Journal: Cell

Article Title: Genome-Scale Identification of SARS-CoV-2 and Pan-coronavirus Host Factor Networks

doi: 10.1016/j.cell.2020.12.006

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Article Snippet: Huh-7.5-Cas9 cells were generated by lentiviral transduction of lentiCas9-Blast (Addgene, cat. #52962) followed by selection and expansion in the presence of 5 μg/ml blasticidin.

Techniques: Virus, Recombinant, SYBR Green Assay, Luciferase, DNA Purification, Plasmid Preparation, CRISPR, Software, Genome Wide, Knock-Out, Inverted Microscopy

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet: ( a ) Vector copy number (VCN) per cell for the integrated guide RNA (gRNA) as well as the gene editors (ABE, CBE, and Cas9) in the respective HUDEP-2 stable cell lines. Primer targeting Cas9 is specific for the gene editors while that targeting U6 promoter is specific for gRNA; primer targeting WPRE is common for both gRNA and gene editors. ( b ) Transcriptome analysis of ABE and CBE expressing HUDEP-2 cells that are represented in pairwise Pearson correlation matrix. The R value for individual boxes is represented by the gradient bar. Individual base conversion efficiency of ABE ( c ) or CBE ( d ) expressing HUDEP-2 cells transduced with gRNA-2 on different days of expansion and differentiation. The base substitution at –115 region of HBG promoter (BCL11A binding site) were analyzed by EditR after sanger sequencing. Flow cytometry analysis of fetal hemoglobin (HbF) and GFP expression in ABE ( e ) or CBE ( f ) expressing HUDEP-2 cells transduced with gRNA-2 during different days of expansion and erythroid differentiation. The expression of GFP is directly proportional to the percentage of cells transduced with gRNA-2. The differentiation profile (CD71+ CD235a+ and CD71+ CD235a- population) of the base edited HUDEP-2 cells expressing ABE ( g ) and CBE ( h ) are represented from day 1 to day 7 of erythroid differentiation, measured by flow cytometry. The decrease in CD71+ CD235- population and increase in CD71+ CD235+ population display progress in erythroid differentiation. Data are expressed as mean ± SEM from three biological replicates.

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques: Plasmid Preparation, Stable Transfection, Expressing, Transduction, Binding Assay, Sequencing, Flow Cytometry

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet: Highly homologous HBG promoter was edited by adenine base editor (ABE), cytosine base editor (CBE), and Cas9 with suitable guide RNAs (gRNAs) that target the well-known BCL11A binding site (–115 transcription start site [TSS]). ( a ) Transduction efficiency of gRNA-2 (for ABE and CBE) or gRNA-7 (for Cas9), percentage of individual base conversion for ABE and CBE (with gRNA-2) and insertions and deletions (indels) for Cas9 (with gRNA-7) before and after erythroid differentiation are represented. The transduction efficiency was analyzed by FACS, the individual base substitution and indel percentage were analyzed by EditR and ICE software respectively after sanger sequencing. ( b ) Flow cytometry analysis of fetal hemoglobin (HbF) and erythroid maturation markers (CD235a and CD71) expression in edited HUDEP-2 cells. The percentage of HbF-expressing cells were analyzed before and after differentiation into erythroblasts. ( c ) Analysis of HBG2 deletion (due to 4.9 kb deletion) by qRT-PCR in the base edited and Cas9 edited HUDEP-2 cells. ( d ) Expression of G gamma-globin chain in ABE, CBE, and Cas9 edited HUDEP-2 cells, measured by RP-HPLC after differentiation into erythroblasts. The data were normalized with respective controls. Data are expressed as mean ± SEM from three biological replicates, asterisks indicate levels of statistical significance (**p < 0.01).

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques: Binding Assay, Transduction, Software, Sequencing, Flow Cytometry, Expressing, Quantitative RT-PCR

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet: ( a ) Schematic representation of the overall screening approach, adenine base editor (ABE) or cytosine base editor (CBE) expressing HUDEP-2 cells were transduced with guide RNA (gRNAs) that target the proximal promoter of the HBG gene. The edited cells were expanded for 8 days. Editing efficiency was evaluated by Sanger sequencing and NGS, while functional analysis was carried out using FACS and qRT-PCR. Top targets from both the ABE and CBE screens with the highest induction of HbF were validated and differentiated to erythroid cells. The differentiated cells were further subjected to FACS, qRT-PCR, RP-HPLC, and HPLC analysis to determine the number of HbF positive cells, HBG expression, individual gamma-globin chains, and fetal hemoglobin levels, respectively. ( b ) Representation of gRNA targeting HBG promoter region in HUDEP-2 cell line, gRNAs targeting –320 bp upstream of transcription start site (TSS) in HBG genes ( HBG1 and HBG2 ) promoter regions are represented in the figure. gRNAs common for HBG1 and HBG2 promoters are represented in blue, while the gRNAs specific to HBG1 promoter are represented in orange color, the primers used for deep sequencing are represented as a red bar. Comparison of transduction efficiency, base editing frequency, and HbF expression in HUDEP-2 cells expressing ABE ( c ) and CBE ( d ) transduced with different gRNAs targeting the HBG proximal promoter. The base edited cells were sequenced by NGS and analyzed for total editing frequency using CRISPResso-2. The transduction efficiency (GFP+ cells) and HbF positive cells were analyzed by FACS.

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques: Expressing, Transduction, Sequencing, Functional Assay, Quantitative RT-PCR, Comparison

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet: The ABE and CBE stable cells transduced with the indicated guide RNAs (gRNAs) were sequenced by NGS (figure ( a ) ABE and ( c ) CBE) and Sanger sequencing (figure ( b ) ABE and ( d ) CBE), the sequencing data were analyzed using CRISPResso-2 and EditR, respectively. The base conversion positions are sequentially represented in the x-axis up to –320 bp from the transcription start site (TSS). The base conversion efficiency of individual bases for each gRNA is represented in the y-axis as the percentage of the desired base converted (A- to-G or C- to-T).

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques: Transduction, Sequencing

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet: K562 cells expressing adenine base editor (ABE) ( a ) or cytosine base editor (CBE) ( b ) were transduced with the indicated guide RNA (gRNA) were sequenced by Sanger sequencing and analyzed using EditR. The gRNAs that showed lower HbF levels with higher editing efficiency were chosen based on the initial screening results. Percentage of HbF positive cells and the transduction efficiency (GFP+) of the respective gRNA for ABE ( c ) and CBE ( d ), measured by flow cytometry.

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques: Expressing, Transduction, Sequencing, Flow Cytometry

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet: HUDEP-2 cells expressing adenine base editor (ABE) or cytosine base editor (CBE) were transduced with the top eight guide RNAs (gRNAs) and analyzed by deep sequencing at the targeted regions in the HBG promoter. The base substitution for ABE ( a ) and CBE ( b ) at single base resolution for the respective gRNA at target site is depicted, the unintended conversion at the target site and conversion beyond protospacer are represented as orange and blue bar, respectively. Comparison of base editing efficiencies of ABE ( c ) or CBE ( d ) at the indicated target sites of HBG1 and HBG2 promoter region in HUDEP-2 cells. The highly homologous HBG1 and HBG2 promoter regions were together amplified and deep sequenced to segregate the specific editing between the HBG1 and HBG2 promoters by using four single nucleotide variations at −271,–307, –317, and –324 position. ( e ) Representation of indel frequency for ABE and CBE stable cells transduced with top eight gRNAs and ( f ) their transduction efficiency. Data are expressed as mean ± SEM from three biological replicates.

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques: Expressing, Transduction, Sequencing, Comparison, Amplification

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet: CRISPResso-2 analysis shows the overall base editing frequencies for individual gRNAs at the target site in ABE ( a ) and CBE ( b ) edited cells. The target region in HBG proximal promoter was PCR amplified and analyzed by next-generation amplicon sequencing. gRNA sequence, PAM site, substitution, insertion, natural variations, deletions, and base position in HBG promoter of HUDEP-2 cells are shown in the respective boxes.

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques: Amplification, Sequencing

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet: ( a ) Transduction efficiency and percentage of individual base conversion for HUDEP-2 -ABE8e stable cells transduced with gRNA-3 or -11 are represented, before and after erythroid differentiation. The transduction efficiency was analyzed by FACS, the individual base substitutions were analyzed by EditR after Sanger sequencing. ( b ) Flow cytometry analysis of HbF and erythroid maturation markers (CD71+ CD235a + population) expression in HUDEP-2-ABE8e stable cells transduced with gRNA-3 or -11. The percentage of HbF-expressing cells were analyzed before and after differentiation into erythroblasts. The differentiation profile was analyzed using CD-235 and CD-71 markers at day 7 of erythroid differentiation. ( c ) RP-HPLC analysis of globin chains after erythroid differentiation. ( d ) Analysis of HBG2 deletion (due to 4.9 kb deletion) by qRT-PCR in HUDEP-2-ABE8e or ABE7.10 stable cells transduced with gRNA-3 and -11. ( e ) Vector copy number (VCN) per cell for the integrated gRNA as well as the ABE8e in the respective HUDEP-2 stable cell lines. Primer targeting Cas9 is specific for the gene editor while that targeting U6 promoter is specific for gRNA; primer targeting WPRE is common for both gRNA and gene editor. Asterisks indicate levels of statistical significance ***p < 0.001.

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques: Transduction, Sequencing, Flow Cytometry, Expressing, Quantitative RT-PCR, Plasmid Preparation, Stable Transfection

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet: ( a ) Base conversions at the top 11 Cas-dependent DNA off-target sites in adenine base editor (ABE) 7.10 stable edited with guide RNA (gRNA)-11, along with the on-target events. The positions of the off-target and on-target loci are represented in their respective chromosome. The frequency of transcriptome-wide cellular levels of A- to- I ( b ), A- to- N ( c ), C- to -U ( d ), and C- to- N ( e ) RNA editing in BE3 stables (CBE), ABE 7.10 stables (ABE), BE3 stables edited with gRNAs-2 or -11, and ABE edited with gRNAs-2 or -11 are represented. The data are mean ± SD of two technical replicates.

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques:

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet: The differential expression of 34 selected genes that are involved in gamma-globin regulation were compared between the unedited HUDEP-2 wild type (WT), cytosine base editor (CBE) control, adenine base editor (ABE) control, edited ABE (guide RNA [gRNA]-2 or -11) and edited CBE (gRNA-2 or -11), and their expressions are represented as heat map.

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques: Quantitative Proteomics, Control

Journal: eLife

Article Title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

doi: 10.7554/eLife.65421

Figure Lengend Snippet:

Article Snippet: Software, algorithm , Benchling , CRISPR gRNA Design Tool | Benchling , , gRNA designing.

Techniques: Recombinant, Expressing, Plasmid Preparation, Selection, cDNA Synthesis, Sequencing, Concentration Assay, Injection, Software, CRISPR, RNA Sequencing, Gene Expression