CRISPR/Sc⁺⁺ -mediated genome editing in rice

Area of Science:

• Molecular Biology
• Plant Science
• Biotechnology

Background:

• Streptococcus canis Cas9 (ScCas9) is an RNA-guided endonuclease used for genome editing.
• Its activity in rice is known to be locus-dependent.
• The protospacer adjacent motif (PAM) specificity for ScCas9 is NNG.

Purpose of the Study:

• To investigate the performance of a modified ScCas9 variant, Sc++, in the rice genome.
• To evaluate Sc++'s genome editing capabilities at different NNG PAM sites.
• To assess the potential of Sc++ for base editing applications in rice.

Main Methods:

• A ScCas9 variant, Sc++, was engineered with a T1227K mutation and loop substitutions.
• The genome editing activity of Sc++ was tested at various NNG PAM sites in rice.
• The nickase activity of Sc++ was utilized for targeted base editing using the rBE73b adenine base editor.

Main Results:

• Sc++ demonstrated broader genome editing compared to the original ScCas9.
• The Sc++ nickase significantly improved targeted base editing in transgenic rice.
• Numerous new OsGS1 alleles were generated, valuable for future rice germplasm screening.

Conclusions:

• The CRISPR/Sc++ system offers enhanced genome editing capabilities for rice.
• Sc++ expands the available genome-editing tools for rice research and crop improvement.
• This technology facilitates the generation of diverse alleles for traits like herbicide resistance.