Boosting genome editing in plants with single transcript unit surrogate reporter systems

CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement.However,the challenge of low editing activity complicates the identification of editing events.In this study,we introduce multiple single transcript unit surrogate reporter(STU-SR)systems to enhance the selection of genome-edited plants.These systems use the same single guide RNAs designed for endogenous genes to edit reporter genes,establishing a direct link between reporter gene editing activity and that of endogenous genes.Various strategies are used to restore functional reporter genes after genome editing,including efficient single-strand annealing(SSA)for homologous recombination in STUSR-SSA systems.STU-SR-base editor systems leverage base editing to reinstate the start codon,enriching C-to-T and A-to-G base editing events.Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in the monocot rice,encompassing Cas9 nuclease-based targeted mutagenesis,cytosine base editing,and adenine base editing.The systems exhibit compatibility with Cas9 variants,such as the PAM-less SpRY,and are shown to boost genome editing in Brassica oleracea,a dicot vegetable crop.In summary,we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.

Drosophila RecQ5 is required for efficient SSA repair and suppression of LOH in vivo

RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination.However,the specific pathway(s)in which it is involved and the underlining mechanism(s)remain poorly understood.We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5(dRecQ5)functions in vivo in homologous recombination-mediated double strand break(DSB)repair.We generated null alleles of dRecQ5 using the targeted recombination technique.The mutant animals are homozygous viable,but with growth retardation during development.The mutants are sensitive to both exogenous DSB-inducing treatment,such as gamma-irradiation,and endogenously induced double strand breaks(DSBs)by I-Sce I endonuclease.In the absence of dRecQ5,single strand annealing(SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion,or non-homologous end joining(NHEJ)when inter-chromosomal homologous sequence is unavailable.Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity(LOH)assays.Together,our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.