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Approaches for the sensitive detection of rare base and prime editing events

Citation

Nguyen Tran, MT and KC, R and Patterson, FM and Liu, G-S and Cook, AL and Hewitt, AW, Approaches for the sensitive detection of rare base and prime editing events, Methods ISSN 1046-2023 (2021) [Refereed Article]

Copyright Statement

2021 Elsevier Inc. All rights reserved.

DOI: doi:10.1016/j.ymeth.2021.01.006

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, and prime editor variants obliges a considered, high-throughput evaluation of these tools for optimized, end-point applications. Herein, we outline novel, cost-effective and scalable approaches for the rapid detection of base editing and prime editing outcomes using gel electrophoresis. For base editing, we exploit primer mismatch amplification (SNP genotyping) for the gel-based detection of base editing efficiencies as low as 0.1%. For prime editing, we describe a one-pot reaction combining polymerase chain reaction (PCR) amplification of the target region with restriction digestion (restriction fragment length polymorphism; RFLP). RFLP enables the rapid detection of insertion or deletion events in under 2.5 h from genomic DNA extraction. We show that our method of SNP genotyping is amenable to both endogenous target loci as well as transfected, episomal plasmid targets in BHK-21 cells. Next, we validate the incidence of base and prime editing by describing Sanger sequencing and next-generation sequencing (NGS) workflows for the accurate validation and quantification of on-target editing efficiencies. 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 Details

Item Type:Refereed Article
Keywords:CRISPR, gene editing, base editing, prime editing
Research Division:Biological Sciences
Research Group:Genetics
Research Field:Genetics not elsewhere classified
Objective Division:Health
Objective Group:Clinical health
Objective Field:Treatment of human diseases and conditions
UTAS Author:Nguyen Tran, MT (Mr Minh Nguyen Tran)
UTAS Author:KC, R (Mr Rajendra KC)
UTAS Author:Patterson, FM (Ms Freya Patterson)
UTAS Author:Liu, G-S (Associate Professor Guei-Sheung Liu)
UTAS Author:Cook, AL (Associate Professor Tony Cook)
UTAS Author:Hewitt, AW (Professor Alex Hewitt)
ID Code:145017
Year Published:2021
Deposited By:Wicking Dementia Research and Education Centre
Deposited On:2021-06-24
Last Modified:2021-09-22
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