Journal: Journal of Microbiology & Biology Education

Article Title: CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and Bioinformatics Laboratory Module for Undergraduate and High School Students

doi: 10.1128/jmbe.00106-21

Figure Lengend Snippet: gRNA design platform. Shown is the process of designing CRISPR gRNAs to target the ADE2 gene using the Benchling web application (free for academic and educational use). (a) First, students design gRNAs against ADE2 by hand, applying their understanding of Streptococcus pyogenes Cas9 activity to identify protospacer-adjacent motif (PAM) sequences, gRNA sequences associated with the PAM, and the Cas9 cut site corresponding to the gRNA. (b) Using the Benchling CRISPR analysis tool, students first define Cas protein and target genome parameters to generate a list of possible gRNAs and then compare on- and off- target scores to choose optimized gRNAs for their experiment.

Article Snippet: Working in groups, students identify some PAMs and their associated gRNA sequences on the ADE2 gene, first by hand ( ) and then by using Benchling’s CRISPR analysis tool.

Techniques: CRISPR, Activity Assay

Journal: Journal of Microbiology & Biology Education

Article Title: CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and Bioinformatics Laboratory Module for Undergraduate and High School Students

doi: 10.1128/jmbe.00106-21

Figure Lengend Snippet: CRISPR-Cas9-mediated genome editing of the ADE2 gene in budding yeast. (a) The ADE2 gene is shown with the direction of transcription indicated by the black arrow. The four different gRNAs utilized in this module (g1, g2, g3, and g4) are depicted with their approximate locations relative to the start of the ORF. Cas9 cleavage of the ADE2 ORF with gRNA g1 can be repaired either through mutagenic NHEJ or through HDR. NHEJ ligates the broken ends with small indels. The indels block further cleavage by Cas9 and can also lead to inactivation of the ORF by a frameshift. HDR with cotransformed donor DNA leads to a precise deletion of the ORF. The molecular outcomes of the editing events can be interrogated by PCR using forward (F) and reverse (R) primers situated upstream and downstream of the ORF, respectively. See Appendix 1E in the supplemental material for more details. (b) Representative plate images of yeast transformations with each ADE2 gRNA with or without donor DNA. A no-gRNA control is used to demonstrate the colony yield obtained in the absence of genome editing. Note the clearly visible red pigment accumulating in colonies transformed with guides 1, 2, and 3, and how the addition of donor DNA impacts the size and color of the edited (red) colonies.

Article Snippet: Working in groups, students identify some PAMs and their associated gRNA sequences on the ADE2 gene, first by hand ( ) and then by using Benchling’s CRISPR analysis tool.

Techniques: CRISPR, Blocking Assay, Control, Transformation Assay

Journal: Genome Biology

Article Title: SETD2 loss-of-function uniquely sensitizes cells to epigenetic targeting of NSD1-directed H3K36 methylation

doi: 10.1186/s13059-025-03483-z

Figure Lengend Snippet: Unbiased genome-wide screening identifies NSD1 as putative SL modifier in SETD2- deficient cells. A Western blot analysis of global H3K36 methylation states in isogenic SETD2 -wildtype/mutant HAP1 cells. B Schematic depiction of CRISPR/Cas9 synthetic lethal screen. C Volcano plot highlighting NSD1 as a synthetic lethal hit (SL index: − 1.76; p value = 2.67e − 06). D Gene ontology analysis of the 127 SL candidates identified in the screen reveal enrichment for factors involved in epigenetic remodeling and DNA damage/repair. E Gene-view schematic illustrating inducible deletion of Setd2 in MEFs through Cre-lox excision of exon 6. F PCR genotyping confirming tamoxifen-inducible Cre activity in the Setd2 flox/flox parental and Setd2 flox/flox ; Nsd1 −/− MEF cell lines. G Crystal violet staining of Setd2 flox/flox and Setd2 flox/flox ; Nsd1 −/− MEF cell lines following treatment with 4-OHT

Article Snippet: Individual sgRNAs for CRISPRi targeting were selected using an in silico CRISPR guide RNA selection tool (Benchling) with corresponding oligos annealed and subcloned by cohesive-end ligation into a lentiviral mU6-(sp)TRACR guide RNA vector following AarI digestion.

Techniques: Genome Wide, Western Blot, Methylation, Mutagenesis, CRISPR, Activity Assay, Staining

Journal: Journal of Cell Science

Article Title: Kinesin-1 transports morphologically distinct intracellular virions during vaccinia infection

doi: 10.1242/jcs.260175

Figure Lengend Snippet: Quantifying the number of kinesin-1 complexes on IMVs and IEVs. (A) Schematic of the intracellular TagGFP2-tagged 60-mer nanocage. Each subunit of the nanocage (grey) is fused with TagGFP2 (green) and FKBP (blue), although this is shown only for one subunit for clarity. FRB (pink) is targeted to the plasma membrane by its palmitoylation and myristoylation sequence and dimerises with FKBP in the presence of the rapamycin analogue AP21967. PDB structures used: 5KP9 , 2Y0G and 4DRI . (B) Representative average-intensity projection images of transiently expressed TagGFP2-tagged nanocages in HeLa cells treated with 500 nM AP21967. Scale bar: 2 µm. (C) Quantification of fluorescence intensities of TagGFP2-tagged 24-, 60-, 120- and 180-mer nanocages. Error bars represent mean±s.d. Linear line of regression is fitted. n =51–114 measurements per nanocage from three independent experiments. (D) Immunoblot analysis of total cell lysates from HeLa wild-type (WT) or TagGFP2–KIF5B CRISPR knock-in (KI) cells using the indicated antibodies. (E) Representative images from time-lapse movie showing the association of kinesin-1 (green) with IMVs (magenta) during moving (red arrowheads) and stationary (blue arrowheads) phases in the HeLa TagGFP2–KIF5B knock-in cell line at 7.5 h post infection with the ΔB5 RFP–A3 virus (see Movie 8 ). Time in seconds is indicated in each image. Scale bar: 2 µm. The graph on the right shows quantification of the TagGFP2–KIF5B:RFP–A3 fluorescence intensity ratio on IMV particles during moving and stationary phases. n =11 virions from two independent experiments. (F) Representative average-intensity projections of endogenously expressed TagGFP2–KIF5B (green) on IEVs or IMVs (magenta) in HeLa TagGFP2–KIF5B knock-in cells 7.5 h post infection with ΔB5 RFP–A3 (left) or WR B5-RFP (right). Scale bar: 2 µm. (G) The left graph shows the mean background-subtracted fluorescence intensity of TagGFP2–KIF5B together with the calculated number of molecules on IMVs and IEVs, superimposed (dotted red lines) on the nanocage calibration plot from C. The table below shows the summary of the readout. SuperPlot (right) showing the number of kinesin-1 complexes associated with IMVs or IEVs from three independent experiments in which 84 and 121 virions were analysed for IMVs and IEVs, respectively. Bars represent mean±s.e.m. Two-tailed unpaired ­Student's t -test was used to determine statistical significance. ** P ≤0.01. (H) SuperPlots showing the background-subtracted antibody intensity signals of KIF5B associated with IMVs (left graph) or IEVs (right graph) in HeLa wild-type (WT) or tagGFP2–KIF5B knock-in (KI) cells. The fold difference between the mean number of KIF5B associated with virions in WT or KI cells is shown. The table summarises the mean number of kinesin-1 complexes associated with IMVs or IEVs in HeLa WT or KI cells after correcting for low levels of tagGFP2–KIF5B expression in the latter. a.u., arbitrary units.

Article Snippet: The guide RNA (gRNA) for KIF5B was designed using a CRISPR design webpage tool ( https://www.benchling.com/ ).

Techniques: Clinical Proteomics, Membrane, Sequencing, Fluorescence, Western Blot, CRISPR, Knock-In, Infection, Virus, Two Tailed Test, Expressing