MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction

Abstract

Powerful and simple, RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. cRiSpR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish, and can be used to target multiple genes simultaneously. One of the challenges associated with the large scale application of this technique to zebrafish is the lack of a cost-effective method by which to identify mutants. to address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing -a robust and cost-effective method to genotype multiple targets in a single reaction. our approach can identify indels in up to four targets from a single reaction, which represents a four-fold increase in genotyping throughput. this method can be used by any laboratory with access to capillary electrophoresis-based sequencing equipment.Following completion of the human genome sequencing project, identification of candidate disease genes has been the focus of much genetic research. With the development of less expensive sequencing technologies, such genes are being discovered at a rapid rate, but functional validation remains slow. Most of the knowledge of gene function has been generated using gene knockout technology in model organisms 1 .The zebrafish (Danio rerio) has become a popular model organism for many reasons including high fecundity, optically transparent embryos and larvae, external development and the ease with which various types of genetic manipulation can be performed 2 . A large number of zebrafish mutants have been generated using a variety of random mutagenesis approaches involving either the chemical mutagen N-ethyl-N-nitrosourea (ENU) or insertional mutagens (Retroviruses, Transposons) 2 . Recent progress in the transformative, targeted, and simple RNA-guided CRISPR/Cas9-based genome editing method has expedited genetic manipulation in many systems, including zebrafish 3-5 . In addition to CRISPR/Cas9, Transcription Activator-like Effector Nuclease(TALENS), and Zinc Finger Nucleases (ZFNs) are other targeted mutagenesis methods being used to generate knockouts for the purposes of developing disease models and understanding disease pathology in zebrafish 2,6 . Recently, the structure-guided endonuclease (SGN) -a DNA-guided genome editing tool that uses flap endonuclease 1 (FEN-1) fused to the Fok1 endonuclease -was also used to generate large deletions in the zebrafish genome 7,8 . While CRISPR/Cas9 is a RNA-guided endonuclease and TALENs and ZFNSs are engineered proteins, the SGN nuclease functions in a similar manner: it induces a double stranded break (DSB) at a DNA target site which is then repaired by an error-prone, non-homologous end-joining method that often leaves insertions and/or deletions during the repair.Because of its simplicity, low-cost and ability to target multiple sites simultaneously, CRISPR/Cas9 is becoming most ...

Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

“…Two targets were analyzed simultaneously in this study, which can be expanded further. Using standard dye sets, up to four different targets sites can be analyzed in one reaction, which was already demonstrated [51]. The method was evaluated in this work by comparison of the obtained results with data from amplicon deep sequencing, which is the most sensitive and informative method with regards to the nature and frequency of mutations [24].…”