CRISPR-Assisted Multiplex Base Editing System in
| Autor: | Tian-Xin Liang, Li-Bing Lu, Jun Sun, Lirong Yang, Jian-Ping Wu |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Histology base editing lcsh:Biotechnology Biomedical Engineering Bioengineering 02 engineering and technology Computational biology Pseudomonas putida KT2440 03 medical and health sciences chemistry.chemical_compound Genome editing lcsh:TP248.13-248.65 CRISPR multiplex genome editing Original Research cytidine deaminase biology Cas9 Cas9 nickase Bioengineering and Biotechnology 021001 nanoscience & nanotechnology biology.organism_classification Pseudomonas putida Protospacer adjacent motif 030104 developmental biology chemistry gene inactivation Uracil-DNA glycosylase 0210 nano-technology Cytosine DNA Biotechnology |
| Zdroj: | Frontiers in Bioengineering and Biotechnology, Vol 8 (2020) Frontiers in Bioengineering and Biotechnology |
| ISSN: | 2296-4185 |
| Popis: | Pseudomonas putida (P. putida) KT2440 is a paradigmatic environmental-bacterium that possesses significant potential in synthetic biology, metabolic engineering and biodegradation applications. However, most genome editing methods of P. putida KT2440 depend on heterologous repair proteins and the provision of donor DNA templates, which is laborious and inefficient. In this report, an efficient cytosine base editing system was established by using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9ppD10A) and the uracil DNA glycosylase inhibitor (UGI). This constructed base editor converts C-G into T-A in the absence of DNA strands breaks and donor DNA templates. By introducing a premature stop codon in target spacers, we successfully applied this system for gene inactivation with an efficiency of 25-100% in various Pseudomonas species, including P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. We engineered an eSpCas9ppD10A-NG variant with a NG protospacer adjacent motif to expand base editing candidate sites. By modifying the APOBEC1 domain, we successfully narrowed the editable window to increase gene inactivation efficiency in cytidine-rich spacers. Additionally, multiplex base editing in double and triple loci was achieved with mutation efficiencies of 90-100% and 25-35%, respectively. Taken together, the establishment of a fast, convenient and universal base editing system will accelerate the pace of future research undertaken with P. putida KT2440 and other Pseudomonas species. |
| Databáze: | OpenAIRE |
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