Enhancing lipid production in plant cells through high-throughput genome editing and phenotyping via a scalable automated pipeline

Abstract

Plant bioengineering is a time-consuming and labor-intensive process, with no guarantee of achieving the desired trait. Here we report a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB). FAST-PB achieves gene cloning, genome editing, and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrate that FAST-PB can streamline the Golden Gate cloning process, with the capacity to construct 96 vectors in parallel. To prove the concept, using FAST-PB, we first found that PEG2050 significantly increases transfection efficiency by over 45%. To validate the pipeline, we established a reporter-gene-free method for CRISPR editing via mutation of HCF136 , affecting cellular chlorophyll fluorescence. Next, we applied this pipeline for lipid production and found that diverse lipids were significantly enhanced up to sixfold through introducing multi-gene cassettes via CRISPR activation, and regenerated plant using this platform. Lastly, we harnessed FAST-PB to achieve high-throughput single-cell lipid profiling through the integration of MALDI-MS with the biofoundry, and differentiated engineered and unengineered cells using the single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering, and change what is possibly using single-cell metabolomics in plants.

Plant phenotyping relevance

植物細胞の高スループット表現型取得を組み込んだ自動化プラットフォームの開発であり、クロロフィル蛍光と単一細胞脂質プロファイリングによる評価が技術的中心の一部となっている。

Code and data availability

The supplied blocks describe the FAST-PB pipeline (automated genome editing plus MALDI-FTICR-MS/LC-MS/MS single-cell lipid phenotyping) but contain no public phenotype/trait dataset, image/sensor data deposit, author analysis code, or trained model with an explicit availability statement or URL. Only supplementary (unl

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