Precision genome editing is highly desired for crop improvement.The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering.A prime editing(PE)approach combinin...Precision genome editing is highly desired for crop improvement.The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering.A prime editing(PE)approach combining a reverse transcriptase(RT)with a Cas9 nickase and a“priming”extended guide RNA(gRNA)has shown a high frequency for precise genome modification in mammalian cells and several plant species.Nevertheless,the applications of the PE approach in dicot plants are still limited and inefficient.We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation.Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems.Further assessment of the activities of the PE components uncovered that the fusion of RT to Cas9 and the structure of PE gRNAs(pegRNAs)negatively affected the cleaving activity of the Cas9 nuclease.The self-complementarity between the primer binding sequences(PBSs)and spacer sequence might pose risks to the activity of the Cas9 complex.However,modifying the pegRNA sequences by shortening or introducing mismatches to the PBSs to reduce their melting temperatures did not enhance the PE efficiency at the MADS-box protein(SlMBP21),alcobaca(SlALC),and acetolactate synthase 1(SlALS1)loci.Our data show challenges of the PE approach in tomato,indicating that a further improvement of the PE system for successful applications is demanded,such as the use of improved expression systems for enriching active PE complexes.展开更多
基金the National Research Foundation of Korea(the Bio&Medical Technology Development Program 2020M3A9I4038352,2020R1A6A1A03044344,2020R1I1A1A01072130,and 2022R1A2C3010331)the Pro gram for New Plant Breeding Techniques(NBT+1 种基金grant PJ016867022022)Rural Development Administration(RDA),Republic of Korea.Author contributions:T.V.V.and J.-Y.K.conceived and designed the research.T.V.V.,N.T.N.,J.K.,S.D.,and J.L.conducted the experiments.T.V.V.and J.-Y.K.analyzed the data.T.V.V.wrote the manuscript.T.V.V.and J.-Y.K.finalized the manuscript.All authors read and approved the manuscript.
文摘Precision genome editing is highly desired for crop improvement.The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering.A prime editing(PE)approach combining a reverse transcriptase(RT)with a Cas9 nickase and a“priming”extended guide RNA(gRNA)has shown a high frequency for precise genome modification in mammalian cells and several plant species.Nevertheless,the applications of the PE approach in dicot plants are still limited and inefficient.We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation.Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems.Further assessment of the activities of the PE components uncovered that the fusion of RT to Cas9 and the structure of PE gRNAs(pegRNAs)negatively affected the cleaving activity of the Cas9 nuclease.The self-complementarity between the primer binding sequences(PBSs)and spacer sequence might pose risks to the activity of the Cas9 complex.However,modifying the pegRNA sequences by shortening or introducing mismatches to the PBSs to reduce their melting temperatures did not enhance the PE efficiency at the MADS-box protein(SlMBP21),alcobaca(SlALC),and acetolactate synthase 1(SlALS1)loci.Our data show challenges of the PE approach in tomato,indicating that a further improvement of the PE system for successful applications is demanded,such as the use of improved expression systems for enriching active PE complexes.
