Xiong et al. Biotechnol Biofuels (2018) 11:172 https://doi.org/10.1186/s13068-018-1170-4 RESEARCH Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique Bin Xiong 1,2,3 , Zhongkang Li 2,3,4 , Li Liu 2,3,4 , Dongdong Zhao 2,3 , Xueli Zhang 2,3* and Changhao Bi 2,3* Abstract Background: Ralstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO 2 fxation, which makes it a potential strain for industrial PHA production and attractive host for CO 2 conver- sion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefcient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efcient and convenient method for R. eutropha genome editing is imperative. Results: An efcient genome editing method for R. eutropha was developed using an electroporation-based CRISPR- Cas9 technique. In our study, the electroporation efciency of R. eutropha was found to be limited by its restriction- modifcation (RM) systems. By searching the putative RM systems in R. eutropha H16 using REBASE database and com- paring with that in E. coli MG1655, fve putative restriction endonuclease genes which are related to the RM systems in R. eutropha were predicated and disrupted. It was found that deletion of H16_A0006 and H16_A0008-9 increased the electroporation efciency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of cas9, enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha. A total of fve genes were edited with efcien- cies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains. Conclusions: We present the frst genome editing method for R. eutropha using an electroporation-based CRISPR- Cas9 approach, which signifcantly increased the efciency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of R. eutropha for PHA production and CO 2 conversion. Keywords: Ralstonia eutropha, Cupriavidus necator, Electroporation, CRISPR, Cas9, Genome editing © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background Ralstonia eutropha H16, also known as Cupriavidus necator H16, is a Gram-negative β-proteobacterium that is ubiquitously present in soil and freshwater environ- ments [1]. It has attracted considerable research inter- est due to its signifcant economic potential [2] and CO 2 fxation ability [3, 4]. Tis facultative chemolithoauto- trophic bacterium is a metabolically versatile organism that can grow well under both lithoautotrophic and het- erotrophic conditions [1]. Under lithoautotrophic con- ditions, it fxes CO 2 via the Calvin–Benson–Bassham (CBB) cycle, which comprises enzymes encoded by the two CBB operons [1]. Te energy used to implement CO 2 fxation and maintain cell growth is generated by energy-conserving hydrogenases, which oxidize molecu- lar H 2 and thereby reduce NAD + to form NADH [1]. Te lithoautotrophic R. eutropha has great potential as Open Access Biotechnology for Biofuels *Correspondence: zhang_xl@tib.cas.cn; bi_ch@tib.cas.cn 2 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People’s Republic of China Full list of author information is available at the end of the article