Revision as of 17:40, 21 October 2019

pBAD-dCas9-J23119-R1+(19bp)

This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 19 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 using a 19bp sgRNA, alleviation of severe arrest and inhibition to the 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.

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 2). 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.

Characterization

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. To precisely record the bacteria growth under stable conditions, a microfluidic chip is developed to adapt to observed features of bacteria. The Microscopic GIFs of bacteria transformed with CRISPRri system adding ssrA tag targeted to R1+(19bp) box from microfluidic system can be seen in BBa_K3081033

Sequence and Features