Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform

Area of Science:

• Plant biotechnology
• Genome engineering
• Molecular biology

Background:

• Developing new genome engineering tools requires testing numerous CRISPR components.
• Plant biotechnology lacks high-throughput methods for iterative design-build-test-learn cycles of gene-editing reagents.

Purpose of the Study:

• To develop a high-throughput platform, ITER (Iterative Testing of Editing Reagents), for optimizing plant genome editing tools.
• To create an efficient LbCas12a-ABE by optimizing its components using the ITER platform.

Main Methods:

• ITER utilizes 96-well arrayed protoplast transfections and high-content imaging for rapid reagent testing.
• Optimization of LbCas12a-ABE involved iterative improvements of five key components: NLS, crRNA, LbCas12a, adenine deaminase, and linker.

Main Results:

• ITER enabled a full optimization cycle within 3 weeks for wheat and maize protoplasts.
• Optimized LbCas12a-ABE activity increased from undetectable levels to 40% on a GFP reporter.
• High base editing efficiency (up to 55%) was achieved in stable wheat transformants, with edits transmitted to progeny.

Conclusions:

• ITER is a sensitive, versatile, and high-throughput platform for accelerating plant genome editing technology development.
• The optimized LbCas12a-ABE and other derived tools demonstrate broad applicability for Cas12a toolbox enhancement.
• ITER is expected to facilitate the creation and optimization of genome editing reagents across diverse plant species.