Journal: Biotechnology for biofuels

Article Title: CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus.

doi: 10.1186/s13068-020-01852-3

Figure Lengend Snippet: Fig. 2 Markerless CRISPR-Cas9-mediated multigene integration in K. marxianus CBS 6556. a Schematic representation of the three-primer colony PCR test and example gels for single and multiple gene integrations into the ABZ1 locus. The length of the up- and down-stream homology regions is 700 bp. b Integration efficiency of single (EGFP), dual (EGFP, DSRED), and triple (EGFP, YFP, and DSRED) genes. c Relative fluorescence intensity of EGFP expressed from different loci as measured by fluorescence microplate reader. d Normalized expression level of fluorescent proteins when produced individually or simultaneously with one or two additional fluorescent proteins. All experiments were performed in biological triplicate. Bars represent the mean, while error bars represent the standard deviation

Article Snippet: CRISPR plasmids (pCRISPR) were constructed using pIW601 (Addgene ID 98907) linearized at PspXI and reassembled with a 60 bp insert containing 20 bp upstream and downstream homology as well as the 20 bp target sequence by Gibson assembly.

Techniques: CRISPR, Fluorescence, Expressing, Produced, Standard Deviation

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

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice

doi: 10.1073/pnas.1615735114

Figure Lengend Snippet: PB-CRISPR vectors and validation by targeting Tet1 and Tet2 in mouse iPS cells. (A) PB-based CRISPR vectors. pCRISPR-sg4, sgRNA-expressing vector with neo gene; pCRISPR-sg5, sgRNA-expressing vector with puromycin gene. (B) pCRISPR-S10, PB plasmid expressing Doxycycline-inducible Cas9; pCRISPR-sg6-Tet1/Tet2, pCRISPR-sg6-based plasmids expressing Tet1 or Tet2 sgRNA. (C) PCR-RFLP analysis of Tet1/Tet2 loci targeted by pCRISPR-sg6-Tet1/Tet2. Expected mutations would eliminate the SacI or EcoRV site in Tet1 and Tet2, respectively. The target regions (∼500 bp) of Tet1 or Tet2 were amplified by PCR. PCR products were digested with corresponding enzymes. Results showed the successful targeting in Tet1-clone 1, Tet1-clone 2, and Tet2-clone 2. (D) Sequencing results of Tet1/Tet2 sgRNA targeted loci. Sequencing results for Tet1-clone 1 revealed a 4-bp deletion in one allele and a 1-bp deletion in another, resulting in elimination of the SacI site. Sequencing results for Tet1-clone 2 revealed mutations in both alleles, with a 3-bp deletion in one and a 1-bp insertion in another, resulting in elimination of the SacI site. Sequencing results for Tet2-clone 2, with an 8-bp deletion in one allele and a 14-bp deletion in another, resulting in elimination of the EcoRV site.

Article Snippet: All plasmids and libraries constructed in the current study will be deposited to Addgene for distribution. pCRISPR-sg4 and pCRISPR-sg5 and pCRISPR-sg6 were constructed by PCR assembly of the U6-sgRNA expression cassette from pX330 ( 7 ), SV40-neo from pIRES2-EGFP (Clontech), puro from pMSCVpuro (BD Biosciences), and ccdB from pStart-K ( 25 ) on a PB backbone from pZGs ( 26 ). pPB-hNRAS G12V was constructed by PCR assembly of NRAS G12V amplified from cDNA, and IRES-EGFP from pIRES2-EGFP on a PB backbone from pZGs ( 26 ).

Techniques: CRISPR, Expressing, Plasmid Preparation, Amplification, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice

doi: 10.1073/pnas.1615735114

Figure Lengend Snippet: Quantitative RT-PCR for transgene expression in mouse liver injected with PB vectors. (A) Schematic maps of PB vectors used in the screening experiment. Mice (n = 3) were injected with pPB-hNRASG12V, pCRISPR-W9-Cdkn2a-sgRNA, and pCAG-PBase. Control mice (n = 3) were injected with saline only. (B) Cas9 expression in mouse liver samples. (C) hNRASG12V expression in mouse liver samples.

Article Snippet: All plasmids and libraries constructed in the current study will be deposited to Addgene for distribution. pCRISPR-sg4 and pCRISPR-sg5 and pCRISPR-sg6 were constructed by PCR assembly of the U6-sgRNA expression cassette from pX330 ( 7 ), SV40-neo from pIRES2-EGFP (Clontech), puro from pMSCVpuro (BD Biosciences), and ccdB from pStart-K ( 25 ) on a PB backbone from pZGs ( 26 ). pPB-hNRAS G12V was constructed by PCR assembly of NRAS G12V amplified from cDNA, and IRES-EGFP from pIRES2-EGFP on a PB backbone from pZGs ( 26 ).

Techniques: Quantitative RT-PCR, Expressing, Injection