Journal: bioRxiv

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

doi: 10.1101/2024.05.29.596527

Figure Lengend Snippet: (A) Gene cloning through the Golden Gate cloning method. (B) Callus transformation. (C) Protoplast isolation from the etiolated maize B73 and N. benthamiana . (D) Protoplast transfection with plasmids. (E) Genomic DNA extraction. The top of each box describes the workflow step while the utilized equipment is shown in smaller font at the bottom of each box.

Article Snippet: A0502: pMOD_A0502 (#91012, Addgene) for CRISPR knockout system in N. benthamiana , B2103: pMOD_B2013 (#91061, Addgene) for CRISPR knockout and activation systems in plants, C0000: pMOD_C0000 (#91081, Addgene) for CRISPR knockout and activation systems in plants, D100: pTRANS_100 (#91198, Addgene) for protoplast system, T230: pTRANS_230 (obtained from Dr. Voytas’s lab ( )) for callus system, A1510: pMOD_A1510 (#91036, Addgene) CRISPR knockout system in maize, A3701: pMOD_A3701 (#91052, Addgene) for CRISPR activation system in N. benthamiana , A4110: pMOD_A4110 (#91056, Addgene) CRISPR activation system in maize.

Techniques: Cloning, Transformation Assay, Isolation, Transfection, DNA Extraction

Journal: bioRxiv

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

doi: 10.1101/2024.05.29.596527

Figure Lengend Snippet: ( A ) Etiolated maize B73 (left) allowed successful protoplast isolation on the automation platform, yielding abundant high-quality protoplast cells (pictured at 40x magnification, middle). Subsequent transfection with the Cas9 vector (p201GFP-Cas9) induced GFP fluorescence in numerous cells (right). ( B ) Protoplast isolation from N. benthamiana leaves (left) using automation yielded abundant and high-quality protoplast cells (pictured at 40x magnification, middle). Protoplasts transfected with the Cas9 vector (p201GFP-Cas9) exhibit GFP fluorescence (right). ( C ) Total intact cell number was counted under three types of PEG (2050, 3350, 4000) treatments in the etiolated maize B73 and N. benthamiana . Biological replicate n = 4. ( D ) Transfection efficiency (Ratio of GFP-expressing and total intact cell numbers) in four days after transfection. Biological replicate n = 4. ( E ) Detection and quantification of chlorophyll fluorescence intensity following HCF136 gene knockout in both maize and N. benthamiana . n = 10. ( F ) Mutation analysis of the HCF136 gene performed using next-generation Sanger sequencing and Inference of CRISPR Editing (ICE) analysis ( ; ) in maize and N. benthamiana . ( G ) Nucleotide sequences identified through ICE analysis following knockout of the HCF136 gene in N. benthamiana callus. ( H ) Overview of the single-cell MALDI workflow for callus-derived protoplasts. ( I ) Protoplast cells in the W5 liquid media were imaged to check the quality before performing MALDI/MS (on the left). Then, using Hoechst staining method to stain the cells, they were imaged (In the middle and on the right). ( J ) Mass spectra with identified lipid species annotated. Error bars represent standard error. Asterisks indicate: *** P < 0.001; ** P < 0.01; * P < 0.05, calculated using two-tailed Welch’s t-test.

Article Snippet: A0502: pMOD_A0502 (#91012, Addgene) for CRISPR knockout system in N. benthamiana , B2103: pMOD_B2013 (#91061, Addgene) for CRISPR knockout and activation systems in plants, C0000: pMOD_C0000 (#91081, Addgene) for CRISPR knockout and activation systems in plants, D100: pTRANS_100 (#91198, Addgene) for protoplast system, T230: pTRANS_230 (obtained from Dr. Voytas’s lab ( )) for callus system, A1510: pMOD_A1510 (#91036, Addgene) CRISPR knockout system in maize, A3701: pMOD_A3701 (#91052, Addgene) for CRISPR activation system in N. benthamiana , A4110: pMOD_A4110 (#91056, Addgene) CRISPR activation system in maize.

Techniques: Isolation, Transfection, Plasmid Preparation, Fluorescence, Expressing, Gene Knockout, Mutagenesis, Sequencing, CRISPR, Knock-Out, Derivative Assay, Staining, Two Tailed Test

Journal: bioRxiv

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

doi: 10.1101/2024.05.29.596527

Figure Lengend Snippet: ( A-B ) Transfection with three types of PEG (2050, 3350 and 4000) in N. benthamiana ( A ) and maize ( B ). Images at the top were captured under bright-field illumination, whereas the images in the second row were acquired using the GFP channel. ( C-D ) GFP expression after transfection with Cas9 vector (p201G Cas9) from first day to fourth day in N. benthamiana ( C ) and maize ( D ). Each day has two images, left is a GFP picture and right is a bright field image.

Article Snippet: A0502: pMOD_A0502 (#91012, Addgene) for CRISPR knockout system in N. benthamiana , B2103: pMOD_B2013 (#91061, Addgene) for CRISPR knockout and activation systems in plants, C0000: pMOD_C0000 (#91081, Addgene) for CRISPR knockout and activation systems in plants, D100: pTRANS_100 (#91198, Addgene) for protoplast system, T230: pTRANS_230 (obtained from Dr. Voytas’s lab ( )) for callus system, A1510: pMOD_A1510 (#91036, Addgene) CRISPR knockout system in maize, A3701: pMOD_A3701 (#91052, Addgene) for CRISPR activation system in N. benthamiana , A4110: pMOD_A4110 (#91056, Addgene) CRISPR activation system in maize.

Techniques: Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

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

doi: 10.1101/2024.05.29.596527

Figure Lengend Snippet: ( A ) Calli harboring the knockout gene HCF136 , generated with the CRISPR knockout system, thrive on MS media supplemented with phosphinothricin (PPT). ( B ) Calli with the overexpression of lipid genes, induced by the CRISPR activation system, grow on MS media supplemented with PPT. ( C ) RT-qPCR results show the overexpression of DGAT1 gene achieved using CRISPR activation in maize protoplast cells. Biological replicate n = 8. ( D ) RT-qPCR results demonstrate the successful overexpression of WRI1 or DGAT1 gene through CRISPR activation in N. benthamiana . Biological replicate n = 4. Error bars represent standard error. Asterisks indicate: *** P < 0.001; ** P < 0.01; * P < 0.05, calculated using two-tailed Welch’s t-test.

Article Snippet: A0502: pMOD_A0502 (#91012, Addgene) for CRISPR knockout system in N. benthamiana , B2103: pMOD_B2013 (#91061, Addgene) for CRISPR knockout and activation systems in plants, C0000: pMOD_C0000 (#91081, Addgene) for CRISPR knockout and activation systems in plants, D100: pTRANS_100 (#91198, Addgene) for protoplast system, T230: pTRANS_230 (obtained from Dr. Voytas’s lab ( )) for callus system, A1510: pMOD_A1510 (#91036, Addgene) CRISPR knockout system in maize, A3701: pMOD_A3701 (#91052, Addgene) for CRISPR activation system in N. benthamiana , A4110: pMOD_A4110 (#91056, Addgene) CRISPR activation system in maize.

Techniques: Knock-Out, Generated, CRISPR, Over Expression, Activation Assay, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

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

doi: 10.1101/2024.05.29.596527

Figure Lengend Snippet: ( A-B ) Lipid classes were measured across four days using LC-MS/MS in N. benthamiana ( A ) and etiolated maize B73 ( B ). Biological replicate n = 6. ( C ) Lipid analysis and quantification after overexpression of 3701-WRI1 and 3701-DGAT, with 3701 as a control in N. benthamiana . Biological replicate n = 4. ( D ) Lipid analysis and quantification after overexpression of 4110-WRI1 and 4110-DGAT, pPTN1586, with 4110 and pPTN1586C as controls in etiolated maize B73. Biological replicate n = 5. In each heatmap, the clustered grey bar at the top represents the control group, while the red or blue bar indicates the overexpression genes. n >= 6. ( E and G ) Quantification of lipid classes through fold change calculation for the overexpression of the WRI1 gene ( E ), and overexpression of the DGAT1 gene ( G ). n >= 5. ( F and H ) Fatty acids quantification through fold change formula for the overexpression of WRI1 gene ( F ), the DGAT1 gene ( H ). n >= 5. ( I-J ) Heatmaps comparing control and experimental samples for lipid data classification and statistical analysis for the overexpression of the WRI1 gene ( I ), and an overexpression of the DGAT1 gene ( J ). ( K-L ) Top ( K ): Shoot and root regeneration from N. benthamiana calli carrying an empty vector 3701. Bottom ( L ): Shoot regeneration from genetically engineered calli (3701-WRI1: Overexpression of WRI1 through CRISPR activation). Error bars represent standard error. Asterisks indicate: *** P < 0.001; ** P < 0.01; * P < 0.05, calculated using two-tailed Welch’s t-test.

Article Snippet: A0502: pMOD_A0502 (#91012, Addgene) for CRISPR knockout system in N. benthamiana , B2103: pMOD_B2013 (#91061, Addgene) for CRISPR knockout and activation systems in plants, C0000: pMOD_C0000 (#91081, Addgene) for CRISPR knockout and activation systems in plants, D100: pTRANS_100 (#91198, Addgene) for protoplast system, T230: pTRANS_230 (obtained from Dr. Voytas’s lab ( )) for callus system, A1510: pMOD_A1510 (#91036, Addgene) CRISPR knockout system in maize, A3701: pMOD_A3701 (#91052, Addgene) for CRISPR activation system in N. benthamiana , A4110: pMOD_A4110 (#91056, Addgene) CRISPR activation system in maize.

Techniques: Liquid Chromatography with Mass Spectroscopy, Over Expression, Control, Plasmid Preparation, CRISPR, Activation Assay, Two Tailed Test