Posted by on July 29, 2020 9:21 am
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scientists to the success of environmental microbial communities for CRISPR gene editing

introduction of mutations, and then observing the phenotypic changes, microbial gene function studies of the common means. However, this method is generally directed to a separable culture of a single species. For those not in the laboratory culture of microorganisms, we know about them is very limited【1】。 In addition, the separation of the culture method can not Research the different species, the interaction between【2】。 Finally, the isolation and culture of microorganisms of the laboratory environment to adapt to, can not fully reflect its in the nature of the real situation of the【3】。 Microbial communities on human health, environmental protection and industrial development has a very important role in the microbial community in an accurate and precise gene editing method will greatly facilitate our understanding of microbial recognition.

recently, the University of California, CRISPR research bull Jennifer Doudna research group at the Preprint serverbioRxivupload entitledTargeted Genome Editing of Bacteria Within Microbial Communitiespaper.< span class="bjh-strong">researchers have developed a new technology, can be of any complex microbial community of a particular species in the in situ site-specific CRISPR gene editing, without the need for pre-separation training.

on the microbial community gene editing first before you know which species can be effectively uptake and integration of exogenous nucleic acid material. Laboratory personnel developed a new method of Environmental Transformation Sequencing(ET-Seq)from the microbial communities in the identification of which bacteria can be effectively based by editing1)。 By ET-Seq method, the researchers also found the most suitable DNA delivery method to be used for the next step of the CRISPR gene editing.< span class="bjh-strong">it is noteworthy that the authors also identified 10 failed in the laboratory isolated and cultured species. ET-Seq results suggest that they may also be gene editing.

next, the authors used CRISPR-Cas Tn17 transposase system for microbial community gene editing, please see BioArt report: Science+Nature: when a CRISPR case on the transposon, site-specific not to insert【4-8】。 Traditional CRISPR-Cas transposase system requires two or more plasmids, while in this study the experimenter of the system is optimized, the development of a DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase(DART)system. As a validation, the laboratory personnel the success of microbial communities in two specific bacterial gene editing. The environmental microbial isolation and culture, transformation and gene editing usually takes the year time, and it is prone to failure. Presented here in situ microbial genetics policy need only a few weeks time, and need not be isolated and cultured.

Figure 1. ET-Seq process

in the future we may need more effective tools to be widely used, long duration of gene targeting to. Traditional antibiotic resistance screening approach may not be appropriate for microbial communities in gene editing, because of the complexity of communities may exist in natural resistance. In order to improve the gene editing efficiency, we also need a more effective nucleic acid delivery system, and further optimization of the forward and reverse screening strategy.

original link:

https://www.biorxiv.org/content/10.1101/2020.07.17.209189v1.full.pdf

author: BioartReports

reference:

1. Steen, A. D. et al. High proportions of bacteria and archaea across most biomes remain uncultured.< span class="bjh-strong">ISME J. (2019) doi:10.1038/s41396-019-0484-y.

2. Pascual-García, A., Bonhoeffer, S. & Bell, T. Metabolically cohesive microbial consortia and ecosystem functioning. Philos. Trans. R. Soc. Lond. B Biol. Sci. 375, 20190245 (2020).

3. Fux, C. A., Shirtliff, M., Stoodley, P. & Costerton, J. W. Can laboratory reference strains mirror ‘real-world’ pathogenesis? Trends Microbiol. 13, 58-63 (2005).

4. Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48-53 (2019).

5. Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S. & Sternberg, S. H. Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature571, 219-225 (2019).

6. Petassi, M. T., Hsieh, S.-C. & Peters, J. E. Guide RNA categorization enables target site choice in Tn7-CRISPR-Cas transposons. bioRxiv 2020.07.02.184150 (2020) doi:10.1101/2020.07.02.184150.

7. Rice, P. A., Craig, N. L. & Dyda, F. Comment on ‘RNA-guided DNA insertion with CRISPR-associated transposases’. Science368, (2020).

8. Strecker, J., Ladha, A., Makarova, K. S., Koonin, E. V. & Zhang, F. Response to Comment on ‘RNA-guided DNA insertion with CRISPR-associated transposases’. Science368, (2020).

Published at Tue, 29 Jul 2020 13:21:32 +0000