Revision as of 17:51, 21 October 2019

pBAD-dCas9-J23119-R1+

This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 20 bp sgRNA targeting to the R1+ DnaA box on E.coli genome replication initiation region, OriC. In natural situations, R1+ is a high affinity box for DnaA binding. By blocking the binding of DnaA protein to R1+ box, severe arrest and inhibition of genome replication initiation is achieved.

For more detailed information, see BBa_K3081058

Design

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 1). 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.

Figure1. Designed targeted box of CRISPRri and observation methods. 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.

Properties

To precisely record the bacteria growth under stable conditions, a microfluidic chip is developed to adapt to observed features of bacteria (Figure 2). 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, 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.

Figure2. Microscopic GIFs of bacteria transformed with CRISPRri system targeted to R1+ box 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)

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. 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.

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 3).

Figure3.Nucleoid staining followed by imaging under laser scanning confocal microscope. 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 4A). 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 4B).

Figure4. 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 4C). 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

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 5). An apparent distinction in nucleo-cytoplasmic ratio can be observed, which suggests that morphology change is not from dCas9 toxicity.

Figure5. 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. The concept of harmless lengthening is further proved by irrelative protein productivity, which will be stated in next session.

Meanwhile, we found morphology of bacteria to be an adjustable and dose-dependent outputs of the system(Figure6). 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.

Figure6. 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.

Sequence and Features