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Approaches for the sensitive detection of rare base and prime editing events
Citation
Nguyen Tran, MP 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, MP (Mr Minh Thuan Nguyen Tran) |
| UTAS Author: | KC, R (Dr 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 |
| Web of Science® Times Cited: | 1 |
| Deposited By: | Wicking Dementia Research and Education Centre |
| Deposited On: | 2021-06-24 |
| Last Modified: | 2021-09-22 |
| Downloads: | 0 |
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