Two-step protocol for extrachromosomal array integration by MosTI in C. elegans using Cas9 protein
Two-step Array Integration Cas9 protocol final.pdf
Protocol for integrating artificial chromosomes in C. elegans
KAUST DepartmentKing Abdullah University of Science and Technology (KAUST), Biological and Environmental Science and Engineering Division (BESE), KAUST Environmental Epigenetics Program (KEEP), Thuwal, 23955- 6900, Saudi Arabia
Biological and Environmental Science and Engineering (BESE) Division
KAUST Grant NumberOSRCRG2020-4388
Permanent link to this recordhttp://hdl.handle.net/10754/690255
MetadataShow full item record
AbstractTransgenic Caenorhabditis elegans harboring large extrachromosomal arrays (several megabases) are relatively easy to generate, but suffer from variable and mosaic expression. Here, we describe a detailed protocol for stable transgene expression by chromosomal integration of arrays at safe-harbor landing sites or at the endogenous unc-119 locus. Our strategy is based on two rounds of injection. The first injection is a DNA-based injection to generate transgenic animals carrying extrachromosomal arrays. The second injection contains Cas9 protein and sgRNA for site-specific array integration at high frequency (44% of injected animals). Although this strategy necessitates two rounds of injections, the advantage is that no Cas9/sgRNA complex is co-integrated. Additionally, the use of an intermediate strain with a fully-formed extrachromosomal inherited array before integration favors integration of large DNA fragments.
CitationEl Mouridi, S., & Frøkjær-Jensen, C. (2023). Two-step protocol for extrachromosomal array integration by MosTI in C. elegans using Cas9 protein. KAUST Research Repository. https://doi.org/10.25781/KAUST-7C055
SponsorsWe thank members of the SGB lab for MosTI testing and feedback. We thank Ramatoulaye Balde for excellent lab support. Research in C.F.J.'s laboratory is supported by KAUST core funding and KAUST's Office of Sponsored Research (OSRCRG2020-4388). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) and WormBase for data and literature curation.