Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

Area of Science:

• Agricultural Science
• Molecular Biology
• Biotechnology

Background:

• CRISPR/Cas9 technology is crucial for precision crop breeding.
• Key optimization targets include minimizing transgene integration and off-target mutations.

Purpose of the Study:

• To develop an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs).
• To assess the efficiency, off-target mutation rate, and transgene-free status of RNP-mediated editing.

Main Methods:

• Utilized CRISPR/Cas9 ribonucleoproteins (RNPs) for genome editing in immature bread wheat embryos.
• Conducted deep sequencing to detect off-target mutations.
• Evaluated the resulting mutant plants for transgene integration.

Main Results:

• The CRISPR/Cas9 RNP protocol is efficient, yielding 4-5 independent mutants from 100 embryos within 7-9 weeks.
• RNP-mediated editing resulted in significantly lower off-target mutations compared to DNA-based CRISPR/Cas9 editing.
• All generated mutants were confirmed to be completely transgene-free.

Conclusions:

• CRISPR/Cas9 RNP mediated genome editing is a highly efficient and precise method for bread wheat.
• This transgene-free approach minimizes off-target effects and holds significant commercialization potential for crop improvement.