@inbook{5bb4bca16e5748dd898b1a405e210a33,
title = "Fast and Quantitative Identification of Ex Vivo Precise Genome Targeting-Induced Indel Events by IDAA",
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{\texttrademark}) 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.",
keywords = "CD34+, CRISPR/Cas9, Ex vivo precise genome targeting, Indel detection by amplicon analysis (IDAA{\texttrademark}), Indel “finger print”, NGS, PBMCs, Primary cells, ProfileIt{\texttrademark}, RNP, Synthetic gRNA",
author = "Saskia K{\"o}nig and Zhang Yang and Wandall, {Hans Heugh} and Claudio Mussolino and Bennett, {Eric Paul}",
year = "2019",
doi = "10.1007/978-1-4939-9170-9_4",
language = "English",
isbn = "978-1-4939-9169-3",
series = "Methods in Molecular Biology",
publisher = "Humana Press",
pages = "45--66",
editor = "Luo, {Yonglun }",
booktitle = "CRISPR Gene Editing",
address = "United States",
}