A Pipeline for Precise and Efficient Genome Editing by sgRNA-Cas9 RNPs inDrosophila
| Autor: | Richard W. Carthew, Yong Jae Kwon, Joseph Q Nguyen, Greg J. Beitel, Shelby A. Blythe, Kevin G. Nyberg |
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| Rok vydání: | 2020 |
| Předmět: |
0303 health sciences
biology Computer science Cas9 Computational biology Balancer chromosome biology.organism_classification 03 medical and health sciences 0302 clinical medicine Genome editing RNA interference CRISPR Drosophila melanogaster Gene 030217 neurology & neurosurgery 030304 developmental biology Subgenomic mRNA |
| DOI: | 10.1101/2020.05.07.080762 |
| Popis: | Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background ofDrosophila melanogasteras well as to otherDrosophilaspecies. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi againsteyes absent. This allows for screening based on eye morphology rather than color. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes. |
| Databáze: | OpenAIRE |
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