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Fast and Quantitative Identification of Ex Vivo Precise Genome Targeting-Induced Indel Events by IDAA

  • Saskia König
  • Zhang Yang
  • Hans Heugh Wandall
  • Claudio MussolinoEmail author
  • Eric Paul BennettEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1961)

Abstract

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 words

Indel detection by amplicon analysis (IDAA™) NGS Ex vivo precise genome targeting PBMCs Indel “finger print” Primary cells CD34+ CRISPR/Cas9 RNP Synthetic gRNA ProfileIt™ 

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Medical Center—University of Freiburg, Institute for Transfusion Medicine and Gene Therapy and Center for Chronic Immunodeficiency at Center for Translational Cell Research (ZTZ)FreiburgGermany
  2. 2.Faculty of Health Sciences, Copenhagen Center for Glycomics (CCG), Department of Cellular and Molecular MedicineUniversity of CopenhagenCopenhagenDenmark
  3. 3.Faculty of Health Sciences, Copenhagen Center for Glycomics (CCG), Department of OdontologyUniversity of CopenhagenCopenhagenDenmark

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