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.