Improvement of multiplex semi-nested PCR system for screening of rare mutations by high-throughput sequencing.

The CRISPR/Cas9 system is an efficient gene-editing method, but it is difficult to obtain mutants for some specific species and special genome structures. A previously reported multiplexed, semi-nested PCR target-enrichment approach, which does not rely on transgenic technology, has been shown to be an effective and affordable strategy for the discovery of rare mutations in a large sodium azide-induced rice population. However, this strategy has the potential for further optimization. Here, we describe an improved multiplex semi-nested PCR target-enrichment strategy with simplified processing procedures, reduced false-positive rates and increased mutation detection frequency (1 mutation/73 Kb).