Abstract
BackgroundThe use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA.ResultsHere, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences.ConclusionsHigh-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0796-9) contains supplementary material, which is available to authorized users.
Highlights
The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells
We designed two pairs of transcription activator-like effector nucleases (TALENs; 1193/1194 and 1195/1196) and two guide RNAs to introduce double-strand break (DSB) at positions ranging from 56 bp to 203 bp from the anthocyanin mutant 1 (ANT1) start codon (Figure S1a, b in Additional file 1)
The 1193/1194 TALEN pair, which cut closest to the start codon, and the two guide RNA (gRNA) along with Cas9 were tested for their ability to create nonhomologous end joining (NHEJ)-induced mutations at the target locus in tomato protoplasts (Figure S2 in Additional file 1)
Summary
The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA. Already in many plant species, loss of function mutations can be created using sequence-specific nucleases that introduce double-strand breaks (DSBs) in coding sequences [1]. Repair of DSBs through homologous recombination (HR), offers a much richer spectrum of possibilities for modifying plant genomes, ranging from introducing single.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have