Gene Replacements and Insertions in Rice via Intron-Targeting CRISPR

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.

For more information, read the article in Nature Plants.