Projects / Programmes
Tools for genome engineering
| Code |
Science |
Field |
Subfield |
| 4.06.00 |
Biotechnical sciences |
Biotechnology |
|
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| Code |
Science |
Field |
| 2.09 |
Engineering and Technology |
Industrial biotechnology |
CRISPR, Lambda Red, TALE, recombineering, genetic engineering, genoe, homologous recombination, point mutation, transcriptional repressor, mutagenesis
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
32254 |
PhD Rok Gaber |
Biotechnology |
Head |
2016 |
52 |
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0104 |
National Institute of Chemistry |
Ljubljana |
5051592000 |
21,007 |
Abstract
Biotechnology is facing a new scientific revolution based on understanding of rules underlying regulation of cellular processes, powerful nucleotide sequencing methods, proteomics, metabolomics and gene synthesis methods. Nowadays whole genomes can be determined by the next generation sequencing methods. We are able to assemble complex pathways into the host genomes; however the real challenge is to reengineer or redesign genomes that could allow introduction of new features for the industrial microorganisms producing sustainable materials, biofuels, fine chemicals or therapeutics. Although the whole genome synthesis has been demonstrated1 it is still extremely demanding, costly and time consuming. A more feasible alternative is to redesign the existing cellular genome and introduce large modifications using introduction of foreign DNA into the defined positions within the genome. Recently developed CRISPR/Cas system for targeted RNA-guided genome disruption (Appendix, Figure 2) represents a breakthrough and a valuable tool. Use of this system has been reported in hundreds of scientific papers in top scientific journals. While CRISPR/Cas system solved the problem of disrupting the selected genes the task of targeted insertion of larger foreign DNA segments into the selected positions of the genome remains a challenge. While CRISPR/Cas system dominates eukaryotic systems, bacterial genome modification is predominantly performed by the Lambda Red recombination system2, which is also most efficient for introduction of point mutations. The problem of an inefficient homologous recombination process at the targeted DNA cleavage sites is addressed in this project proposal. We propose that the recombination efficiency will be significantly increased by the recruitment of the mutagenic DNA and proteins of the recombination machinery to the target DNA cleavage site within the genome. We plan to achieve this goal by preparing fusions of the programmable Cas9 endonuclease with the bacteriophage Lambda recombination protein Beta or Escherichia coli RecA recombinase (Fig. 3). RecA binds to the single stranded DNA (ssDNA) that occurs after the genome damage and forms filaments that coat the ssDNA. RecA fused to the Cas9 will serve to recruit the additional molecules of RecA. Similarly, Beta protein of the lambda Red complex binds to the ssDNA and its fusion to the Cas9 should increase the local concentration of the components of the recombination machinery at the target genomic site. We expect that the recruitment of the recombination machinery at specific site in the genome will augment the homologous recombination and insertion of the heterologous DNA into the target position of the genome.
Alternatively we plan to achieve recruitment of the mutagenic DNA to the target site by extending the 3´ of the sgRNA which will be complementary to the mutagenic ssDNA. The complementary pairing to the mutagenic DNA will align it in the position for the replacement (Fig. 5). Similarly TALE DNA binding protein designed to bind the mutagenic double stranded DNA and fused to the Cas9 will localize it to desired recombination site (Fig. 4). We expect that those approaches should increase the efficiency of the heterologous DNA insertion due to the increased local concentration of the mutagenic DNA. Some of the methods will be applied both to the bacterial as well as mammalian cells.
Additionally we plan to transiently suppress the endogenous E. coli genes that hinder recombination using designed transcription repressors, enabling more efficient recombination in wild type strains (Fig. 1). Different sizes of DNA inserts and simultaneous multiple insertions into the genome will be tested.
We expect that the improved targeted insertion of the large DNA segments into the selected positions of the genome will represent an extremely powerful tool for genome remodelling with strong potential for biotechnological and therapeutic applications.
Significance for science
Since the inception of the CRISPR/Cas technology as a tool for genome engineering it has been a subject of hundreds of scientific papers in top scientific journals (at least 20 papers in Cell, Science and Nature family journals in 2013-2015) and has been adopted by countless academic and industrial research departments as the developmental tool. Additionally several startup companies emerged which are dedicated to the use and development of this technology. However efficient insertion of selected ectopic DNA sequences into the defined target positions of the host genomes, both bacterial and mammalian is still rather problematic. Significant improvement in the homologous recombination efficiency achieved by further improvement of the CRISPR/Cas technology based on the concepts proposed in this project will open new prospects for biotechnology, particularly for the design of the biotechnological production organisms and also for medical applications. Further improvement of the CRISPR/Cas-mediated homologous recombination would be appreciated by the scientists in the field especially in applications where selection/counter-selection process cannot be used to select recombinants out of the wild type population. Our proposed idea is original and has to our best knowledge not been investigated yet. This technology could be implemented in several fields of biotechnology and molecular biology, for engineering organisms as cell factories for the biotechnological production of pharmaceuticals, biofuels, food additives, biomaterials but also for the engineering cellular logic and many other applications. We expect to publish in high ranked journals and previously file the patent applications.
Significance for the country
The topic of the proposed research is highly relevant for the industry in the field of biotechnology, biopharmaceuticals, cell-based therapy and synthetic biology in general. Particular the problem of targeted genetic modification of the existing industrial microbial strains which is relevant for the Slovenian, European and worldwide biotechnological industry is addressed in this project (the problem is explained in Chapter 11, proposed solution is given in Work Package 1). Reduction of the time needed to the perform genome modifications of the industrial strains as well as the possibility of simultaneous multiple modifications could play an important role in increasing the productivity and reducing the costs of biotechnological and pharmaceutical industry. That could increase the profit of industry and in the long term also reduce the cost of the pharmaceuticals. In addition to the industrial research process, the academic researchers would also benefit from more effective methods for modification of prokaryotic and eukaryotic genomes. Qualitatively more extensive genome redesign and reduced the needed to perform the genome modification technical would enable scientists to focus more on the main topic of their research. That could lead to increased number of scientific discoveries, and would in long term lead to higher prosperity of the society. Although this is a basic research project it is expected to generate innovations with potential as intellectual property. The project leader has more than two years of experiences in cooperation with industrial partners (described in Chapter 9.3) and the potential intellectual property generated in this project could be integrated into further industrial collboration or could represent the basis for starting a spinoff company engaged in the preparation of customer designed strains of various industrial microorganisms.
