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Abstract

Precision chemistry entailing user-directed nucleotide substitutions and template-specified repair can be facilitated by base editing and prime editing, respectively. Recently, the diversification of adenine, cytosine, andprime editor variants obliges a considered, high-throughput evaluation of these tools for optimized, end-pointapplications. Herein, we outline novel, cost-effective and scalable approaches for the rapid detection of baseediting and prime editing outcomes using gel electrophoresis. For base editing, we exploit primer mismatchamplification (SNP genotyping) for the gel-based detection of base editing efficiencies as low as 0.1%. For primeediting, we describe a one-pot reaction combining polymerase chain reaction (PCR) amplification of the targetregion with restriction digestion (restriction fragment length polymorphism; RFLP). RFLP enables the rapiddetection of insertion or deletion events in under 2.5 h from genomic DNA extraction. We show that our methodof SNP genotyping is amenable to both endogenous target loci as well as transfected, episomal plasmid targets inBHK-21 cells. Next, we validate the incidence of base and prime editing by describing Sanger sequencing andnext-generation sequencing (NGS) workflows for the accurate validation and quantification of on-target editingefficiencies. Our workflow details three different methods for the detection of rare base and prime editing events,enabling a tiered approach from low to high resolution that makes use of gel electrophoresis, Sanger sequencing,and NGS.

Item Type:	Article
Authors/Creators:	Nguyen Tran, MT and KC, R and Patterson, FM and Liu, G-S and Cook, AL and Hewitt, AW
Keywords:	CRISPR, gene editing, base editing, prime editing
Journal or Publication Title:	Methods
Publisher:	Academic Press Inc Elsevier Science
ISSN:	1046-2023
DOI / ID Number:	10.1016/j.ymeth.2021.01.006
Copyright Information:	© 2021 Elsevier Inc. All rights reserved.
Related URLs:	Google Scholar: Liu, G-S UTAS Profile: Liu, G-S UTAS Profile: Cook, AL Google Scholar: Hewitt, AW UTAS Profile: Hewitt, AW
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