Journal: Cell reports

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis

doi: 10.1016/j.celrep.2021.110008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Briefly, a guide RNA (5′-GCAGATGTAGTGTTTCCACA-3′) targeting the open reading frame in the immediate vicinity of the stop codon was cloned into the Cas9 expression vector pX330-U6-Chimeric_BB-CBh-hSpCas9 (gift from Feng Zhang; ), Addgene plasmid #42230).

Techniques: Derivative Assay, Recombinant, Electron Microscopy, Transfection, Expressing, Plasmid Preparation, Software, Cell Analysis, Gas Chromatography, Mass Spectrometry

Journal: bioRxiv

Article Title: A strategy for genome-wide seamless tagging of human protein-coding genes

doi: 10.1101/2025.03.04.641506

Figure Lengend Snippet: a , Overview of the CRISPR-Cas9 editing strategy to seamlessly integrate DNA cassettes as fusions to human protein-coding genes and ways in which the results have been evaluated. b , Schematic of knock-in vectors and overview of gene editing. The system comprises of three plasmids: plasmid 1, a “donor vector” that encodes an antibiotic resistance gene as a selection marker fused to P2A self-cleaving peptide (purple), plasmid 2, a “donor cleaving vector” that carries the Scramble-gRNA (Sc-gRNA) (orange) and Cas9 variant SpCas9-NG (black), and plasmid 3, a “locus-specific vector” that encodes a library of gRNAs targeting 5’ and 3’ ends of protein-coding sequences of 18,804 human genes that defines the genomic target to be modified by SpCas9-NG (brown).

Article Snippet: The plasmid coding Cas9-NG (34) was purchased from Addgene (PX330-SpCAS9-NG).

Techniques: CRISPR, Knock-In, Plasmid Preparation, Selection, Marker, Variant Assay, Modification

Journal: bioRxiv

Article Title: A strategy for genome-wide seamless tagging of human protein-coding genes

doi: 10.1101/2025.03.04.641506

Figure Lengend Snippet: a , Design of donor-cleaving plasmid. The U6 promoter-based expression cassette followed by the Sc-gRNA (orange), gRNA scaffold (blue) and termination signal sequence (red). b , Design of gRNA-CRISPR-library plasmid. The U6 promoter-based expression cassette followed by the library gRNAs targeting 18,804 human genes including their splice variants (cyan), gRNA scaffold (blue), capture sequence (pink) and termination signal sequence (red). c , Efficiency of NHEJ-based strategies using two different gRNA cleaving vectors. The efficiency of CRISPR/Cas9-induced NHEJ-mediated DNA integration was tested by targeting five human protein-coding genes to insert four types of minicircle (MC) donor vectors targeted by distinct guide RNAs directed against different PAM sequences, TGG, AGG, or CGG (MC-Sc-gRNA-TGG, MC-Sc-gRNA-AGG, MC-VKG1-gRNA-TGG and MC-VKG1-gRNA-CGG).

Article Snippet: The plasmid coding Cas9-NG (34) was purchased from Addgene (PX330-SpCAS9-NG).

Techniques: Plasmid Preparation, Expressing, Sequencing, CRISPR

Journal: bioRxiv

Article Title: A strategy for genome-wide seamless tagging of human protein-coding genes

doi: 10.1101/2025.03.04.641506

Figure Lengend Snippet: We designed a genome-wide resource of two gRNA libraries consisting of gRNA that 1, target PAMs resulting in the only in-frame double strand breaks; 2, minimal off-target Cas9 cleavage; 3, maximal on-target Cas9 cleavage, 4, avoidance of homopolymer stretches (e.g., AAAA, GGGG) and 5, GC content between 20 to 80%. Following the above criteria, our libraries consist of a minimum of 1 and maximum of 8 gRNAs per splice variant of human genes.

Article Snippet: The plasmid coding Cas9-NG (34) was purchased from Addgene (PX330-SpCAS9-NG).

Techniques: Genome Wide, Variant Assay

Journal: bioRxiv

Article Title: A strategy for genome-wide seamless tagging of human protein-coding genes

doi: 10.1101/2025.03.04.641506

Figure Lengend Snippet: a , Visualization of mixed pools of cells in which endogenous proteins are fused to mClover3 using our CRISPR/Cas9-NHEJ-based strategy. Cells vary in fluorescence intensity and patterns of expression suggesting tagging of many proteins with varying abundances and different subcellular localizations. Scale bar is 50µm. b , NGS results indicate that ∼80 % of the human genes were tagged 5’ or 3’ to protein-coding sequences in the genome of HEK293T cells. c , The graph shows the distribution of abundance for all proteins expressed in HEK293T versus successfully or unsuccessfully detected targets; boxes represent 25th, 50th, and75th percentiles, and whiskers represent 1.5 times the interquartile range. Median is indicated by a white line. Outliers are not shown. Statistical significance p-value is 5.5 × 10 -28 and is calculated by Student’s t-test. Data indicates that low protein abundance posed a significant challenge to successful tagging, as the proteins that were not successfully tagged displayed low or no expression levels in HEK 293T cells. d , NGS results indicate that 89.7% of the essential genes were tagged 5’ or 3’ to protein-coding sequences in the genome. e , the distribution of abundance of essential proteins expressed in HEK 293T versus successfully or unsuccessfully tagged genes; boxes represent 25th, 50th, and 75th percentiles, and whiskers represent 1.5 times the interquartile range. Median is indicated by a white line. Statistical significance p-value is 1.02 × 10 -2 and is calculated by Student’s t-test. Data indicates that low protein abundance posed a challenge to successful tagging of essential genes, as the proteins that were not successfully tagged displayed low expression levels in HEK 293T cells. f , comparison of annotated protein localizations between PRISM and the Human Protein Atlas (HPA) datasets (37). The diagram features colored bands that correspond to groups of proteins with similar localization annotations between our data set and HPA. The width of each band is proportional to the number of proteins in the group. g , imaging analysis of individual successful targets. mClover3 fluorescence intensity and subcellular localization vary widely for each gene.

Article Snippet: The plasmid coding Cas9-NG (34) was purchased from Addgene (PX330-SpCAS9-NG).

Techniques: CRISPR, Fluorescence, Expressing, Comparison, Imaging

Journal: bioRxiv

Article Title: A strategy for genome-wide seamless tagging of human protein-coding genes

doi: 10.1101/2025.03.04.641506

Figure Lengend Snippet: V isualization of each single cell in which endogenous proteins are fused to mClover3 using our CRISPR/Cas9-NHEJ-based strategy. We observed variations in fluorescence intensity and patterns of expression, suggesting successful tagging of multiple proteins with varying abundances and different subcellular localizations. Since single cells were collected randomly, some proteins may appear more than once. However, if they were collected from the same plate, they were discarded from the analysis, assuming that they could come from the same original tagged cells. Conversely, if they were collected from different plates, they were kept in the analysis since they could be another confirmation of robust tagging, as they show the same localization and fluorescent intensity.

Article Snippet: The plasmid coding Cas9-NG (34) was purchased from Addgene (PX330-SpCAS9-NG).

Techniques: CRISPR, Fluorescence, Expressing