Fast and Quantitative Identification of Ex Vivo Precise Genome Targeting-Induced Indel Events by IDAA
Recent developments in gene targeting methodologies such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionized approaches for gene modifications in cells, tissues, and whole animals showing great promise for translational applications. With regard to CRISPR/Cas9, a variety of repurposed systems have been developed to achieve gene knock-out, base editing, targeted knock-in, gene activation/repression, epigenetic modulation, and locus-specific labeling. A functional communality of all CRISPR/Cas9 applications is the gRNA-dependent targeting specificity of the Cas9/gRNA complex that, for gene knock-out (KO) purposes, has been shown to dictate the indel formation potential. Therefore, the objective of a CRISPR/Cas9 KO set up is to identify gRNA designs that enable maximum out-of-frame insertion and/or deletion (indel) formation and thus, gRNA design becomes a proxy for optimal functionality of CRISPR/Cas9 KO and repurposed systems. To this end, validation of gRNA functionality depends on efficient, accurate, and sensitive identification of indels induced by a given gRNA design. For in vitro indel profiling the most commonly used methods are based on amplicon size discrimination or sequencing. Indel detection by amplicon analysis (IDAA™) is an alternative sensitive, fast, and cost-efficient approach ideally suited for profiling of indels induced by Cas9/gRNA with similar sensitivity, specificity, and resolution, down to single base discrimination, as the preferred next-generation sequencing-based indel profiling methodologies. Here we provide a protocol that is based on complexed Cas9/gRNA RNPs delivered to primary peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals followed by quantitative IDAA indel profiling. Importantly, the protocol described benefits from a short “sample-to-data” turnaround time of less than 5 h. Thus, this protocol describes a methodology that provides a suitable and effective solution to validate and quantify the extent of ex vivo CRISPR/Cas9 targeting in primary cells.
Key wordsIndel detection by amplicon analysis (IDAA™) NGS Ex vivo precise genome targeting PBMCs Indel “finger print” Primary cells CD34+ CRISPR/Cas9 RNP Synthetic gRNA ProfileIt™
We thank Vasili Korol and Ilia A. Solov’yov from the University of Southern Denmark, Department of Physics, Chemistry and Pharmacy, for development of ProfileIt™ and Camilla Andersen from Copenhagen Center for Glycomics, Department of Odontology, University of Copenhagen, for excellent technical assistance. This work was supported by the Witten/Herdecke University internal research promotion Grant No. IFF2017-12, the German Duchenne Foundation “Aktion Benni & Co.” starting grant to E.E.-S., the Danish National Research Foundation [DNRF107], the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 765269, and the German Federal Ministry of Education and Research (BMBF). Z.Y. received support from the Lundbeck Foundation and H.H.W. received support from ERC-2017-COG Type of action: ERC-COG; 772735; GlycoSkin.
Conflict of Interest Statement: E.P.B. declares that a patent application covering the IDAA™ method is pending, and acts as scientific advisor for Cobo Technologies Aps.
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