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: eLife

Article Title: SON and SRRM2 are essential for nuclear speckle formation

doi: 10.7554/eLife.60579

Figure Lengend Snippet: ( A ) The strategy for the CRISPaint generated endogenous truncating mutations (0-to-10) accompanied by the TagGFP2 (depicted as GFP for simplicity) fusion are shown. ( B ) The sizes of SRRM2 truncated GFP fusion proteins are displayed. ( C ) Protein purified using a GFP-trap pull-down from lysates of corresponding stable HAP1 cell lines carrying the truncated SRRM2 alleles are run on PAGE. Western blotting of SRRM2 using an antibody generated against the common N-terminus is used to show the amount of loaded protein on the gel. SC35 blot shows a significant reduction in signal intensity of SRRM2-tr5 and a complete loss of signal from SRRM2-tr6 to tr10. ( D ) GFP-trap pull-down performed on lysates from wild-type, tr0 and tr10 HAP1 cells enrich for SRRM2 in tr0 cells, indicating the GFP-tagged allele is specific to SRRM2 and is detected by also SC35 blot (Lanes 1 and 2 inputs compared to Lanes 5 and 6 on the upper and lower left-side blots). SRRM2 also co-purifies two other NS-associated proteins; SRRM1 and RBM25 (Lane 6 on lower right-side blots). SRRM2-tr10 is not detected by SC35 but the pull-down efficiency (Lane 7 on upper left-side blot) and loading is validated by SRRM2 (Lanes 3 and 7 on lower left-side blot) and GFP blots (Lanes 3 and 7 on upper left-side blot). ( E ) Total cell lysates from wild-type, tr0 and tr10 HAP1 cells are run on 4–12% polyacrylamide gel and blotted with SC35 reveal the high-molecular weight (~300 kDa) as the most intense band and the absence of signal in tr10 cell lines validates that this band represents SRRM2 (filled arrow head). Longer exposure of the blot reveals a weak cross-reactivity with a 35 kDa protein, most likely to be SRSF7, around 35 kDa (empty arrow head).

Article Snippet: Recombinant DNA reagent , CRISPaint Gene Tagging Kit , Addgene , Cat. #: 1000000086, RRID: Addgene_1000000086 , .

Techniques: Generated, Purification, Western Blot, High Molecular Weight

Journal: eLife

Article Title: SON and SRRM2 are essential for nuclear speckle formation

doi: 10.7554/eLife.60579

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , CRISPaint Gene Tagging Kit , Addgene , Cat. #: 1000000086, RRID: Addgene_1000000086 , .

Techniques: Recombinant, Sequencing, Software

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