Journal: Cell Reports Methods

Article Title: CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

doi: 10.1016/j.crmeth.2022.100318

Figure Lengend Snippet: SARS-CoV-2 epitope mapping by CasPlay (A) A peptide library tiling the SARS-CoV-2 proteome (excluding ORF1ab) from a previous study ( Barber et al., 2021 ) was presented by CasPlay. (B) CasPlay experiments were performed using serum samples from 8 samples collected prior to December 2020 (“pre-COVID”), 8 patients following COVID-19 infection (“convalescent”), 8 individuals prior to receiving a vaccine, and the same 8 individuals between 2 weeks and 3 months after receiving the second dose of an mRNA vaccine. gRNA barcode enrichment analysis by CasPlay revealed epitope regions in the spike and/or nucleocapsid proteins recognized by patient antibodies in convalescent and vaccinated patient samples. Enrichment is calculated as normalized output gRNA sequencing reads divided by pre-enriched normalized sequencing reads, with data averaged across 4 gRNA barcode replicates and two independent experimental replicates (see for further details).

Article Snippet: Then, approximately 100 ng of purified recombinant SARS-CoV-2 spike protein (Sino Biological 40589-V08H4 was added to the microarray and incubated at 23°C for 1 h. After washing twice with 40 μL PBST, 1:100 anti-SARS-CoV-2 spike CR3022 human IgG antibody (Cell Signaling 37475) was added to the microarray and incubated at 23°C for 1 h. The microarray was then washed twice with PBST and then incubated with 1:40 Alexa 647-conjugated anti-human IgG Fc antibody (Biolegend 410714) at 23°C for 1 h. The microarray was then visualized using a Genepix 4300A microarray scanner, and fluorescence intensities were extracted and analyzed as previously described ( ; ; ; ).

Techniques: Infection, Sequencing

Journal: Cell Reports Methods

Article Title: CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

doi: 10.1016/j.crmeth.2022.100318

Figure Lengend Snippet: CasPlay is compatible with full-length protein display and applications (A) Synthetic antibodies were expressed as C-terminal fusions to dCas9 with unique gRNA barcodes. A nanobody recognizing a peptide from β-catenin ( Braun et al., 2016 ; Traenkle et al., 2015 ) and an scFv recognizing the spike protein from SARS-CoV-2 ( Wang et al., 2021 ) were paired with unique gRNAs. (B) Target antigens of the synthetic antibodies were adsorbed to a plate surface and then incubated with the dCas9-antibody fusions. Primers specific to the gRNA sequences were then used to RT-PCR the gRNAs associated with each antibody to detect binding of the antibody to its target antigen. (C) Densitometry of RT-PCR amplicons on an agarose gel reveal specific retention and detection of each synthetic antibody only in the presence of its respective analyte, indicating synthetic antibody functionality. n = 3 independent replicates for each condition. Mean with SD plotted.

Article Snippet: Then, approximately 100 ng of purified recombinant SARS-CoV-2 spike protein (Sino Biological 40589-V08H4 was added to the microarray and incubated at 23°C for 1 h. After washing twice with 40 μL PBST, 1:100 anti-SARS-CoV-2 spike CR3022 human IgG antibody (Cell Signaling 37475) was added to the microarray and incubated at 23°C for 1 h. The microarray was then washed twice with PBST and then incubated with 1:40 Alexa 647-conjugated anti-human IgG Fc antibody (Biolegend 410714) at 23°C for 1 h. The microarray was then visualized using a Genepix 4300A microarray scanner, and fluorescence intensities were extracted and analyzed as previously described ( ; ; ; ).

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Agarose Gel Electrophoresis

Journal: Cell Reports Methods

Article Title: CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

doi: 10.1016/j.crmeth.2022.100318

Figure Lengend Snippet:

Article Snippet: Then, approximately 100 ng of purified recombinant SARS-CoV-2 spike protein (Sino Biological 40589-V08H4 was added to the microarray and incubated at 23°C for 1 h. After washing twice with 40 μL PBST, 1:100 anti-SARS-CoV-2 spike CR3022 human IgG antibody (Cell Signaling 37475) was added to the microarray and incubated at 23°C for 1 h. The microarray was then washed twice with PBST and then incubated with 1:40 Alexa 647-conjugated anti-human IgG Fc antibody (Biolegend 410714) at 23°C for 1 h. The microarray was then visualized using a Genepix 4300A microarray scanner, and fluorescence intensities were extracted and analyzed as previously described ( ; ; ; ).

Techniques: Synthesized, Recombinant, Plasmid Preparation, Expressing, Software, Microarray

Journal: Journal of Human Immunity

Article Title: Ribosomal RNA processing impairments in a B cell immunodeficient patient with WDR75 variants

doi: 10.70962/jhi.20250061

Figure Lengend Snippet: U2OS cells with inactivation of the WDR75 gene locus and ectopic expression of WDR75 p.T46I present with altered pre-rRNA processing. (A) Sanger sequences of gDNA at the site (guide sequence) of CRISPR/Cas9 modification of different U2OS clones ectopically expressing either WDR75 WT or WDR75 p.T46I. (B) Alignments of sequencing profiles from TOPO TA cloned RT-PCR products of U2OS cells modified by CRISPR/Cas9. U2OS cells were treated with CHX 100 µg/ml for 2 h before extracting the RNA. (C) Northern blot analysis of U2OS cells ectopically expressing a pWPI EV, WDR75 WT, or WDR75 p.T46I (M1). The WDR75 gene locus was inactivated (see A and B) in WT- and WDR75 p.T46I-expressing U2OS cells. Indicated radiolabeled probes were used to detect pre-rRNA precursors and mature rRNAs. Statistical analysis presented in C was performed using paired Student’s t test; *P < 0.05; **P < 0.01. Source data are available for this figure: .

Article Snippet: KO score for each sequence was evaluated using Inference of CRISPR Edits (RRID:SCR_024508) software (Performance Analysis, 2019. v3.0; Synthego).

Techniques: Expressing, Sequencing, CRISPR, Modification, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Northern Blot

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Systematic comparison of barcoding strategies used in the category of molecular barcodes.

Article Snippet: CRISPR-associated approach , Sequence-based barcodes , Customized peptide libraries are designed to encode a unique 20 bp nucleic acid sequence used as the gRNA barcode , - , - , S , n/a , COVID-19 samples (convalescent, pre-vaccine and post-vaccine) , Cas9 display (CasPlay) system (GenePix 4300A microarray scanner); Illumina NextSeq 500 (single-end 150 bp) used to sequenced dCas9-fusion library , GenePix Pro 7; Cutadapt v2.5 ) and customized commend lines , Multiplex samples , Evaluation of vaccine-induced antibody reactivities from the SARS-CoV-2 proteome , .

Techniques: Software, Sequencing, Multiplex Assay, CRISPR, Plasmid Preparation, Microarray, Binding Assay, Amplification, Ligation, DNA Sequencing, Multiplexing, Generated, Staining, Flow Cytometry, High Throughput Screening Assay, Inhibition, Blocking Assay, Conjugation Assay, RNA Sequencing Assay, Transmission Assay, Incubation, Diagnostic Assay, Next-Generation Sequencing, Infection