Recombineering

Recombineering (recombination-mediated genetic engineering) is a method of genetic engineering in bacterial genomes that involves homologous recombination. Recombineering changed the way genetic engineering was done because it eliminated the need to cut DNA with restriction enzymes. In recombineering, linear DNA donor substrates, such as PCR products or synthetic single-stranded oligonucleotides (ssDNA oligos), are introduced into bacteria to genetically engineer bacterial chromosomes and bacterial episomes. DNA fragments are designed with the desired DNA sequence change and flanking regions that are homologous to the target DNA. Phage-encoded recombination enzymes recombine the introduced linear DNA at the target DNA site. Recombineering is used for genetic modification such as gene knockout, gene replacement, deletions, point mutations, addition of tags and construction of reporter fusions. Electroporation is a common method used to introduce the linear DNA donor substrate into bacterial strains that have recombination functions.

Bacteria strains that have recombination functions contain bacteriophage recombination genes that protect linear DNA from degradation and enable recombination. Recombination is turned on through a temperature-sensitive repressor. Temporarily changing the bacterial culture temperature releases the repression and allows transient expression of the bacteriophage integrated into the bacterial genome ( λ prophage), including those genes that supply recombination function. Recombineering was initially developed in Escherichia coli, but its use has been adapted to non-model bacteria.

Major developments in recombineering were made by Donald L. Court at the National Cancer Institute/Frederick Cancer Research and Development Center, Frederick, Maryland, USA. Foundational publications on recombination-mediated genetic engineering came out in 1998 from Kenan C. Murphy at University of Massachusetts and Francis Stewart at TU Dresden, Germany.

In CRISPR-assisted recombineering, CRISPR assists the selection of successful recombineering events that change the target sequence and negatively selects cells in which the desired homologous recombination has not occurred. Negative selection occcurs by CRISPR-Cas9 induced double stranded breaks which are lethal in bacteria where non-homologous end-joining (NHEJ) is not very effective.

No-SCAR (Scarless Cas9 Assisted Recombineering) is a method of genetic engineering that uses a single step without the use of selectable antibiotic resistance marker that needs to be removed. As with previous CRISPR-assisted methods, Cas9 counter-selection is used to enrich for point mutations. Rather than requiring co-transformation of the guide RNA encoding plasmid and the mutation encoding oligonucleotides, the no-SCAR system was designed to stably maintain cas9 and sgRNA plasmids. The technique does not leave unwanted changes to the genome, known as scars.

CRISPR-enabled trackable genome engineering (CREATE) links guide RNA to homologous repair cassettes so that editing is combined with barcoding to track genotype-phenotype relationships.

Multiplex automated genome engineering (MAGE) is a derivative of recombineering in which many genomic loci in a single cell or population of cells are introduced to generate genetic diversity. MAGE is used to introduce targeted genetic changes in order to understand genotype-phenotype relationships. MAGE and related platforms, CRMAGE and CREATE, are used to monitor the allelic frequency of amino-acid mutants in a population of selective pressure or growth with the goal of discovering amino acid substitutions that impact cellular fitness. CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) is an optimized form of MAGE.

CRISPR-based oligo recombineering (CORe) was developed for proteome-wide assessment of the contribution of amino-acids to protein function in cells. CORe is used to complement chemoproteomic technologies that do not discern how chemically reactive sites contribute to protein function and provide a way to prioritize drug targets.

Retron Library Recombineering (RLR), developed by researchers at the Wyss Institute for Biologically Inspired Engineering (Harvard), generates millions of mutations simultaneously with barcoding of mutant cells. Retrons, segments of bacterial DNA that undergo reverse transcription, produce ssDNA fragments. Mutant ssDNA and a single-stranded annealing protein are introduced into cells, which causes the mutant DNA to be incorporated during DNA replication.