Homologous Recombination

Depending on the project, the ability to target the genetic insert to a specific location could be of utmost importance. In the wildtype N. crassa, both homologous recombination and non-homologous end rejoining machinery are present. Non-homologous end rejoining results in an essentially random insertion of the genetic insert in the N. crassa genome. This is not very useful for synthetic biology, however. Homologous recombination is much more precise and predictable, as the procedure relies on the similarity of DNA sequences to target the insertion. Normally this machinery is used in the crossing over events during meiosis.

By knocking out the non-homologous end rejoining machinery, as has been done in mus strains of N. crassa, one is able to target the desired homologous site via homologous recombination at very high frequencies. In a study by Yuuko Ninomiya and al., it was found that by having a 1000 bp homologous region on either side of the cassette to be inserted, when the cassette was inserted into the genome, it was inserted into the targeted region 100% of the time.

This gives Neurospora crassa mus strains a very powerful propensity for genetic manipulation. High transformation efficiencies, ease of transformation, and accurate genetics clearly show that N. crassa should be utilized more in the field of synthetic biology.