Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome

Abstract

The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.

Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

“…Engineered nuclease-induced mutations are detected by various methods, which include mismatch-sensitive Surveyor or T7 endonuclease I (T7E1) assays 23 , RFLP analysis 24 , fluorescent PCR 25 , DNA melting analysis 26 and Sanger and deep sequencing. The T7E1 and Surveyor assays are widely used but often underestimate mutation frequencies because the assays detect heteroduplexes (formed by the hybridization of mutant and wildtype sequences or two different mutant sequences); they fail to detect homoduplexes formed by the hybridization of two identical mutant sequences.…”

A highly effective TALEN-mediated approach for targeted gene disruption in Xenopus tropicalis and zebrafish

“…More recently, specific genomic DSB has also been successfully generated by using RNA-guided Cas9 nuclease, providing an alternative approach for genome editing [4,5]. To date, genome editing mediated by these engineered nucleases has been successfully implemented in cells and in a number of organisms including Xenopus tropicalis and zebrafish [6][7][8][9][10][11][12][13][14][15][16][17][18][19].…”

The zebrafish eye—a paradigm for investigating human ocular genetics

“…Almost all TALEN RNA-injected wild-type embryos displayed mutant non-pigmented cells in the RPE, highlighting the efficiency with which TALENs can induce directed mutations in the zebrafish. 15 CRISPR-Cas is a microbial adaptive immune system that uses targeted nucleases to initiate double-strand breaks in foreign genetic elements. CRISPR RNAs that guide the Cas9-ribonucleoprotein complex to the target sequence can be injected into the zebrafish embryo and designed to target any 20 nucleotide genomic sequence to achieve phage or plasmid DNA cleavage with high specificity.…”