Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli
| Autor: | Ilaria Zanella, Guido Grandi, Michele Tomasi, Enrico König, Matteo Parri, Francesca Zerbini, Elena Caproni, Carmela Irene, Luca Frattini, Davide Fraccascia, Alberto Grandi, Luisa Ganfini, Laura Fantappiè |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
030106 microbiology lcsh:QR1-502 Bioengineering Bacterial genome size Biology CRISPR-Cas9 High-throughput genome editing Synthetic biology Escherichia coli Gene Deletion Genome Bacterial Homologous Recombination Mutagenesis RNA Guide Recombinases Synthetic Biology CRISPR-Cas Systems Gene Editing Multigene Family Biotechnology Applied Microbiology and Biotechnology Recombineering lcsh:Microbiology 03 medical and health sciences Genome editing CRISPR Guide RNA Genetics Genome Cas9 Research Bacterial Robustness (evolution) 030104 developmental biology RNA Guide RNA Guide Kinetoplastida |
| Zdroj: | Microbial Cell Factories Microbial Cell Factories, Vol 16, Iss 1, Pp 1-18 (2017) |
| ISSN: | 1475-2859 |
| Popis: | Background The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a “brute force” validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions. Results We first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 “dispensable” genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions. Conclusions This work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0681-1) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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