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Review
. 2022 May 11;13(5):857.
doi: 10.3390/genes13050857.

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

Blake Carrington  1 , Kevin Bishop  1 , Raman Sood  1
Affiliations
Review

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

Blake Carrington et al. Genes (Basel). .

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.

Keywords: AS-PCR; CRISPR/Cas9; HMA; HRMA; LDR; RFLP; fluorescent PCR; indels; qPCR; zebrafish.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
A schematic depicting main steps and output for methods that identify indels by direct analysis of PCR products. Genomic DNA containing an indel (marked in red) is PCR amplified and directly analyzed using either of the methods listed here. PAGE is straightforward with size separation of PCR products by running on polyacrylamide gels. HMA is performed by denaturation and reannealing of PCR products to form homo- and/or heteroduplexes which are separated by PAGE gels. Fluorescent PCR involves use of a fluorescently labeled primer and PCR products are run on a capillary electrophoresis instrument. In HRMA, PCR is performed in the presence of a fluorescent dye that binds dsDNA. As the PCR products melt the dye is released and fluorescence is measured, resulting in a melt curve which corresponds to the genotype. A 3-primer qPCR is performed with primers that flank the indel site with a third primer that overlaps the indel site. PCR is performed on a qPCR instrument and the relative quantity of the smaller PCR product is compared to the larger PCR product to determine the genotypes in the sample.
Figure 2
Figure 2
A schematic depicting main steps and output for indel detection methods that require further processing of PCR products before analysis. Sanger sequencing can be used for analysis of both somatic and germline samples followed by either manual or computational analysis of chromatograms. T7EI assay is performed by denaturation and reannealing of PCR products followed by T7EI digestion. Heteroduplexes are digested with T7EI while homoduplexes stay intact. Digested products are run on a gel and heterozygous samples can be differentiated while WT and homozygous sample run at the same size and cannot be distinguished from each other. Next generation sequencing (NGS) is carried out with a kit for library preparation, cluster generation, and sequencing of clusters. Sequence reads are then aligned and analyzed with bioinformatics tools.
Figure 3
Figure 3
Indel detection methods that require the mutant allele to be known. We used a 2 bp deletion (delAA) as an example to illustrate these methods in this figure. (A) Allele-specific PCR (AS-PCR) is performed using forward primers that are allele specific and each has a unique fluorophore (red and blue stars). Fluorescence can be detected following the PCR with a plate reader or in real time with a qPCR instrument. (B) RFLP assay is conducted by digestion of PCR products with a restriction enzyme that will only recognize the WT or mutant allele. After digestion samples are run on a gel and genotypes can be determined based on bands which correlate to if digestion occurred or not. (C) Multiplex ligation detection is carried out post-PCR amplification. LDR primers consisting of two allele-specific primers and a common primer are annealed to the PCR products. Primers are then ligated together if the common primer and allele specific primer are perfectly adjected to each other. The ligated products are run on a PAGE gel to determine genotypes. Red and blue circles denote different fluorophores used for WT and mutant alleles.
Figure 4
Figure 4
A schematic of the workflow to establish and characterize indel mutants with the applicable indel detection methods for somatic analysis, founder screening or genotyping listed underneath. Green boxes represent when indels are not known while the blue box is when an indel mutation has been established. Methods that can be used at all steps of mutant generation and characterization are highlighted in red.
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