Abstract
Advances in robotic system-assisted genome editing techniques and computer-aided design tools have significantly facilitated the development of microbial cell factories. Although multiple separate software solutions are available for vector DNA assembly, genome editing, and verification, by far there is still a lack of complete tool which can provide a one-stop service for the entire genome modification process. This makes the design of numerous genetic modifications, especially the construction of mutations that require strictly precise genetic manipulation, a laborious, time-consuming and error-prone process. Here, we developed a free online tool called GEDpm-cg for the design of genomic point mutations in C. glutamicum. The suicide plasmid-mediated counter-selection point mutation editing method and the overlap-based DNA assembly method were selected to ensure the editability of any single nucleotide at any locus in the C. glutamicum chromosome. Primers required for both DNA assembly of the vector for genetic modification and sequencing verification were provided as design results to meet all the experimental needs. An in-silico design task of over 10,000 single point mutations can be completed in 5 min. Finally, three independent point mutations were successfully constructed in C. glutamicum guided by GEDpm-cg, which confirms that the in-silico design results could accurately and seamlessly be bridged with in vivo or in vitro experiments. We believe this platform will provide a user-friendly, powerful and flexible tool for large-scale mutation analysis in the industrial workhorse C. glutamicum via robotic/software-assisted systems.
Highlights
Industrial biomanufacturing, using well-tailored microbial cell factories with economically competitive titers, synthesis rates and yields (TRY), offers a potentially green and economical alternative to current petroleum-based chemical synthesis (Clomburg et al, 2017)
The development of microbial cell factories has been greatly facilitated by computer-aided design tools (Appleton et al, 2017b; Hillson et al, 2019; Carbonell et al, 2020), among which design tools for genome editing play an important role in liberating biologists from laborious, repetitive and error-prone design work (Montague et al, 2014; Quintin et al, 2016; Wang et al, 2019b)
Most genetic modification design tools were developed to handle a specific module for a single phase of the editing process that will be more programmable, such as CHOPCHOP (Montague et al, 2014) for designing CRISPR guide RNAs, PrimeDesign (Hsu et al, 2021) for designing engineered guide RNAs, Merlin (Quintin et al, 2016) for designing ssDNAs, as well as j5 (Hillson et al, 2012) and Raven (Appleton et al, 2014) for designing DNA assembly primers
Summary
Industrial biomanufacturing, using well-tailored microbial cell factories with economically competitive titers, synthesis rates and yields (TRY), offers a potentially green and economical alternative to current petroleum-based chemical synthesis (Clomburg et al, 2017). Most industrial workhorses are developed without in-depth genetic knowledge by random mutagenesis strategies such as adaptive laboratory evolution and chemical/physical mutagenesis (Ikeda, 2003; Zhang et al, 2014; Sandberg et al, 2019). Since point mutations (single nucleotide substitutions, insertions or deletions) are the predominant mutation type identified in industrial/evolved strains (Kvitek and Sherlock, 2013; Lang et al, 2013), large-scale point mutation analysis is highly desired for further understanding the genetic basis responsible for the evolved C. glutamicum phenotypes (Bailey et al, 2002; Nielsen and Keasling, 2016). A robotic system-assisted C. glutamicum automation genome editing platform (MACBETH) has been developed, with the capacity to generate thousands of single nucleotide mutant strains per month (Wang Y. et al, 2018), which opens the possibility for future robot-assisted large-scale point mutation editing. As a CRISPR/Casdeaminase-mediated base editing platform, MACBETH was unable to edit all the nucleotides of interest due to the limitation of genome-targeting scope, editing window, and base transition capability (Wang Y. et al, 2019)
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