Sequence-specific nucleases have been exploited to create targeted gene
knockouts in various plants. However, replacing a fragment and even
obtaining gene insertions at specific loci in plant genomes remain
challenging. Chinese Academy of Sciences' Jun Li, along with a group of
researchers, describes an efficient intron-mediated site-specific gene
replacement and insertion that generate mutations using non-homologous end
joining (NHEJ) using CRISPR/Cas9.
Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a
donor DNA template including the same pair of sgRNA sites, the team achieved
gene replacements in the rice gene 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS) at a frequency of 2.0%. The team also obtained targeted
gene insertions at a frequency of 2.2% using a sgRNA targeting one intron
and a donor DNA template including the same sgRNA site.
Rice plants harboring the OsEPSPS gene with the intended substitutions were
also found to be glyphosate-resistant. Furthermore, the site-specific gene
replacements and insertions were inherited by the next generation. These new
approach can be used to replace targeted gene fragments and insert DNA
sequences into specific genomic sites in rice and other plants.
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