Journal: The CRISPR Journal

Article Title: Bacterial Retrons Enable Precise Gene Editing in Human Cells

doi: 10.1089/crispr.2021.0065

Figure Lengend Snippet: Retrons enable homology-directed repair (HDR) in K562 and 293T BFP reporter cells. (A) Schematic showing the principle of BFP reporter cell line, adapted from figure 3A of Richardson et al.22 (B) Schematic of plasmid design. (C) The percentage of BFP– cells, indicating a lower bound on the SpCas9 cutting efficiency in BFP-to-GFP conversion K562 reporter cells after being transfected with different DNA components. (D) The percentage of GFP+ cells, indicating the HDR editing efficiency in BFP-to-GFP conversion K562 reporter cells after being transfected with different DNA components. In (C) and (D), data are presented as the mean ± SD of three biological replicates. Mean values are shown above bars. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; p ≥ 0.05, n.s. Statistical analysis was carried out using one-way ANOVA. NTC, non-transfected control; Cas9 only, cells transfected with the plasmid without gRNA or any retron; gBFP + Cas9, cells transfected with the plasmid expressing both gBFP and Cas9; gBFP-An + Cas9, cells transfected with the plasmid expressing gBFP, An, and Cas9; gBFP + Cas9-An ssDNA, cells co-transfected with the plasmid expressing gBFP and Cas9, and the synthesized An ssDNA; gBFP + Cas9-At ssDNA, cells co-transfected with the plasmid expressing gBFP and Cas9, and the synthesized At ssDNA; gBFP-An + Cas9-Sa163, cells co-transfected with the plasmid expressing gBFP, An, and Cas9-Sa163; gBFP-An + Cas9-Ec86, cells co-transfected with the plasmid expressing gBFP, An, and Cas9-Ec86. “An” and “At” are ssDNA donor sequences of BFP-to-GFP conversion (Supplementary Note S2). Flow cytometry plots related to (C) and (D) are presented in Supplementary Figure S2F. Color images are available online.

Article Snippet: Genes encoding SpCas9 and BFP were obtained from previously reported plasmids (Addgene plasmid #64323, #64216, #64322, Ralf Kühn lab 18 ; mCherry was amplified from Addgene plasmid # 60954, Jonathan Weissman lab).

Techniques: Plasmid Preparation, Transfection, Control, Expressing, Synthesized, Flow Cytometry

Journal: The CRISPR Journal

Article Title: Bacterial Retrons Enable Precise Gene Editing in Human Cells

doi: 10.1089/crispr.2021.0065

Figure Lengend Snippet: Retrons produce msDNA in human cells. (A) Schematic showing construct design for the quantitative polymerase chain reaction (qPCR) assay: gRNA and msr-msd expression was driven by the human U6 promoter; human codon–optimized reverse transcriptase (RT) and SpCas9 expression was driven by the CBh promoter; donor templates were inserted into the replaceable regions of msd. T2A and P2A, self-cleaving peptides. (B) Relative abundance of msDNA produced by different retrons. DNA amplified from the same volume of OBZ206 single-stranded DNA (ssDNA) at 1 × 10–7 ng/μL was set as onefold to calculate the relative abundance. Data are presented as the mean ± standard deviation (SD; n = 2). Statistical analysis was carried out using one-way analysis of variance (ANOVA). NTC, non-transfected control. Cas9 only, cells transfected with the plasmid without gRNA or any retron. gRNA + Cas9, cells transfected with the plasmid expressing both gRNA and Cas9. w/o msr/msd, cells transfected with plasmid that co-express gRNA, Cas9, and RT, but no msr/msd was inserted. Other labels in the x-axis indicate cells transfected with plasmids that carry different retron sequences. Color images are available online.

Article Snippet: Genes encoding SpCas9 and BFP were obtained from previously reported plasmids (Addgene plasmid #64323, #64216, #64322, Ralf Kühn lab 18 ; mCherry was amplified from Addgene plasmid # 60954, Jonathan Weissman lab).

Techniques: Construct, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription, Produced, Amplification, Standard Deviation, Transfection, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Novel reporter of the PINK1-Parkin mitophagy pathway identifies its damage sensor in the import gate

doi: 10.1101/2025.02.19.639160

Figure Lengend Snippet: Genome-wide screens with the MFN2-Halo reporter identify activators and facilitators of the PINK1-Parkin mitophagy pathway. (A) Schematic illustrating the PINK1-Parkin pathway reporter MFN2-Halo. (B) Representative confocal images of HeLaMFN2-Halo+mCh-Parkin cells transduced with a cassette co- expressing a dual sgRNA targeting MFN2 and TagBFP2 to mark transduced cells (white outline). Scale bar = 20 µm. (C) Flow cytometry of HeLaMFN2-Halo cells. **** p ≤ 0.0001 Error bars mean +/- SD. N = 3 independent experiments. (D) Representative immunoblots of endogenously tagged and untagged MFN2 alleles (MFN2-Halo and MFN2-UT, respectively) of same cells in (1B). N = 5 replicates on at least 2 occasions. (E) Flow cytometry of HeLaMFN2-Halo+mCh-Parkin cells +/- AO treatment. **** p ≤ 0.0001 Error bars mean +/- SD. N = 3 independent experiments. (F) Schematic illustrating the FACS based genome-wide CRISPRi screening strategy. Six screens were performed in total, using three different cell lines with or without exposure to OXPHOS inhibitors. (G) Stacked bar graph represents the proportion of nuclear gene perturbations encoding mitochondrial (mito) vs. non-mitochondrial proteins (not mito). (H) Dot plots of gene scores (product of log2 fold change and adjusted -log10 p-value) from screens described in (F). (I) One-sided volcano plots for screens described in (F).

Article Snippet: Cells were transduced with the dual- sgRNA library from the Weissman Lab (Dual sgRNA CRISPRi Library 1-2 was a gift from Jonathan Weissman Addgene #187246) at an MOI of 0.3 based off the virus titer .

Techniques: Genome Wide, Transduction, Expressing, Flow Cytometry, Western Blot