Robust CRISPR/Mb2Cas12a genome editing tools in cotton plants

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• CRISPR-Cas systems are powerful tools for genome editing.
• Cas12a variants offer unique properties for gene modification.
• Efficient and versatile genome editing in cotton is crucial for crop improvement.

Purpose of the Study:

• To evaluate the efficiency and accuracy of the CRISPR/Mb2Cas12a system in cotton.
• To assess the temperature tolerance and PAM site flexibility of Mb2Cas12a.
• To develop a multiplex genome editing system using Mb2Cas12a in cotton.

Main Methods:

• Demonstration of CRISPR/Mb2Cas12a system in cotton.
• Testing of Mb2Cas12a efficiency at target sites.
• Evaluation of Mb2Cas12a performance across a temperature range (22°C–32°C).
• Analysis of Mb2Cas12a editing at relaxed VTTV PAM sites.
• Development of a multiplex editing system using a single crRNA cassette.

Main Results:

• Achieved over 90% editing efficiency at target sites in cotton.
• Mb2Cas12a showed significant tolerance to temperatures between 22°C and 32°C.
• Expanded genome editing range by approximately 2.6-fold compared to LbCas12a due to relaxed VTTV PAM recognition.
• Successfully developed a multiplex system for simultaneous editing of eight target sites.

Conclusions:

• The CRISPR/Mb2Cas12a system is highly efficient and accurate for cotton genome editing.
• Mb2Cas12a offers enhanced flexibility in PAM site recognition and temperature tolerance.
• The developed multiplex system provides a powerful tool for complex genetic modifications in cotton.