4.6 Targeted Deletion Techniiues 63I-SceI- stimulated scarless resolution. In principle, addition of new deletions to
the final host can be accomplished in a multiplex, iterative fashion. This might
allow the combination of the best deletion candidates, selected due to faster
growth.
4.6.2.4 New Variations on Deletion Construction
Several overlapping studies demonstrate an approach which couple the CRISPR/
Cas9 system with λ-Red-mediated recombineering [86–90]. In contrast to pre-
vious strategies, it does not rely on chromosomal integration and subsequent
removal of selectable markers. Since E. coli lacks the nonhomologous end-
joining (NHEJ) repair system, double-stranded chromosomal breaks are highly
lethal, unless rescued by providing a bridging template DNA segment. This
strategy requires targeted double-stranded DNA cleavage by Cas9 and λ-Red-
mediated genomic integration of a homologous template DNA carrying the
desired deletion. The donor DNA can be either single or double stranded and
might be introduced as a plasmid or in a linear form. Chromosomal cleavage
not only facilitates recombination but also provides strong counterselection
against the wild-type cells; therefore the efficiency of this tool can be very high,
up to 100%.
CRISPR/Cas9-derived nickases were also used to generate targeted deletions
between genomic repeats [91]. They showed that creating single-stranded chro-
mosomal incisions by mutant Cas9 nucleases are not lethal; moreover, it facili-
tates the intramolecular recombination between repetitive elements.
Dual-targeted nicking in IS element repeats generated two deletions in one step,
removing a total of 133 kbp from the genome.
The CRISPR/Cas9 coupled with NHEJ system from mycobacteria enables
rapid and continuous creation of large deletions without applying selection
markers or homologous DNA template [92, 93]. First, CRISPR/Cas9-targeted
double-stranded breaks are generated flanking the desired deletion. Next, the
NHEJ proteins seal the DNA ends in an imprecise way and thus rescue the cells.
Using this powerful technique, deletion of a 123 kbp genomic fragment was
demonstrated [93].
Another way of using CRISPR/Cas nucleases to facilitate λ-Red-mediated
genome editing is to provide long linear DNA fragments by cleaving bacte-
rial artificial chromosomes (BACs) in vivo. Both the BAC cleavage and the
genomic recombination processes are selected for using appropriately placed
positive/negative selection markers. This method, referred to as replicon exci-
sion for enhanced genome engineering through programmed recombination
(REXER), has been used to replace a 230 kbp-long genomic segment of E. coli
and could be a promising technique for the stepwise re-coding of the complete
chromosome [16].
A related strategy referred to as multiple essential genes assembling (MEGA)
applies the I-SceI endonuclease to release a linear DNA fragment from a circular
plasmid in vivo [94]. The released fragment comprises all essential genes corre-
sponding to the targeted genomic region. Subsequent cleavage of the I-SceI sites
inserted into the chromosome generates a double-strand break that facilitates
the replacement of the genomic region with the essential gene cluster by λ-Red
recombination.