28 2 Trackable Multiplex Recombineering (TRMR) and Next-Generation Genome Design Technologies
mechanism, such as E. coli). However, cells containing a mutation in the PAM
site are protected from DNA cleavage [42].
Each CREATE cassette includes the target site in a gene and a proximal PAM
sequence that are selected for mutagenesis. Since in most cases the PAM falls
within the open reading frame, it is silently mutated, so the amino acid sequence
is not altered. To allow multiplex editing, the PAM-specific corresponding gRNA
coding sequence is co-synthesized with the target site editing oligo and cloned
into an editing vector, which also serves as a target-specific barcode. This design
enables the creation of barcoded libraries composed of tens of thousands of cells,
with each genome having a single amino acid edit. Hence, TRMR or T^2 RMR and
CREATE can be used in conjunction, with TRMR or T^2 RMR identifying impor-
tant genes under specific conditions and CREATE allowing for the engineering
of those genes for optimal results. These two technologies could additionally be
combined in the future (this would require new technology allowing for an
increase in the length of targeting oligos that can be synthesized on a microchip).
Adding the main T^2 RMR elements to the CREATE cassette design can allow
higher versatility in editing. Gene expression tuning can be coupled to gene edit-
ing, enabling the investigation of expression in conjunction with point muta-
tions. The ability to cycle these edits for the generation of multiple diverse
genotypes will help researchers to isolate desired complex traits that combine
both protein sequence and expression level.2.6 Conclusions vi Contents
Recent advances in DNA synthesis and the development of standardized genetic
parts have greatly increased genome engineering capabilities. TRMR and T^2 RMR
allow a single researcher to modify an entire genome in a single day and map
which alleles are responsible for a desired phenotype. This ability to fine-tune
expression levels, particularly when combined with other technologies for mak-
ing point mutations or combinatorial mutations, will allow researchers to quickly
and easily engineer strains for maximal production of, or tolerance to, any
compound.Definitions
Recombineering Genetic engineering using homologous recombination
TRMR Trackable multiplex recombineering
Recombineering substrate A single- or double-stranded piece of DNA that is
to be inserted into the target’s genome via recombineering
λ-Red system Using λ-Red phage proteins to enable homologous recombina-
tion in bacteria
Molecular barcode Unique, short nucleotide sequence used to identify and
track a specific gene or piece of DNA
Synthetic DNA Artificial DNA that is synthesized; does not have to be based on
naturally occurring DNA sequence