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. 2020 May 19;6:30. doi: 10.1038/s41421-020-0164-0

Fig. 2. Design and construction of Tf1-splicing reporter system for retrotransposition.

Fig. 2

a Schematic representation of life cycle for Tf1-splicing reporter. The NEO gene was placed in Tf1 genome upstream of 3′-LTR, but would transcribe in the opposite orientation to Tf1 transcription (denoted by a blue arrow). Four different artificial introns were designed to be inserted in NEO gene, respectively, aligned in the same orientation to that of Tf1. Scissors denotes Cas12a-targeting sequences of NEO gene formed from splicing. nmt1 nmt1 promoter, Gag gag protein, PR protease, RT reverse transcriptase, IN integrase, NEO G418 resistance gene. b Schematic representation of four NEO gene constructs with artificial intron (black letters) in different insertion sites and their splicing products. The sequences formed via splicing are targeted by designed crRNAs (denoted to the right). The five PAM regions for Cas12a targeting are underlined in orange.