Journal: Current protocols in molecular biology

Article Title: Use of the CRISPR-Cas9 system in Drosophila cultured cells to introduce fluorescent tags into endogenous genes

doi: 10.1002/cpmb.112

Figure Lengend Snippet: (A) Diagram of a theoretical gene target and results of knock-in using ssDNA Drop-In method (Basic Protocol 1) and CRISPaint method (Basic Protocol 2). FP, fluorescent protein open reading frame (ORF); T2A, self-cleaving peptide ORF; PuroR, puromycin resistance ORF. (B) Comparison of standard plasmid-based donor method for tagging with a fluorescent protein ORF with ssDNA Drop-In and CRISPaint methods.

Article Snippet: Cas9+ Drosophila cells, such as S2R+-MT::Cas9 ( Viswanatha et al., 2018 ) (DGRC #268) Schneider’s Media ( Reagents & Solutions ) Schneider’s Media with Puromycin ( Reagents & Solutions ) Frame selector plasmids ( pCFD3-frame_selector_ (0,1,or 2) (Addgene #s 127553–127555; DGRC#s 1482–1484) CRISPaint donor plasmid(s) (See Addgene Kit # 1000000086; pCRISPaint-mNeonGreen-T2A-PuroR cannot be distributed through Addgene and is available directly from Hornung lab; ( Schmid-Burgk et al., 2016 )) Optional control sgRNA, pCFD3-Act5c (Addgene #130278; DGRC #1492) Genomic DNA extraction kit, such as a Zymo Quick-DNA MiniPrep Kit (Zymo Research #D3024) PCR polymerase and buffer such as High Fidelity Phusion Polymerase (NEB #M0530) and 5X Buffer dNTP Mixture (2.5 mM each) Ethidium Bromide Agarose Fluorescent Microscope Image analysis software such as CellProfiler ( Carpenter et al., 2006 ) Eppendorf Tubes Tabletop Centrifuge PCR Machine Spectrophotometer, such as a NanoDrop microvolume spectrophotometer Standard agarose gel electrophoresis apparatus Target selection, knock-in design, and isolation of single-cell clones list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 For each target gene, identify a sgRNA target site in the 3’ coding sequence.

Techniques: Knock-In, Comparison, Plasmid Preparation

Journal: Current protocols in molecular biology

Article Title: Use of the CRISPR-Cas9 system in Drosophila cultured cells to introduce fluorescent tags into endogenous genes

doi: 10.1002/cpmb.112

Figure Lengend Snippet: A. Conversion table for target gene cut frame and CRISPaint frame selector sgRNA. B. Screenshot of output of the Find CRISPRs tool (www.flyrnai.org/crispr3/web). Arrow, column in a search results table showing the target gene sgRNA cut frame. The example sgRNA shown was used to generate His2Av-mNeonGreen knock-in cell line in Bosch et al. 2019. C. Schematic of gene targeting when using the three CRISPaint frame selector sgRNAs (0, 1, 2).

Article Snippet: Cas9+ Drosophila cells, such as S2R+-MT::Cas9 ( Viswanatha et al., 2018 ) (DGRC #268) Schneider’s Media ( Reagents & Solutions ) Schneider’s Media with Puromycin ( Reagents & Solutions ) Frame selector plasmids ( pCFD3-frame_selector_ (0,1,or 2) (Addgene #s 127553–127555; DGRC#s 1482–1484) CRISPaint donor plasmid(s) (See Addgene Kit # 1000000086; pCRISPaint-mNeonGreen-T2A-PuroR cannot be distributed through Addgene and is available directly from Hornung lab; ( Schmid-Burgk et al., 2016 )) Optional control sgRNA, pCFD3-Act5c (Addgene #130278; DGRC #1492) Genomic DNA extraction kit, such as a Zymo Quick-DNA MiniPrep Kit (Zymo Research #D3024) PCR polymerase and buffer such as High Fidelity Phusion Polymerase (NEB #M0530) and 5X Buffer dNTP Mixture (2.5 mM each) Ethidium Bromide Agarose Fluorescent Microscope Image analysis software such as CellProfiler ( Carpenter et al., 2006 ) Eppendorf Tubes Tabletop Centrifuge PCR Machine Spectrophotometer, such as a NanoDrop microvolume spectrophotometer Standard agarose gel electrophoresis apparatus Target selection, knock-in design, and isolation of single-cell clones list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 For each target gene, identify a sgRNA target site in the 3’ coding sequence.

Techniques: Knock-In

Journal: bioRxiv

Article Title: Characterization of Pro-Fibrotic Signaling Pathways using Human Hepatic Organoids

doi: 10.1101/2023.04.25.538102

Figure Lengend Snippet: (A) Top: A diagram of the CRISPaint system used to insert P2A-Clover at the 3’ end of COL1A1 in an iPSC line. After blasticidin selection, cells were cloned to generate the COL1A1-P2A-Clover iPSC line. Bottom : The self-cleaving P2A peptide enables COL1A1 expressing cells to be labelled with a fluorescent intracellular protein (Clover). ( B ) The collagen producing cells in a HO produced from COL1A1-P2A-Clover iPSCs are labelled with an intracellular fluorescent Clover protein. ( C ) Left: An overlay of bright field and fluorescent images of differentiating COL1A1-P2A Clover HO cultures reveals that the number of Clover + cells increase between days 3 and 12. Scale bar: 50 μm. Right : Confocal images show nuclei (DAPI stained) and Clover + fluorescent cells in day 20 COL1A1-P2A-Clover organoids. The yellow dashed circles indicate bile ducts. Clover + cells are not found within the bile ducts but are in other areas of the organoid. Scale bar: 50 μm. ( D ) Bright field (top) and immunofluorescence (bottom) images of day 21 COL1A1 -P2A Clover HOs that were treated on day 13 with no addition (NC), 50 ng/ml TGFβ1 or 50 ng/ml PDGFβ. Both of these growth factors induced a marked increase in COL1A1 + cells. Scale bars, 50 μm. ( E ) SHG analysis of the collagen fibers formed in human HOs. Top: Cross-sectional views of collagen fibers (cyan) and nuclei (magenta) in day 21 control organoids, or day 21 organoids that were treated on day 13 with 50 ng/ml TGFβ1 or 50 ng/ml PDGF. Control organoids (left) have isolated regions with relatively thin collagen fibers (cyan). In contrast, the TGFβ1 or PDGF-treated organoids form a network of thick collagen fibers that extend throughout the entire organoid. Collagen producing cells (green) can also be seen in these images. Scale bars, 50 μm. Bottom: A quantitative comparison of collagen fiber area in SHG images was performed for control, TGFβ1- or PDGF-treated hepatic organoids (n = 5 organoids per category) on day 21. There is a statistically significant increase in total collagen abundance (% of collagen fiber area relative the total area of the imaged organoids) in the organoids after exposure to TGFβ1 (*, p < 0.05) or PDGF (**, p <0.01, Welch’s t-test). There was also a statistically significant increase in the abundance of thick collagen fibers (i.e., those fibers > 3 um diameter) in the TGFβ1 and PDGF-treated hepatic organoids.

Article Snippet: The CRISPR-assisted insertion tagging system (CRISPaint) plasmid (pCRISPR-HOT-Clover-BlastR) was obtained from Addgene (Plasmid #138569; http://n2t.net/addgene:138569 ).

Techniques: Selection, Clone Assay, Expressing, Produced, Staining, Immunofluorescence, Control, Isolation, Comparison