The ongoing genomic revolution continues to present the field of biomedical research with major, albeit welcome, challenges. While a wealth of genomic data continues to be gathered at an ever-increasing pace, a gene's sequence information alone is often not intrinsically useful. The essential challenge is how to most efficiently derive clinically-important knowledge about gene function from the data. Answering this challenge will require more robust methods for correlating changes in an organism's genome with the resulting phenotype. The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system has recently emerged as a promising, versatility and ease-of-use approach for targeted genetic alterations. The CRISPR/Cas9 approach uses synthetic guide RNA (sgRNA) specifically designed to anneal to target DNA. Recently, we have shown that the CRISPR/Cas9 system can be extremely efficient, generating biallelic mutations in the F0 at high frequency (>90% in some cases). With Xenopus, the ability to microinject hundreds of synchronous, in vitro-fertilized embryos represents a significant advantage over other systems for generating mutations in F0. We intend to use the Xenopus tropicalis model system for this proposal, as it is a true diploid organism (unlike X.laevis) and its complete genome sequence data is now available. We also will develop a deletion method to eliminate a large genomic region, and a homologous recombination method to allow insertion of a DNA fragment at a desired locus within the genome. Existence of such a tool for DNA deletion and insertion is critical to providing a deeper understanding of gene function (the basis of improved disease models) than can be derived from inactivation studies alone. Use of the frog for development and refinement of the tool will be far more rapid and cost-effective than would be the case in mouse, for example.
The CRISPR/Cas9 system relies upon the formation of double-stranded hybrids between synthetic guide RNA (sgRNA) and genomic DNA to confer sequence specificity, facilitating rapid design and delivery of targeted cleavage constructs. With Xenopus, the ability to microinject hundreds of synchronous, in vitro-fertilized embryos represents a significant advantage over other systems for generating mutations in F0. We intend to use the Xenopus tropicalis model system and develop a deletion method to eliminate a large genomic region, and a homologous recombination method to allow insertion of a DNA fragment at a desired locus within the genome.
| Blitz, Ira L (2018) Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus. J Vis Exp : |
| Blitz, Ira L; Fish, Margaret B; Cho, Ken W Y (2016) Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes. Development 143:2868-75 |
| Nakayama, Takuya; Blitz, Ira L; Fish, Margaret B et al. (2014) Cas9-based genome editing in Xenopus tropicalis. Methods Enzymol 546:355-75 |
