One-step genotyping method in CRISPR based on short inner primer-assisted, tetra primer-paired amplifications

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Title
One-step genotyping method in CRISPR based on short inner primer-assisted, tetra primer-paired amplifications
Author(s)
Nan Ee Lee; Dae In Ha; Jeong Heon KoYong-Sam Kim
Bibliographic Citation
Molecular and Cellular Probes, vol. 55, pp. 101675-101675
Publication Year
2021
Abstract
Base editors and prime editors induce precise DNA modifications over one or several nucleotides in eukaryotic cells. The T7E1 assay has been widely adopted for the assessment of genome editing, but it has several limitations in the applications for prime editing and base editing due to low sensitivity, inaccuracy and additional disadvantages. Here, we propose a short inner primer-assisted, tetra primer-paired amplification (SIPATA) method as an alternative to T7E1 analysis. SIPATA is a PCR-based method in which two long outer and two short (15 nt) inner primers are used for the amplification of a specific genotype in the presence of Hot start-Taq. One of the inner primers carries a 3'-terminally wild-type nucleotide sequence, and the other carries a post-editing sequence. Under optimized conditions, SIPATA enabled sensitive and accurate genotyping of single-nucleotide conversions by base editors and prime editors. Furthermore, SIPATA could be applied to trace low levels of DNA modifications achieved by HDR-mediated gene correction or chimerism during the generation of model animals. Multiplexed genotyping was also possible without compromising those multifaceted analytical advantages of SIPATA. Our findings demonstrate that SIPATA offers a robust, fast and sensitive genotyping platform for single-nucleotide variations in a variety of CRISPR applications.
Keyword
Base editingCRISPRGenotypingPrime editingSIPATASNP
ISSN
0890-8508
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/j.mcp.2020.101675
Type
Article
Appears in Collections:
Synthetic Biology and Bioengineering Research Institute > Genome Editing Research Center > 1. Journal Articles
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