Recombineering and MAGE

Abstract

Recombination-mediated genetic engineering, also known as recombineering, is the genomic incorporation of homologous single-stranded or double-stranded DNA into bacterial genomes. Recombineering and its derivative methods have radically improved genome engineering capabilities, perhaps none more so than multiplex automated genome engineering (MAGE). MAGE is representative of a set of highly multiplexed single-stranded DNA-mediated technologies. First described in Escherichia coli, both MAGE and recombineering are being rapidly translated into diverse prokaryotes and even into eukaryotic cells. Together, this modern set of tools offers the promise of radically improving the scope and throughput of experimental biology by providing powerful new methods to ease the genetic manipulation of model and non-model organisms. In this Primer, we describe recombineering and MAGE, their optimal use, their diverse applications and methods for pairing them with other genetic editing tools. We then look forward to the future of genetic engineering.

Fig. 1 |. Single-stranded and double-stranded recombineering and MAGE.

a | In single-stranded recombineering, single-stranded DNA (ssDNA; typically synthetic oligodeoxynucleotides (oligos)) is the carrier of new genetic information. First, ssDNA that is targeted to the lagging strand of the replication fork is electroporated into a cell. Once inside the cell, ssDNA is thought to be bound by an exogenously expressed phage single-stranded DNA-annealing protein (SSAP) and annealed at the replication fork through a specific interaction between the SSAP and the host bacterial single-stranded DNA-binding protein (SSB). The annealed ssDNA then primes synthesis of the nascent genome, incorporating user-defined modifications in the process. After the first round of replication there is one wild-type copy of the genome and one chimeric copy. A second round of replication would then afford one mutant genome and three wild-type copies. This supports the theory that recombineering efficiency should top out at 25%, although higher efficiencies have recently been demonstrated, with editing presumably occurring over multiple rounds of genomic replication. b | Recombineering using double-stranded DNA (dsDNA) as the template works much the same, except that an additional phage protein is required. An exogenously expressed phage exonuclease degrades one strand of a dsDNA cassette, loading the SSAP onto the exposed strand. This ssDNA strand is then annealed at the lagging strand of the replication fork and recombineering proceeds as in part a. Often, dsDNA will be designed to contain a selectable marker, as integration of a long strand is much less efficient than small modifications. c | Oligos can precisely create insertions, mismatches or deletions in genomic DNA. These can have various uses when targeted at different genetic elements. d | Because efficiency is around an order of magnitude higher when targeting the lagging strand of the replication fork, it is important to understand which replichore a target modification is being directed to. This will determine whether to target the positive (+) strand or the negative (−) strand. e | Multiplex automated genome engineering (MAGE) offers two conceptual advances: a pool of diverse oligos is used, and many cycles of editing are conducted to saturate mutations within a population. Applications of MAGE range from whole-genome recoding and allele tracking to mutagenesis of gene clusters and saturating mutagenesis of a single gene. OriC, genomic origin of replication.

Fig. 2 |. Optimizing the ARF in bacteria.

There are several factors that help improve the allelic recombination frequency (ARF), which are applicable across most bacterial species. a | Oligodeoxynucleotides (oligos) are designed containing phosphorothioate (PT) bonds, shown as asterisks. b | A population of bacteria are transformed with oligos through electroporation. The percentage of cells successfully transformed presents an upper bound for the ARF. Once inside the host cell, all free single-stranded DNA (ssDNA) is bound by bacterial single-stranded DNA-binding protein (SSB). c | Host nucleases degrade ssDNA in most bacteria, so protection with PT bonds is important. Two PT bonds at the 5′ end of the oligo usually provide adequate protection. d | Optimization of protein production can improve the ARF significantly. Some factors to consider are codon optimization, promoter strength and ribosome binding site strength. e | Orders of magnitude improvement can be gained by expressing a host-optimized single-stranded DNA-annealing protein (SSAP). A specific interaction with a host SSB determines SSAP compatibility. f | After a modification has been made to the genomic DNA, a mismatched base pair will be present. There are several possible strategies to prevent error correction by endogenous mismatch repair (MMR) machinery. Transient expression of a dominant-negative MutL (MutL-DN) causes chain termination and failure to recruit MutH. This optimal route is depicted here, but alternatives are available including MutH or MutS disruption, and the modification of multiple (4+) consecutive bases, which would then not be efficiently recognized by MutS. g | Serial enrichment for efficient recombineering (SEER) is a method to identify a host-optimized SSAP. A diverse library of SSAP variants is encoded on a plasmid, transformed into the target bacterial host, enriched through a series of antibiotic selections and deep sequenced to identify the most successful SSAP variants. Oligos used in the enrichment steps each contain an allelic modification to a host gene that confers resistance to a common antibiotic. Ab R, antibiotic resistance gene; NGS, next-generation sequencing; Ori, origin of replication.

Fig. 3 |. Eukaryotic MAGE.

a | Schematic of a eukaryotic multiplex automated genome engineering (eMAGE) locus. An origin of replication (ori) is cloned immediately upstream of a locus of interest and a selectable marker so that the leading and lagging strands of either are predictable. The selectable marker used in this illustration is URA3, with an in-frame stop codon indicated in red. Correction of this stop codon allows cells to survive in the absence of supplemented uracil or uridine. The correction of this in-frame stop codon with an oligodeoxynucleotide (oligo) can be selected for, which enriches populations that have successfully made this modification for incorporation of oligos that confer targeted modifications (purple) into the adjacent locus of interest. b,c | Recombination pathways compete for oligo incorporation. Replication fork stalling or collapse can activate one of several DNA damage tolerance pathways or require fork restart. Rad51 (grey circles) is recruited to a stalled or collapsed replisome, mediating strand invasion and fork restart (b). Rad52 (grey hexamers) is involved in a recombination salvage mechanism, whereby annealing can occur at stalled replication forks (c). This Rad52-directed mechanism is the presumed recombination pathway responsible for oligo incorporation in eMAGE. We present a picture of either mechanism, as mechanistic details of eukaryotic recombineering remain to be worked out.

Fig. 4 |. Reading out MAGE results.

a | Multiple allele-specific colony PCR is a method for quickly identifying edited clonal populations. First, three primers are designed for each targeted modification. Forward primers bind to either the wild-type (wild-type primer) or edited (Mut primer) DNA, whereas a third reverse primer (universal primer) will be paired with both forward primers. Disambiguation is strongest when the 3′-terminal base of the forward primers is designed to anneal to the targeted base modification. Here, the Mut primer is depicted to have a pink terminal base that pairs successfully with the mutated red base, whereas the wild-type primer has a grey terminal base that does not pair, blocking elongation of the primer by DNA polymerase in the PCR reaction. After numerous multiplex automated genome engineering (MAGE) cycles, the edited population is plated out for single colonies, and two separate PCR reactions are run for each colony (wild type + universal and Mut + universal). On an electrophoresis gel, a DNA band should appear only for the allele that is present in the clonal population. Multiple alleles can be combined into a single PCR reaction if the amplicons are designed to have different lengths so that they are easily differentiated by gel electrophoresis. Colony 6, with four mut bands, has successfully incorporated all of the targeted allelic modifications, whereas every other colony shows at least one wild-type band (wt). b | Amplicon-based next-generation sequencing (NGS) for screening and selecting targeted mutations introduced by MAGE-based strategies such as MAGE sequencing or directed evolution with random genomic mutations (DIvERGE; pictured here). Illumina NGS libraries can be easily created in two PCR steps from a population of edited or edited and then enriched cells. First, amplicon PCR mixes a population of cells separately with primers to amplify each targeted locus (green, yellow or blue) and at the same time affix an adapter sequence. Amplicon PCR reactions are run separately for each targeted locus, but the population of cells is pooled in the reaction. Second, barcoding PCR is run on each amplified locus to add primers that bind to the adapter region and affix a unique barcode and sequences for binding to a flow cell. Oligo, oligodeoxynucleotide.

Fig. 5 |. Library-scale genome diversification using MAGE-seq and DIvERGE.

a | Multiplex automated genome engineering with amplicon deep sequencing (MAGE-seq) is based on scanning codon mutagenesis, wherein an oligodeoxynucleotide (oligo) with a degenerate NNK codon is designed for each codon within a targeted gene region. This batch of oligos is pooled and delivered as a library, subjected to multiple MAGE cycles and then a desired phenotype is screened or selected for. The population of enriched cells is then genotyped by amplicon deep sequencing. b | In directed evolution with random genomic mutations (DIvERGE), soft-randomized oligos are designed to tile a targeted genomic locus. As the soft-randomized oligos are incorporated, they will introduce mutations at random, and, depending on the allelic recombination frequency (ARF), this will enable a highly elevated mutation rate to be targeted with precision throughout the genome.

Fig. 6 |. Advanced techniques pair MAGE with other tools.

a | CRISPR multiplex automated genome engineering (CRMAGE) operates via a two-plasmid system. Cas9 and the recombineering proteins Dam, a single-stranded DNA-annealing protein (SSAP) and RecX are expressed on an episomally maintained vector. A second vector contains trans-activating crispr RNA (tracrRNA) and a CRISPR array with genomic-targeting (green) and self-targeting (grey and brown) CRISPR RNAs (crRNAs). First, oligodeoxynucleotide (oligo) editing templates are transformed into cells, these templates are incorporated into the host genome by recombineering and the successfully edited cells are selected for with induction of the CRISPR targeting system. Cas9/short guide RNA (gRNA) fails to recognize edited target sequence but creates double-strand breaks in unedited targets, resulting in cell death and selection for edited cells. b | Oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting (ORBIT) can create genetic knockouts or fusions depending on the integrating plasmid selected and the oligo design. To fuse green fluorescent protein (GFP) to a gene of interest, an oligo encoding an attP (red) site and a plasmid containing GFP (green), a hygromycin resistance marker (blue) and an attB site (yellow) are co-transformed into a cell. After successful incorporation of the oligo by recombineering, Bxb1 integrase (Int) integrates the GFP plasmid at the attP site, creating a carboxy-terminal gene fusion of GFP to the target gene (grey). A similar strategy can be used to perform targeted gene deletion. c | Replicon excision for enhanced genome engineering through programmed recombination (REXER) efficiently integrates long synthetic DNA into Escherichia coli genomes. A bacterial artificial chromosomes (BAC) (grey) containing an edited template (red) is transformed into E. coli. CRISPR–Cas9 is then expressed and excises the edited template along with the −2 (sacB; orange) and +2 (CamR; green) selection markers from the transformed BAC. In step 1, homology arms facilitate replacement of genomic DNA (black) and the −1 (rpsL; yellow) and +1 (KanR; blue) selection markers with both negative and positive selection pressures. Step 2 uses a new BAC with a new editing template and the −1, +1 markers to replace the previously incorporated −2, +2 markers. This process can be repeated for de novo synthesis of a synthetic E. coli genome (genome stepwise interchange synthesis (GENESIS)). abR, antibiotic resistance marker; Ori, origin of replication.

Fig. 7 |. Retrons allow recombineering without exogenously delivered DNA.

a | Synthetic cellular recorder integrating biological events (SCRIBE) uses retrons to produce multicopy satellite DNA (msDNA) in vivo, which is used as a substrate for recombineering. The presence of an induction molecule triggers expression of a reverse transcriptase (RT; orange) and a retron transcript (msr/msd). The msd portion of the retron transcript is reverse transcribed by the RT to create msDNA. This msd region can be modified to include a customized loop that carries a user-defined mutant allele. A single-stranded DNA-annealing protein (SSAP) then mediates incorporation of the msDNA into the nascent copy of the genome at the lagging strand of the replication fork. b | Error-prone T7 RNA polymerase (RNAP) can be used to produce mutagenized msr-msd transcripts. These transcripts are reverse transcribed to produce a library of msDNA editing templates that contain random mutations for continuous evolution of a target sequence. c | Retron library recombineering is a technique for genome-scale reverse genetics. Synthetic or natural DNA variation is encoded into the retron msd element on a plasmid library. This plasmid library is transferred to a population of bacteria, the retron directs a mutation into the genome and the population is taken through a screen or selection. The prevalence of each allele within a population can be tracked by sequencing of a plasmid amplicon containing the retron msd. NSG, next-generation sequencing; ssDNA, single-stranded DNA.