Rapid and low-cost strategy for detecting genome-editing induced deletion: A single-copy case

Genome editing techniques have been implemented in human daily lives, which has created a high demand for the development of new gene-edited product analysis methods. Conventional assays are time-consuming, labor-intensive, and costly. This paper proposes a rapid and low-cost strategy for detecting genome-editing induced deletion which works by integrating rapid-multiplex ligation-dependent probe amplification (MLPA) with a dual-lateral flow nucleic acid biosensor (LFNAB) cascade in a single-copy case. A rapid-MLPA was first introduced to the LFNAB system as a replacement for the conventional PCR for enhanced specificity and accuracy. A dual-LFNAB was applied for the detection of genome-editing induced deletion without any additional instrumentation or complex operation. After optimization, we achieved the specific detection of wildtype alleles and deletion alleles in spiked samples with a detection limit of 0.4 fM, which is comparable to that of electrophoresis-based detection assays and fluorescent biosensors. To confirm the validity and feasibility of our strategy, we assayed two pork samples from two WUZHISHAN pigs successfully. By comparing the detection results from next-generation sequencing analysis, we found that the proposed cascade demonstrates at least 20-fold shorter assay time and at least 100-fold less assay cost. To this effect, the proposed method is a rapid and low-cost solution to sample-to-answer detection of genome-editing induced deletion and shows remarkable potential in regards to international trade, transparency, and freedom of choice.