title: An optimized TALEN application for mutagenesis and screening inDrosophila melanogaster.
abstract: Transcription activator-like effector nucleases (TALENs) emerged as powerful tools for locus-specific genome engineering. Due to the ease of TALEN assembly, the key to streamlining TALEN-induced mutagenesis lies in identifying efficient TALEN pairs and optimizing TALEN mRNA injection concentrations to minimize the effort to screen for mutant offspring. Here we present a simple methodology to quantitatively assess bi-allelic TALEN cutting, as well as approaches that permit accurate measures of somatic and germline mutation rates inDrosophila melanogaster. We report that percent lethality from pilot injection of candidate TALEN mRNAs into Lig4 null embryos can be used to effectively gauge bi-allelic TALEN cutting efficiency and occurs in a dose-dependent manner. This timely Lig4-dependent embryonic survival assay also applies to CRISPR/Cas9-mediated targeting. Moreover, the somatic mutation rate of individual G0 flies can be rapidly quantitated using SURVEYOR nuclease and capillary electrophoresis, and germline transmission rate determined by scoring progeny of G0 outcrosses. Together, these optimized methods provide an effective step-wise guide for routine TALEN-mediated gene editing in the fly.
authors.affiliation: Graduate Program in Neurobiology of Disease; Mayo Graduate School; Mayo Clinic ; Rochester, MN, USA.
authors.forename: Han B
authors.initials: HB
authors.lastname: Lee
authors.affiliation: Yale University School of Medicine ; New Haven, CT, USA.
authors.forename: Zachary L
authors.initials: ZL
authors.lastname: Sebo
authors.affiliation: Department of Biochemistry and Molecular Biology; Mayo Clinic ; Rochester, MN, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Peng
authors.affiliation: Department of Biochemistry and Molecular Biology; Mayo Clinic ; Rochester, MN, USA ; Division of Gastroenterology and Hepatology; Mayo Clinic ; Rochester, MN, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Guo
keywords: TALEN; TALENs, Transcription activator-like effector nucleases; TALEs, TAL effectors; ZFNs, Zinc Finger Nucleases; CRISPR, Clustered Regularly Interspersed Short Palindromic Repeats; Cas9, CRISPR-associated; RVDs, repeat-variable diresidues; DSBs, double-stranded breaks; NHEJ, non-homologous end joining; HR, homologous recombination; RFLP, restriction fragment length polymorphism; HRMA, high resolution melt analysis.; engineered endonuclease; genome engineering; mutagenesis; screening