High-throughput construction of gene deletion cassettes for generation of Neurospora crassa knockout strains.
The availability of complete genome sequences for a number of biologically important fungi has become an important resource for fungal research communities. However, the functions of many open reading frames (ORFs) identified through annotation of whole genome sequences have yet to be determined. The disruption of ORFs is a practical method for loss-of-function gene analyses in fungi that are amenable to transformation. Unfortunately, the construction of knockout cassettes using traditional digestion and ligation techniques can be difficult to implement in a high-throughput fashion. Knockout cassettes for all annotated ORFs in Neurospora crassa were constructed using yeast recombinational cloning. Here, we describe a high-throughput knockout cassette construction method that can be used with any fungal transformation system.
Department of Genetics, Dartmouth Medical School, Hanover, NH, USA.
Patrick D
PD
Collopy
Hildur V
HV
Colot
Gyungsoon
G
Park
Carol
C
Ringelberg
Christopher M
CM
Crew
Katherine A
KA
Borkovich
Jay C
JC
Dunlap
D004271:DNA, Fungal / Q000235:genetics*; D018274:Electroporation / Q000379:methods*; D017353:Gene Deletion; D016681:Genome, Fungal; D009492:Neurospora crassa / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics*; D014170:Transformation, Genetic
D004271:DNA, Fungal