Precise gene replacement in rice by RNA transcript-templated homologous recombination

Area of Science:

• Plant molecular biology
• Genome editing technologies
• Biotechnology

Background:

• Efficient delivery of donor repair templates (DRT) to the nucleus is a major challenge for gene replacement in plants via homology-directed DNA repair (HDR).
• In vivo production of RNA templates for transcript-templated HDR (TT-HDR) is hindered by primary transcript processing and cytosolic transport, making them unavailable for nuclear HDR.

Purpose of the Study:

• To develop a novel method for efficient nuclear delivery of RNA templates for transcript-templated homology-directed repair (TT-HDR) in plants.
• To demonstrate the application of this method for precise gene editing in rice.

Main Methods:

• Coupling CRISPR-Cpf1 with a CRISPR RNA (crRNA) array flanked by ribozymes and a DRT flanked by ribozymes or crRNA targets to generate self-processing primary transcripts.
• Utilizing DNA-free ribonucleoprotein complexes containing recombinant Cpf1, crRNAs, and DRT transcripts for gene editing.
• Employing transcript-templated homology-directed repair (TT-HDR) for precise gene modification.

Main Results:

• The developed system produces primary transcripts that self-process to release crRNAs and DRT within the nucleus.
• Successful replacement of the rice acetolactate synthase (ALS) gene with a mutated version using the DNA-free ribonucleoprotein complex and TT-HDR.
• Generation of stable rice lines with two desired mutations in the ALS gene through TT-HDR.

Conclusions:

• This novel CRISPR-Cpf1-based system overcomes previous limitations in delivering RNA templates for nuclear HDR in plants.
• The method enables efficient and precise gene replacement and modification in plants, paving the way for advanced crop improvement.
• Transcript-templated HDR (TT-HDR) using self-processing RNA transcripts represents a powerful tool for plant genome engineering.