2.5 Complementing Technologies 272.5 Complementing Technologies
2.5.1 MAGE
The TRMR and T^2 RMR approach of targeting all genes simultaneously in a
trackable manner may prove beneficial for selecting candidate genes for other
downstream techniques in the pursuit of improved production of chemicals
and for strain development. The advantage TRMR and T^2 RMR provide is the
potential discovery of genes whose involvement in any specific pathway is cur-
rently impossible to predict using computational or other methods. For exam-
ple, gene candidates can be derived from a tolerance experiment, as mentioned
earlier. However tolerance may be increased even further by combining several
such mutations via multiplex automated genome engineering (MAGE) [40] or
by using another combinatorial, recursive multiplex recombineering tech-
nique. Not only do such combinations dramatically increase the mutational
space, but combinatorial experiments also must consider epistatic effects
among the combined mutations, which are extremely difficult to predict a pri-
ori [25]. Additionally, the question of which candidate genes should be included
in the second-step MAGE experiment is far from trivial. Intuitively, one might
pick the top performing genes for combinatorial experiments. However this
approach, termed the “greedy approach,” might result in reaching a local
maximum in the potential fitness landscape rather than the desired global
maximum. Current computational efforts are being carried out to tackle these
challenges.
2.5.2 CREATE
To date, TRMR and T^2 RMR have only been used for modifying the expression
level of genes. However, work done on the engineering of biocatalysts has shown
that in some cases a single point mutation can alter the catalytic activity of an
enzyme (reviewed in [41]), suggesting that big advances can come from subtle
changes. Barcoded editing at the single nucleotide polymorphism (SNP) level
will therefore lead to even faster improvements in strain engineering and path-
way optimization.
Recent technologies were designed to address this need for higher-resolution
genome editing, namely, creating point mutations within an open reading frame.
The first generation of these ideas took advantage of the newly discovered
CRISPR/Cas9 systems to increase editing efficiency and introduce single edits
[42–44]. Multiplex editing of numerous sites quickly followed [34]. Similar to
TRMR and T^2 RMR, CREATE utilizes the λ-Red recombineering system and
array-based DNA synthesis to create rationally designed edits. Here, however,
the CRISPR/Cas9 system is used for increasing editing efficiency and for the
removal of non-edited genomes. CRISPR/Cas9-based editing technologies take
advantage of the RNA-guided endonuclease activity of the Cas9 protein [45].
This activity depends on a dinucleotide GG protospacer adjacent motif (PAM),
leading to a site-specific double-strand break in the cell’s genomic DNA and sub-
sequent cell death (in cells deficient of an efficient double-strand break repair