A comprehensive all-in-one CRISPR toolbox for large-scale screens in plants

Area of Science:

• Plant molecular biology
• Genome engineering
• Functional genomics

Background:

• CRISPR-Cas technologies are powerful for single-gene editing but limited for large-scale screens in plants.
• Developing efficient tools for high-throughput genetic screens is crucial for plant science.

Purpose of the Study:

• To develop a comprehensive, all-in-one CRISPR toolbox for diverse genome engineering applications in plants.
• To establish high-throughput methods for large-scale CRISPR screens in both monocot and dicot species.

Main Methods:

• Development of 61 all-in-one T-DNA expression vectors for Cas9, Cas12a, cytosine base editing (CBE), adenine base editing (ABE), and CRISPR-Act3.0.
• Evaluation of vectors in rice and tomato protoplasts.
• Construction of single guide RNA (sgRNA) pools for high-throughput sgRNA selection.
• Application in targeted mutagenesis screens (Acetolactate Synthase) and gene activation screens (Flowering Locus T).

Main Results:

• Demonstrated broad and reliable applicability of the all-in-one vectors in rice and tomato protoplasts.
• Established a high-throughput approach for selecting high-activity sgRNAs.
• Identified novel alleles for herbicide resistance via targeted mutagenesis.
• Successfully identified potent sgRNAs for FLOWERING LOCUS T activation, leading to early flowering in Arabidopsis thaliana.

Conclusions:

• The developed CRISPR toolbox significantly advances the capability for large-scale genetic screens in plants.
• This resource facilitates both loss-of-function and gain-of-function studies, offering broad applications in plant research and crop improvement.