A Comprehensive Review of Indel Detection Methods for Identification of Zebrafish Knockout Mutants Generated by Genome-Editing Nucleases

Abstract

The use of zebrafish in functional genomics and disease modeling has become popular due to the ease of targeted mutagenesis with genome editing nucleases, i.e., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These nucleases, specifically CRISPR/Cas9, are routinely used to generate gene knockout mutants by causing a double stranded break at the desired site in the target gene and selecting for frameshift insertions or deletions (indels) caused by the errors during the repair process. Thus, a variety of methods have been developed to identify fish with indels during the process of mutant generation and phenotypic analysis. These methods range from PCR and gel-based low-throughput methods to high-throughput methods requiring specific reagents and/or equipment. Here, we provide a comprehensive review of currently used indel detection methods in zebrafish. By discussing the molecular basis for each method as well as their pros and cons, we hope that this review will serve as a comprehensive resource for zebrafish researchers, allowing them to choose the most appropriate method depending upon their budget, access to required equipment and the throughput needs of the projects.

Systematic Comparison of Computational Tools for Sanger Sequencing-Based Genome Editing Analysis

“…Several strategies have been developed to analyze indel frequencies; however, each strategy has specific limitations [5][6][7]. Mismatch cleavage assays, high-resolution melting analysis (HRMA), and heteroduplex mobility assays are based on the cleavage or modified migration of heteroduplexes formed by mutated and wild-type (wt) DNA strands [8][9][10].…”