Recombineering in Non-Model Bacteria

Abstract

The technology of recombineering, in vivo genetic engineering, was initially developed in Escherichia coli and uses bacteriophage-encoded homologous recombination proteins to efficiently recombine DNA at short homologies (35 to 50 nt). Because the technology is homology driven, genomic DNA can be modified precisely and independently of restriction site location. Recombineering uses linear DNA substrates that are introduced into the cell by electroporation; these can be PCR products, synthetic double-strand DNA (dsDNA), or single-strand DNA (ssDNA). Here we describe the applications, challenges, and factors affecting ssDNA and dsDNA recombineering in a variety of non-model bacteria, both Gram-negative and -positive, and recent breakthroughs in the field. We list different microbes in which the widely used phage λ Red and Rac RecET recombination systems have been used for in vivo genetic engineering. New homologous ssDNA and dsDNA recombineering systems isolated from non-model bacteria are also described. The Basic Protocol outlines a method for ssDNA recombineering in the non-model species of Shewanella. The Alternate Protocol describes the use of CRISPR/Cas as a counter-selection system in conjunction with recombineering to enhance recovery of recombinants. We provide additional background information, pertinent considerations for experimental design, and parameters critical for success. The design of ssDNA oligonucleotides (oligos) and various internet-based tools for oligo selection from genome sequences are also described, as is the use of oligo-mediated recombination. This simple form of genome editing uses only ssDNA oligo(s) and does not require an exogenous recombination system. The information presented here should help researchers identify a recombineering system suitable for their microbe(s) of interest. If no system has been characterized for a specific microbe, researchers can find guidance in developing a recombineering system from scratch. We provide a flowchart of decision-making paths for strategically applying annealase-dependent or oligo-mediated recombination in non-model and undomesticated bacteria. © 2022 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: ssDNA recombineering in Shewanella species Alternate Protocol: ssDNA recombineering coupled to CRISPR/Cas9 in Shewanella species.

Keywords: RecET; annealase; non-model bacteria; recombineering; λ Red.

Figure 1.. Overview of ssDNA recombineering in Shewanella species.

Design and procure ssDNA oligos to make the desired genetic changes. Obtain the plasmid pX2SW3Bet, which expresses the annealase W3 Beta under the arabinose inducible P_BAD promoter. Make electrocompetent Shewanella cells induced for the annealase and introduce the mutagenic ssDNA oligos by electroporation, to generate the targeted mutation/s. Following the necessary cell recovery period, select bacterial colonies on kanamycin, and analyze possible recombinants using PCR. Verify candidates with DNA sequencing. This process can be repeated for additional recombination cycles.

Figure 2.. Overview of ssDNA recombineering coupled to CRISPR/Cas9 in Shewanella species.

For ssDNA recombineering coupled to a CRISPR/Cas9 counter-selection, design and procure ssDNA oligos to make the desired genetic changes. Obtain plasmid pX2C9pLacW3Beta, which encodes the W3 beta annealase under control of the constitutive P_Lac promoter, and Cas9 under control of the arabinose inducible P_BAD promoter. Modify the pACYC’ plasmid by inserting the desired sgRNA sequence for Cas9 targeting. Make electrocompetent cells and introduce the mutagenic ssDNA oligos and the sgRNA plasmid by electroporation. The annealase, which does not require induction, will generate the targeted mutations. Following the necessary cell recovery period, which allows time for the recombination to occur, plate the cells on kanamycin solid media supplemented with arabinose. The arabinose will induce the Cas9 protein, which will cleave non-recombinant DNA molecules, providing a counter-selection. Recombinants are analyzed using PCR and sequence verified. This process can be repeated for additional recombination cycles.

Figure 3.. Flow chart for determining approach to take for developing recombineering in a non-model bacterium.

Systems for species in which a recombineering system has been characterized should be prioritized for testing. Test the annealase alone for ssDNA recombineering before testing the annealase/exonuclease pair for dsDNA recombineering. Other distantly related systems can be tested as well if resources allow. For species with no known or functional recombineering system, researchers should first attempt to characterize a homologous system from or associated with the strain or species of interest. If no electroporation protocol is available for the host of interest, researchers may be able to develop a protocol either using plasmids or ssDNA. If genetic parts (inducible promoter, transcription terminator, low-copy plasmid ori, selective marker) are not available for the host of interest, the researchers will need to identify and characterize them. See Figure 4 for an overview of the genetic parts and tools needed.

Figure 4.. Genetic parts and tools needed for annealase-independent oligo-mediated recombination, ssDNA recombineering, and dsDNA recombineering.

a) A means for introducing linear recombinogenic ssDNA or dsDNA into the cells is needed, preferably electroporation. b) The simplest approach is oligo-mediated recombination, which requires only ssDNA oligos. c) For ssDNA recombineering, expression of a phage annealase is also required. d) In addition to recombinogenic dsDNA, recombineering with dsDNA necessitates the expression of both an annealase and its related exonuclease. More details may be found in the text.

Figure 5.. Experimental workflow for testing multiple recombineering systems in a non-model species.

Genomic sequence of the host of interest is required for designing recombinogenic ssDNA oligos and dsDNA donor templates. An efficient transformation protocol is also needed for introducing the linear DNA. Once a recombineering system has been identified using available databases and homology search functions such as BLAST, the researcher should first test the functionality of ssDNA recombination with the annealase (also indicated here as SSAP) of choice. Once ssDNA recombineering is established, the annealase (SSAP)/exonuclease pair can be co-expressed and tested for dsDNA recombination by attempting to introduce a selectable marker such as an antibiotic resistance gene. Confirm the insertion using PCR. As always, recombinants to be used in downstream applications should be sequenced. When multiple rounds of recombineering are performed, Illumina-based whole genome sequencing is ideal since prolonged laboratory handling can result in single nucleotide polymorphisms.