Benchmarks www.BioTechniques.com 275 Vol. 64 | 2018 No. 6 Benchmarks Benchmarks Our approach implements a PAGE technique that is known to provide resolution of as small as 1 bp in 1000 bp [1,2] as an inexpensive and robust screening approach for identifying CRISPR-Cas9-in- duced mutations in zebrafish. The CRISPR-Cas9 genome editing technique is widely used in many labs, especially in the zebrafsh community [3–5]. In our experience, the rate-limiting step when using this technology is the screening of zebrafsh for CRISPR-Cas9-induced mutations. Several techniques describing the identifcation of CRISPR-Cas9-induced mutations have been reported, each with their own strengths and limitations [6–14]. T7 Endonuclease I (T7E1) and Surveyor Mismatch Cleavage Assays, both PCR- and molecular-based assays, are effcient in identifying mismatched DNA at a specifc locus; however, these assays also detect single-nucleotide polymorphisms (SNPs). SNPs are prevalent in the zebrafsh genome, and in our hands, use of the T7E1 assay leads to false-positive results for our genes of interest. High-resolution melting analysis (HRMA) and derivative melting curves require a quantitative PCR machine that can be expensive to implement if the equipment and software are not already in a laboratory. Furthermore, the deriv- ative melting curve assay is best used to detect mutations that have a change of greater than 15 bp in nucleic acid length at the target site [10]; however, the median CRISPR-Cas9-induced indel size ranges from 4–9 bp depending on the length of the single-stranded guide RNA (sgRNA) [15]. Sequencing is defnitive in identifying indels of any size but can be expensive and slow for a primary screening approach. In our laboratory, CRISPR-Cas9 is used as a tool to create and establish mutants for specifc genes of interest in zebrafsh. To facilitate screening, we tested neutral PAGE as a rapid and sensitive method for identi- fying CRISPR-Cas9 mutants. There are assays that use PAGE to identify CRISPR- Cas9-induced mutations in zebrafish, mice and human cells; however, these assays require heteroduplex formation prior to PAGE [13,14]. We reasoned that it would be possible to directly run PCR products via PAGE both to identify new mutations and to genotype zebrafsh with known mutations based solely upon a size difference in amplicon length rather than through formation and detection of heteroduplexes or enzymatic cleavage of DNA mismatches. The detection of small changes in nucleotide length, such as those of a typical CRISPR-Cas9 indel, requires a high-percent polyacrylamide gel. We use gels containing a 15% concen- tration of acrylamide monomer to obtain suf fcient resolving power in amplicons ranging from 25–150 bp [2]. Importantly, we fnd that an acrylamide monomer to N,N′ -methylenebisacrylamide crosslinker A PAGE screening approach for identifying CRISPR-Cas9-induced mutations in zebrafish Ariel J VanLeuven 1 , Sungdae Park 2 , Douglas B Menke 2 & James D Lauderdale 1,2 1 Department of Cellular Biology, University of Georgia, 724 Biological Sciences Building, Athens, GA 30602- 2607, USA; 2 Department of Genetics, University of Georgia, Paul D Coverdell Center, 500 D.W. Brooks Drive, Room 250, Athens, GA 30602, USA; 3 Faculty in the Neuroscience Division of the Biomedical & Health Sciences Institute (BHSI), University of Georgia, Paul D Coverdell Center, 500 D.W. Brooks Drive, Athens, GA 30602-7394, USA BioTechniques 64:275-278 (June 2018) 10.2144/btn-2018-0012 Keywords: Danio rerio • genotyping • polymerase chain reaction (PCR) The introduction of CRISPR-Cas9 technology for targeted mutagenesis has revolutionized reverse genet- ics and made genome editing a realistic option in many model organisms. One of the diffculties with this technique is screening for mutations in large numbers of samples. Many screening approaches for identify- ing CRISPR-Cas9 mutants have been published; however, in practice these methods are time consuming, expensive, or often yield false positives. This report describes a PCR-based screening approach using non- denaturing PAGE. This approach does not depend on the formation of heteroduplexes and reliably detects changes as small as 1 base-pair (bp) in nucleic acid length at the target site. This approach can be used to identify novel mutations and is also useful as a routine genotyping method. METHOD SUMMARY Our approach implements a PAGE technique that is known to provide resolution of as small as 1 bp in 1000 bp as an inexpensive and robust screening approach for identifying CRISPR-Cas9-induced mutations in zebrafish. In this approach, we PCR amplify a small region (<150 bp) encompassing the CRISPR-Cas9 target site and the PCR product is then directly run on a 10 × 8 cm, 15% polyacrylamide gel at 200 Volts for 2–2.5 h. Using this approach, we routinely detect 1–14 bp indels without relying on the formation of heteroduplexes prior to PAGE. For reprint orders, please contact: reprints@futuremedicine.com