Abstract
The development of gene editing technologies, especially the CRISPR-Cas9 system, has been pivotal for understanding the functional role of proteins. Rapid and efficient genotyping methods are necessary to screen for generated mutations and streamline the isolation of homozygotes. CRISPR-Cas9 system targeting a single site in the gene typically results in small indels. Many genotyping methods utilize the heteroduplex that is formed when wild-type and mutant amplicons with small indels anneal during PCR creating a bubble due to mismatched strands. These methods include T7 endonuclease/Cel-I assay, high resolution melting (HRM) analysis, and heteroduplex mobility assay (HMA). Our protocol explains a simple, two step method of a mixing HMA (mHMA) to identify homozygous mutants, a modification of the previously published HMA. We have utilized the mHMA for screening and genotyping numerous CRISPR generated models. The mHMA method to differentiate homozygous wild type from homozygous mutant animals eliminates - •DNA sequencing, even with small indels that can be difficult to discern on a gel.•additional enzymatic reaction steps, such as with the T7EI/Cel-I assay.•specialized equipment and analysis tools, such as with HRM analysis.
Highlights
Biochemistry, Genetics and Molecular Biology Genotyping method for CRISPR-Cas9 generated homozygous mutations Mixing heteroduplex mobility assay for screening homozygous mutants S
5X tris-borate-EDTA (TBE) buffer 40% acrylamide 10% ammonium persulfate solution (APS) Tetramethylethylenediamine (TEMED) Hand casting system Electrophoresis chamber and power supply 10X TBE running buffer DNA loading dye DNA ladder (100 bp DNA ladder) DNA stain Gel imaging system (ultraviolet (UV) imaging) Note: Chemicals and other components can be used from any reliable company
Genomic DNA samples used in this study were made available from animals that were generated under an approved Institutional Animal Care and Use Committee (IACUC) protocol at the University of Alabama at Birmingham
Summary
Polymerase chain reaction (PCR) tubes (strip format), 0.2mL Designed primer set for CRISPR target site (forward and reverse primers) Taq 2X Master Mix (M0270 L; New England Biology, Ipswich, MA). Nuclease-free water Thermocycler system Micropipettes Polyacrylamide gels (precast or handcast); reagents below if hand casting: 5X tris-borate-EDTA (TBE) buffer 40% acrylamide 10% ammonium persulfate solution (APS) Tetramethylethylenediamine (TEMED) Hand casting system (if making handcast gels) Electrophoresis chamber and power supply 10X TBE running buffer DNA loading dye DNA ladder (100 bp DNA ladder) DNA stain (ethidium bromide or alternative) Gel imaging system (ultraviolet (UV) imaging) Note: Chemicals and other components can be used from any reliable company. No animals were directly used in this study. Genomic DNA samples used in this study were made available from animals that were generated under an approved Institutional Animal Care and Use Committee (IACUC) protocol at the University of Alabama at Birmingham. G0- mutagenized generation (e.g. animals obtained from embryos injected with CRISPR-Cas nucleases). G1- first generation following mutagenesis (G0 x wild type) F1- first filial or inbreeding generation
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