CRISPR/Cas9-mediated mutagenesis in the sea lamprey Petromyzon marinus: a powerful tool for understanding ancestral gene functions in vertebrates.
Lamprey is one of only two living jawless vertebrates, a group that includes the first vertebrates. Comparisons between lamprey and jawed vertebrates have yielded important insights into the origin and evolution of vertebrate physiology, morphology and development. Despite its key phylogenetic position, studies of lamprey have been limited by their complex life history, which makes traditional genetic approaches impossible. The CRISPR/Cas9 system is a bacterial defense mechanism that was recently adapted to achieve high-efficiency targeted mutagenesis in eukaryotes. Here we report CRISPR/Cas9-mediated disruption of the genes Tyrosinase and FGF8/17/18 in the sea lamprey Petromyzon marinus, and detail optimized parameters for producing mutant F0 embryos. Using phenotype and genotype analyses, we show that CRISPR/Cas9 is highly effective in the sea lamprey, with a majority of injected embryos developing into complete or partial mutants. The ability to create large numbers of mutant embryos without inbred lines opens exciting new possibilities for studying development in lamprey and other non-traditional model organisms with life histories that prohibit the generation of mutant lines.
Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Boulder, CO 80309, USA.
Tyler
T
Square
Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Boulder, CO 80309, USA.
Marek
M
Romášek
Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Boulder, CO 80309, USA Department of Zoology, Comenius University in Bratislava, Bratislava 84215, Slovakia.
David
D
Jandzik
Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Boulder, CO 80309, USA Department of Pediatrics, University of Colorado, Denver, Aurora, CO 80045, USA.
Maria V
MV
Cattell
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Boulder, CO 80309, USA.
Michael
M
Klymkowsky
Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Boulder, CO 80309, USA daniel.medeiros@colorado.edu.
Daniel M
DM
Medeiros
D000818:Animals; D001483:Base Sequence; D019521:Body Patterning; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D020869:Gene Expression Profiling; D005838:Genotype; D017403:In Situ Hybridization; D007798:Lampreys / Q000235:genetics*; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D010802:Phylogeny; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors
D005346:Fibroblast Growth Factors; D014442:Monophenol Monooxygenase
CRISPR; Evolution; Lamprey; Vertebrate