Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish
- Hoshijima, K., Jurynec, M.J., Klatt Shaw, D., Jacobi, A.M., Behlke, M.A., Grunwald, D.J.
- Developmental Cell 51(5): 645-657.e4 (Journal)
- Registered Authors
- Grunwald, David, Hoshijima, Kazuyuki, Jurynec, Michael
- CRISPR-Cas9 mutagenesis, F0 screen, deletion mutations, genome editing, targeted mutagenesis, zebrafish genetics
- MeSH Terms
- CRISPR-Cas Systems*
- Embryo, Nonmammalian/metabolism
- Gene Deletion*
- Gene Editing/methods*
- Loss of Function Mutation
- 31708433 Full text @ Dev. Cell
Hoshijima, K., Jurynec, M.J., Klatt Shaw, D., Jacobi, A.M., Behlke, M.A., Grunwald, D.J. (2019) Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish. Developmental Cell. 51(5):645-657.e4.
Inconsistent activity limits the use of CRISPR-Cas9 in zebrafish. We show supernumerary guanine nucleotides at the 5' ends of single guide RNAs (sgRNAs) account for diminished CRISPR-Cas9 activity in zebrafish embryos. Genomic sequences can be targeted consistently with extremely high efficiency using Cas9 ribonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that perfectly matches the protospacer target site. Following injection of zebrafish eggs with such RNPs, virtually every copy of a targeted locus harbors an induced indel mutation. Loss of gene function is often complete, as F0 embryos closely resemble true null mutants without detectable non-specific effects. Mosaicism is sufficiently low in F0 embryos that cell non-autonomous gene functions can be probed effectively and redundant activities of genes can be uncovered when two genes are targeted simultaneously. Finally, heritable deletion mutations of at least 50 kbp can be readily induced using pairs of duplex guide RNPs targeted to a single chromosome.
Genes / Markers
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes