Abstract
BackgroundThe generation of point mutations is a major tool for evaluating the roles of specific nucleotides or amino acids within the regulatory or functional landscape. However, examination of these mutations in vivo requires the generation of animals carrying only the relevant point mutations at the endogenous genomic loci, which is technically challenging. The CRISPR-Cas9 based genome editing greatly facilitates the generation of such genetically modified animals; however, most of the described methods use double-strand DNA (dsDNA) as the donor template. The dsDNA plasmids frequently undergo undesired integration events into the targeted genomic locus. The use of a single-strand oligodeoxynucleotide (ssODN) as the donor template prevents this complication and is therefore the preferred choice for introducing point mutations, as well as short sequences such as protein tags.ResultsWe successfully applied the CRISPR-based white co-conversion strategy with a ssODN template, instead of the originally described dsDNA plasmid, to create genetically modified Drosophila melanogaster strains. We used the technique to easily introduce point mutations in two distinct chromosomes. Using the generated flies, we were able to demonstrate the in vivo importance of the respective mutations. For the Nucleoporin107 (Nup107) gene, the 1090G > A mutation was confirmed to affect ovarian development, while for the tinman (tin) gene, the regulatory role of the downstream core promoter element (DPE) was demonstrated within the developing Drosophila melanogaster embryo.ConclusionsThe described approach has facilitated the successful generation of point mutations in two different chromosomes, by two different labs. Distinct phenotypes associated with the newly-generated genotype were identified, thus exemplifying the importance of investigating the in vivo role of specific nucleotides. In addition, detailed guidelines, recommendations and crossing schemes are provided in order to support the generation of additional genetically modified animals by the scientific community.
Highlights
The generation of point mutations is a major tool for evaluating the roles of specific nucleotides or amino acids within the regulatory or functional landscape
White co-conversion involves the injection of three DNA components into the Cas9-expressing strain (Additional file 1: Figure S1): 1) pCFD4d plasmid harboring two guide RNA (gRNA) targeting the white gene and the locus of interest, respectively. 2) pUC57- white [coffee] plasmid serving as a donor template for the white locus
The distinct molecular repair events can be readily distinguished by eye color - red denotes no change, white eyes indicate non-homologous end joining (NHEJ), and coffee eyes indicate homologydirected repair (HDR) (Fig. 1a)
Summary
The generation of point mutations is a major tool for evaluating the roles of specific nucleotides or amino acids within the regulatory or functional landscape Examination of these mutations in vivo requires the generation of animals carrying only the relevant point mutations at the endogenous genomic loci, which is technically challenging. Mutations of multiple nucleotides are typically used to demonstrate their contribution to promoter or enhancer activity, while single base pair alterations were typically used to demonstrate the function and regulation of enzymes, transcription factors and signal transducers These studies were mainly conducted in vitro, using exogenously provided DNA. Examination of these mutations in vivo, in the context of the whole animal, requires the generation of a genetically modified animal carrying only the relevant point mutations at the endogenous genomic locus. The in vivo context or relevance of point mutations that were previously analyzed in vitro, often remains unexplored
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