Genotyping strategies for detecting CRISPR mutations in polyploid species: a case study-based approach in hexaploid wheat

As a versatile tool for genome engineering, CRISPR-Cas9 has been revolutionizing the field of molecular biology, biotechnology, and crop improvement. By precisely targeting pre-selected genomic sites, CRISPR-Cas9 primarily induces insertions or deletions (indels) of variable size. Despite the significant advance in the technology per se, detecting these indels is the major and difficult part of the CRISPR program in polyploid species, like wheat, with relatively low mutation rates. A plethora of methods are available for detecting mutations, but no method is perfect for all mutation types. In this case study, we demonstrated a new, protocol for capturing length polymorphism from small indels using a nested PCR approach. This new method is tractable, efficient, and cost-effective in detecting and genotyping indels >3-bp. We also discussed the major genotyping platforms used in our wheat CRISPR projects, such as mismatch cleavage assay, restriction enzyme assay, ribonucleoprotein assay, and Sanger sequencing, for their advantages and pitfalls in wheat CRISPR mutation detection.

Fast and reliable sgRNA efficiency testing using HIReff

CRISPR/Cas9 is the method of choice for gene editing like the endogenous knock-in of sequences in order to investigate protein function, abundance or intracellular localization. One of the crucial steps in the preparation of CRISPR/Cas9-mediated knock-ins is the design of sgRNAs, which need to be tested carefully in order to minimize off-target binding and reach highest cleavage efficiency. Usually, sgRNA is evaluated via mismatch cleavage assays, like Surveyor or T7 endonuclease 1 assay. We demonstrate that these methods are often highly cost- and time-intensive with a low sensitivity and high fail rate. As an alternative, we present a new HITI-based sgRNA efficiency (HIReff) test to precisely evaluate sgRNA efficiency. HIReff is based on a sophisticated integration vector with on-site generation of a linear donor fragment that allows a comparably easy read-out via fluorescence signal and integrates several internal controls. Next to a quantifiable sgRNA assessment, HIReff provides additional information on the 'gene/protein to be studied' abundance, subcellular localization and promoter activity and allows derivation of fluorescence protein labeled clonal lines. We highlight benefits of HIReff in comparison to commonly used enzyme-based assays and demonstrate improved practicability and high sensitivity, while being less time-, labor- and cost-intensive at the same time. Our results suggest HIReff as a fast and easy-to-use alternative for sgRNA efficiency testing.

Comparison of T7E1 and Surveyor Mismatch Cleavage Assays to Detect Mutations Triggered by Engineered Nucleases

Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases.