Alt r genome editing detection kit

Genomic DNA was isolated using NucleoSpin Tissue (catalog# 740952, Macherey-Nagel, Allentown, PA, USA) from cells treated with LV_crMYOC, AAV2_crMYOC, LV_Null and AAV2_Null. Untreated cells were used as experimental control. MYOC, which is a target of selected gRNA was amplified by PCR. PCR product was denatured and reannealed using the Alt-R Genome Editing Detection Kit protocol (catalog# 1075932, Integrated DNA Technologies, Coralville, Iowa, USA). This generated mismatched heteroduplex DNA products containing strands with CRISPR/Cas9-induced indel reannealed to wild-type strands or different indel. The heteroduplexes were subsequently detected using T7 endonucleases (T7E1), that cleaved the mismatched DNA. The resulting cleaved products were analyzed by gel electrophoresis.

For the detection of mutagenesis at target sites in each Cas12a RNP‐introduced E. gracilis, a T7 Endonuclease I (T7EI) assay was performed using an Alt‐R Genome Editing Detection Kit (Integrated DNA Technologies). Genomic DNA template from E. gracilis was extracted using a Kaneka Easy DNA Extraction Kit version 2 (KANEKA, Tokyo, Japan). DNA fragments including the EgGSL2 target sites were amplified with Tks Gflex DNA Polymerase (Takara Bio, Shiga, Japan) using the EgGSL2 Check F‐R primer set (Table S1). T7EI treatment was carried out according to the manufacturer's protocol using the amplified DNA fragments. Digested DNA fragments were analysed by agarose gel electrophoresis.

To detect the mutation resulting from DSB repair through the NHEJ pathway, a T7E1 assay was performed as described previously [23 (link)]. T7EI assay detect on-target genome editing and estimate editing efficiency using T7 endonuclease I (T7EI), which can cleave a heteroduplex DNA having mutation in any strand. In the T7EI assay, CRISPR-induced mutant gene is amplified by PCR. The PCR products are denatured and reannealed to allow heteroduplex formation between wild-type DNA and CRISPR–mutated DNA. Mutations are then detected using T7EI, which recognizes and cleaves mismatched DNA heteroduplexes. T7EI assay results are analyzed by visualizing cleavage products and full-length amplicons by gel electrophoresis. In this study genomic DNA was isolated from ChiLCV-challenged plants treated either with gRNA-Cas9 or with mock vector (control) samples collected at 4 dpi and was used as the template for PCR using primers ChiLCV-C1/V1q F and ChiLCV-IR R (Table 1). Amplicons of the ChiLCV genomic fragment obtained from those samples were denatured, renatured, and treated with T7EI using the Alt-R^™ Genome Editing Detection Kit (Integrated DNA Technologies) following the manufacturer’s protocol. Results were evaluated using agarose gel electrophoresis. A control heteroduplex DNA sample provided in the kit served as positive control for the assay.

T1 plants were screened for mutations using a T7 endonuclease 1 (T7E1) assay. DNA was extracted from 3-week-old T1 plants by heating a 1 mm leaf disk in 25 μL Extract-N-Amp extraction solution (E7526) at 95 °C for 10 minutes and then adding 25 μL PCR Diluent (E8155, MilliporeSigma, St. Louis, MO). Primers were selected to amplify an approximately 1000 bp region flanking the Cas9 target site (Supplemental Table S1). PCR was carried out using Phire Green Hot Start II Mastermix (ThermoFisher Scientific, Waltham, MA) under manufacturer-recommended conditions. For the T7E1 assay, the Alt-R Genome Editing Detection Kit (Integrated DNA Technologies, Coralville, IA) was used according to manufacturer specifications. In any samples with the presence of non-wildtype amplicons, PCR products were purified (Wizard SV Gel and PCR Clean-Up System, Promega Corporation, Madison, WI) and sent for Sanger sequencing at the Cornell Biotechnology Resource Center (Cornell University, Ithaca, NY). T2 seeds collected from plants with confirmed target site mutations were screened for the absence of fluorescence and the presence of a homozygous mutation at the target site using Sanger sequencing. T3 seeds collected from these non-transgenic, homozygous mutant plants were used for further analyses.

Prnp fragments were amplified using AccuPrime Taq DNA Polymerase, High Fidelity (Invitrogen, LS12346086; see S1 Table for primer sequences). After agarose gel electrophoresis, DNA concentrations were estimated by comparison to the GeneRuler 1 kb Plus DNA Ladder; band intensities were quantified using ImageJ. Hybridization and T7E1 incubation steps were performed according to the Alt-R Genome Editing Detection Kit instructions (Integrated DNA Technologies, 1075931), except that incubation with T7E1 was shortened to 30 min. Reactions were stopped by addition of EDTA to ~25 mM.