Detection of Multi-Base Mutation by Genome Editing Using MultiNA™

Introduction

With the advent of genome editing technology, specific genes can now be knocked out and inserted in target genes. However, in confirming the results of genome editing, it is necessary to check whether the mutation is on-target or not. Although direct sequence analysis is the most reliable method, assays involving large numbers of samples are costly. The heteroduplex mobility assay (HMA) is a simple method in which wild- and mutant-type duplexes are analyzed by electrophoresis and on-target editing is confirmed by the difference in mobility between the homoduplex and heteroduplex strands. The HMA method has gained wide acceptance as a tool that enables easy confirmation of genome editing results by using a fully-automatic electrophoresis system. However, due to differences in multiple bases (from 1 to 5 base pairs), it is sometimes impossible to ascertain on-target editing by HMA because the difference in mobility is cancelled out by the 3-dimensional structures of the homoduplexes and heteroduplexes. In such cases, confirmation is possible by the enzyme mismatch cleavage method using the enzymes called T7 endonuclease I (T7EI) and Cel 1. These enzymes possess activity that recognizes and cleaves mismatches in doublestranded DNA. Because the enzymes can also recognize mismatches of multiple bases, this method is effective for detecting mutations that cannot be detected by HMA. This article introduces an example of an analysis in which quick and simple detection of mismatch cleavage by T7EI was possible by using a Shimadzu microchip electrophoresis system.

August 19, 2020 GMT