figure is the real picture of agar plate under illumination of blue light. The location of the white arrow is the donor cells. Receiver cells are marked by number one to six.
GFP gene is exchanged for dCas9 with companion of constantly-expressed single guide RNA to establish the qs-CRISPRri system. Through the donor-receiver system, we realized spatial-level control of cell growth (Figure 17). Receiver colonies located nearer to the donor grow much slower than farther ones, which is observed in either dropping or smearing plate.
Figure 17. Spatial-level growth control of donor-receiver split qs-CRISPRri system. For both two agar plates, the colony located at the center is the donor cells.
Furthermore, a complete quorum sensing system-implanted strain is tested with microfluidic system. Distinct from arabinose-induced CRISPRri system, decrease in cell division rate shows an ultrasensitive behavior (Figure 18 GIF). The cells were found to almost stop division and only lengthen its own shape all of a sudden, which we believe to be attributed to accumulation of AHL. Since cell growth is exponential at early stage, AHL concentration would increase rapidly. As AHL concentration reaches a certain threshold, cell genome replication is interrupted by binding of dCas9 and cell cycle extends largely. This enables E. coli to rapidly autoregulates its own growth state, and provides a potential concept of self-tuning medical-used bacteria without external interference.
Figure 18.Microfluidic videos of E. coli transformed with the complete quorum sensing CRISPRri system. During the first stage, morphology and growth rate of all cells keep in normal. However, at specific timepoint, most of the cells suddenly stop cell division, start elongation and end up with hundreds of micrometers. This ultrasensitive phenomenon is never observed in arabinose-induced CRISPRri system.
We hope to construct a self-regulating system, but in our experiment, the expression of LuxI is still artificially induced by IPTG. The only reason why we do not use a constant promoter is that we try to avoid over-inhibition of cell growth during molecular cloning. In fact, it can be replaced by any constant promoters with proper strength to realize full-automatic regulation of growth in real application.
The CRISPRri system can work well to control the growth of E. coli Nissle 1917 strain.
Escherichia coli strain Nissle 1917 (EcN) is a remarkable probiotic bacterium, which has been well researched over decades. Considering the non-pathogenicity of Nissle strain, it is widely used as a chassis organism in current industrial applications and biological therapies. Considering that the oriC sequence of E. coli Nissle is identical to that in Top10, we hope to demonstrate if the function of CRISPRri system in the new E. coli strain is same as it in Top10.
We transformed CRISPRri system to E. coli Nissle 1917. Through the microfluidic imaging system, we observed that with the treatment of 0.25% arabinose, the proliferation of bacteria in the experimental group, whose sgRNA targets on the M box in oriC region, was significantly slower than control groups (Figure 19A). Quantitative results showed that the average cell number doubling time of the experimental group (M+) was greater than 2 hours, while the control group (polyA) only need about 40 minutes to complete cell division (Figure 19B).
As mentioned before, irrelative protein productivity can be largely enhanced in bacteira with CRISPRri system. We co-transform GFP-expression plasmid and CRISPRri system into one cell. We found the fluorescent expression intensity of bacteria in the experimental group (M+) increased significantly after treatment with arabinose inducer compared with no arabinose treatment, but the control group did not have such a phenomenon (Figure 19C GIF).
Figure 19.(A) Cell number doubling time accessed from image processing in Nissle. (B) GFP production per cell mass unit in Nissle bacteria. CRISPRri system targeted to M+ is co-transformed with a constantly-expressed GFP plasmid. Fluorescent intensity and OD600 are both measured by a microplate reader. The transformed strain is transferred to M9 medium after overnight cultivation. Three hours after transferring, the bacterial solution is diluted into medium with different arabinose concentration. The ratio of FI to OD600 shown here is at the timepoint of 6 hours. (C) Characterization of CRISPRri system in Nissle, with dCas9 targeted to M+ or polyA boxes.
The CRISPRri system can be used to fine-tune the plasmid copy number
Using plasmids of different replication origins is the most common approach to tune the copy number of a specific gene, however, through which only coarse and discrete tuning can be achieved rather than fine-tuning. Recently, some researchers engineered the replication origin to make the plasmid copy number tunable in a continuous manner, but it is limited to such a specific kind of plasmid and hard to promote. Basing on the fact that our CRISPRri system can control the replication of genome DNA, we hoped to harness its power for plasmid’s DNA replication control.
We used a low copy number plasmid of pSC101 origin to harbor our system and a medium-copy plasmid of p15A origin as the target. The expression of mRFP in the target plasmid was measured to quantify the plasmid copy number. Four target sites on p15A origin are rationally selected (Figure 20A). When either strand of the replication initiation site (+1) is targeted, mRFP expression shows a decreasing trend, among which targeting the strand that replication activator (RNA II) binds to shows a greater effect. The copy number can be also upregulated when targeting the template strand of the replication inhibitor (RNA I), which shows the flexibility of our system (Figure 20B). Overall, our system enables fine-tuning of different kinds of commonly used plasmid in a continuous manner.
Figure 20. CRISPRri can be used to fine-tune the plasmid copy number. The schematic diagram of the p15A plasmid copy number control system. (B) Plasmid copy number can be downregulated or upregulated when different sites are targeted, in which RNA I and ini (+) show the best results.
Discussion
We have developed a novel toolbox to bridge overall states of bacteria with synthetic gene circuits that combine the inner mechanism of DNA replication and inherent programmability of CRISPR system. By exploiting the different target sites on genome replication origin, we establish a convenient approach for rapid in-vivo scan for those DNA boxes with potentially important physiological process. This approach is able to extend to other functional region of genome, especially for those sequences with unclear biological essence.
Based on the scanning results, we offer multiple strategies to realize control over complex behaviors and properties of bacteria, including cell cycle, morphology and protein production. Through this multi-input CRISPRri system, bacteria with any wanted growth states can be reached with user-defined adjustment that enables the system to potentially applied in laboratory and industry.
Considering the difficulty of too much artificial interference in human body, we modified the system by combining it with quorum sensing system and realized spatial and automatic regulation, which offers possibility in its medical use.To further demonstrate the potential of our system being used in medical scenario, the CRISPRri system is transplanted into E.coli Nissle strain. We validate that cell number doubling time, morphology, and production of unnecessary proteins were controlled in a similar manner as that of TOP10. In this way, we confirm that our system can help achieve control over division, adhesion to tumor, and drug production of therapeutic bacteria.
The portability of the system is further proved by joining with other functional system in subsequent demonstration. In this process of developing and optimizing the system, we constructed a measurement platform of precisely recording and analyzing important parameters relative to overall states of bacteria and any of the methods can be supported by another. High robustness and precision of our measurement system has helped us build a quantitative insight into the inner link among different general state parameters of E. coli, which provides a potential characterization and evaluation framework to extend to any artificial bacteria system.
Reference
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