Fast and Quantitative Identification of Ex Vivo Precise Genome Targeting-Induced Indel Events by IDAA
Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
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.
Original language | English |
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Title of host publication | CRISPR Gene Editing |
Editors | Yonglun Luo |
Number of pages | 22 |
Publisher | Humana Press |
Publication date | 2019 |
Pages | 45-66 |
ISBN (Print) | 978-1-4939-9169-3 |
ISBN (Electronic) | 978-1-4939-9170-9 |
DOIs | |
Publication status | Published - 2019 |
Series | Methods in Molecular Biology |
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Volume | 1961 |
ISSN | 1064-3745 |
- CD34+, CRISPR/Cas9, Ex vivo precise genome targeting, Indel detection by amplicon analysis (IDAA™), Indel “finger print”, NGS, PBMCs, Primary cells, ProfileIt™, RNP, Synthetic gRNA
Research areas
ID: 216866944