Methods enabling genomic point mutagenesis are the most versatile tools in genome engineering since they can be easily adapted to other modifications such as deletions, insertions and replacements (10
). A prerequisite for such a method is that the selection/counterselection marker should not leave any scar in the chromosome. This requirement precludes the use of FLP and Cre recombinases that are very popular in performing chromosomal deletions. There have been two distinct strategies described for performing such precise mutations in yeast using PCR products. The first strategy embeds a selection marker within a long DR (7
). Presence of the DR can cause spontaneous deletion of the selection marker at low frequencies when grown in permissive conditions. Unfortunately, the long DR can cause aberrant integration of the mutagenesis cassette, and the low frequency of excision combined with imprecise excision events, makes the method relatively inefficient. The other strategy based on the delitto perfetto
) addresses some issues and is very efficient at introducing point mutations with few false positives. However, this method requires two separate transformation steps, one to delete the undesired allele and a second to introduce the desired mutation, as well as a replica plating step, possibly to maximize efficiency (10
In order to streamline genome engineering, we sought to create a quick and efficient method to create precise chromosomal mutations. To this end we have developed a method called MIRAGE that can create precise alterations in the chromosome in a single transformation step. In this method, the mutagenesis cassette is designed to have an IR of selection marker(s) embedded within a 25-bp DR. Flanking these are regions of homology that target the cassette to the desired site in the chromosome.
By using an IR in the mutagenesis cassette, we have overcome low excision frequency limitations of the strategy introduced by Langle-Rouault and Jacobs (7
). This may be due to any combination of four roles that the IR may play—first, it encodes the marker(s) to select/counterselect for the desired genotype. Second, it introduces chromosomal instability that results in its own excision from the chromosome at extremely high frequencies, nullifying the need for growth on permissive medium for expediting its loss. However, to minimize contamination from surrounding non-transformants, colony purification on SC−Ura medium may be performed without any adverse effect. Third, its excision from the chromosome results in a double strand break (11
) that may create a hotspot for recombination. And fourth, it may bring the flanking DR in close proximity allowing for very precise homologous recombination between relatively short sequences. Indeed, Langle-Rouault and Jacobs (7
) used DR between 50 bp and 60 bp to ensure homologous recombination (albeit with low efficiency) between distant sequences in the chromosome, whereas in our method DR as short as 25 bp are highly proficient at recombination.
Cassette preparation takes 2 days due to the requirement for high-efficiency overnight ligation. However, compared to the two-step methods like delitto perfetto
, MIRAGE requires only a single transformation step and no replica plating, making it a quicker method, which is particularly useful when dealing with slow-growing mutants. Additionally, while we have shown selection/counterselection based on Ura/FOA, other markers such as Trp/5-fluoroanthranilic acid (FAA) (16
), Lys/aminoadipate (17
) or any combination thereof, could also be used.
We have shown that inverted repeat, an unstable genetic element, can be employed as a useful genetic tool to perform one-step modifications to the S. cerevisiae
genome with high efficiency. This method does not require the use of helper plasmids, making it widely applicable. Also of note is that yeast seemed to propagate a relatively long IR (~4.5 kb) as stably as a shorter IR (~2.2 kb), while actively transcribing the genes contained. This is particularly interesting in the light that E. coli
is conferred nonviable when IR of >240 bp is introduced in the chromosome (18
). It would also be interesting to test whether this method can be adapted for use in other systems, including mammalian and bacterial cells, since DR bracketed IR should, in theory, induce double-strand breaks in these chromosomes upon excision.