Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish.

Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration.

Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor.

We determined the crystal structure of a bifunctional group I intron splicing factor and homing endonuclease, termed the I-AniI maturase, in complex with its DNA target at 2.6 A resolution. The structure demonstrates the remarkable structural conservation of the beta-sheet DNA-binding motif between highly divergent enzyme subfamilies. DNA recognition by I-AniI was further studied using nucleoside deletion and DMS modification interference analyses. Correlation of these results with the crystal structure provides information on the relative importance of individual nucleotide contacts for DNA recognition. Alignment and modeling of two homologous maturases reveals conserved basic surface residues, distant from the DNA-binding surface, that might be involved in RNA binding. A point mutation that introduces a single negative charge in this region uncouples the maturase and endonuclease functions of the protein, inhibiting RNA binding and splicing while maintaining DNA binding and cleavage.

In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional group I intron-encoded protein, I-AniI.

The AnCOB group I intron from Aspergillus nidulans encodes a homing DNA endonuclease called I-AniI which also functions as a maturase, assisting in AnCOB intron RNA splicing. In this investigation we biochemically characterized the endonuclease activity of I-AniI in vitro and utilized competition assays to probe the relationship between the RNA- and DNA-binding sites. Despite functioning as an RNA maturase, I-AniI still retains several characteristic properties of homing endonucleases including relaxed substrate specificity, DNA cleavage product retention and instability in the reaction buffer, which suggest that the protein has not undergone dramatic structural adaptations to function as an RNA-binding protein. Nitrocellulose filter binding and kinetic burst assays showed that both nucleic acids bind I-AniI with the same 1 : 1 stoichiometry. Furthermore, in vitro competition activity assays revealed that the RNA substrate, when prebound to I-AniI, stoichiometrically inhibits DNA cleavage activity, yet in reciprocal experiments, saturating amounts of prebound DNA substrate fails to inhibit RNA splicing activity. The data suggest therefore that both nucleic acids do not bind the same single binding site, rather that I-AniI appears to contain two binding sites.