Journal: Immunity

Article Title: The Interferon-Stimulated Gene RIPK1 Regulates Cancer Cell Intrinsic and Extrinsic Resistance to Immune Checkpoint Blockade

doi: 10.1016/j.immuni.2022.03.007

Figure Lengend Snippet: A. Experimental workflow for double-gene deletion AsCas12a CRISPR screening. B. Scatter plot of median Log2 fold-change of crRNAs associated with the indicated target gene after Cas12a-mediated co-deletion of Rosa26 control (WT) or Ripk1 (Ripk1null). Fold-change is calculated between in vitro and in vivo timepoints. Targets preferentially depleted in WT (red), Ripk1null (beige), or both (blue), or targets preferentially enriched in Ripk1null (orange) are highlighted and have a P-value < 0.05 (see Methods). Also shown are Log2 fold-change for individual crRNAs (red bars) for significant hits overlaid on the distribution for all crRNAs. C. Select targets identified in (B) projected onto a schematic of the TNF signaling pathway in Ripk1 WT (top) and Ripk1null (bottom) cancer cells. Highlighted gene targets (non-opaque) are depleted in WT or enriched in Ripk1null tumors and illustrate inferred signaling bias for each genotype. D-E. Expression and quantitation of NF-kB and MAPK pathway proteins (n=2–3) (D) and NF-kB transcriptional reporter activity (representative of 3 independent experiments) (E) in WT or Ripk1null B16 cancer cells after treatment with 100 ng/ml murine TNF. F. CASP3 cleavage after TNF stimulation of TSA WT or Ripk1null cells for the indicated times under serum-free conditions. G. In vitro dose response of TNF-mediated killing with 1 ug/ml cycloheximide for 24 hours for WT or Ripk1null B16 and TSA cells measured by normalized viability (representative of 2–3 independent experiments). P-values for time course was determined by repeated measures ANOVA. For dose response and reporter assay, a non-linear model was fitted and significance determined by comparison to a reduced model using ANOVA.

Article Snippet: For double-gene deletion CRISPR screening, a dual-crRNA library was cloned into a AsCas12a crRNA expression vector pRG212 (EFS-GFP-P2A-Neo-U6-crRNA, Addgene #149722).

Techniques: CRISPR, Control, In Vitro, In Vivo, Expressing, Quantitation Assay, Activity Assay, Reporter Assay, Comparison

Journal: Immunity

Article Title: The Interferon-Stimulated Gene RIPK1 Regulates Cancer Cell Intrinsic and Extrinsic Resistance to Immune Checkpoint Blockade

doi: 10.1016/j.immuni.2022.03.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For double-gene deletion CRISPR screening, a dual-crRNA library was cloned into a AsCas12a crRNA expression vector pRG212 (EFS-GFP-P2A-Neo-U6-crRNA, Addgene #149722).

Techniques: Control, Virus, Recombinant, Protease Inhibitor, Membrane, Lysis, Transfection, Luciferase, Cell Viability Assay, Mutagenesis, Staining, Sequencing, RNA Sequencing, Derivative Assay, Expressing, CRISPR, Plasmid Preparation, Retroviral, Scaffolding, Software

Journal: Immunity

Article Title: The Interferon-Stimulated Gene RIPK1 Regulates Cancer Cell Intrinsic and Extrinsic Resistance to Immune Checkpoint Blockade

doi: 10.1016/j.immuni.2022.03.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For double-gene deletion CRISPR screening, a dual-crRNA library was cloned into a AsCas12a crRNA expression vector pRG212 (EFS-GFP-P2A-Neo-U6-crRNA, Addgene #149722).

Techniques: Control, Virus, Recombinant, Protease Inhibitor, Membrane, Lysis, Transfection, Luciferase, Cell Viability Assay, Mutagenesis, Staining, Sequencing, RNA Sequencing, Derivative Assay, Expressing, CRISPR, Plasmid Preparation, Retroviral, Scaffolding, Software

Journal: Nucleic acids research

Article Title: Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.

doi: 10.1093/nar/gkae016

Figure Lengend Snippet: Figure 2. CRISPR-Cas12m activity in E. coli cells. (A) Plasmid DNA interference assay in E. coli . To assess transformation efficiency, each E. coli transformant sample was serially diluted (10 ×) and grown on Kn supplemented media at 37 ◦C o v ernight. Effectiv e DNA interference of the targeted plasmid resulted in a reduction in colony forming units (CFU). Data are presented as mean ( n = 3). KanR – kanam y cin resistance, ori – origin of replication, dAsCas12a – AsCas12a R u vC activ e site mutant (D908A). (B) Experimental w orkflo w of the superfolder GFP (sfGFP) fluorescence interference assay in E. coli . (C) sfGFP fluorescence interference experiment in E. coli . To assess transformation efficiency and sfGFP expression, each E. coli transformant sample was serially diluted (10 ×) and grown overnight at 37 ◦C on the arabinose-supplemented media. Effective DNA binding by dAsCas12a and GoCas12m resulted in reduction of sfGFP fluorescence while CFU remained unchanged. NT – non-targeting control. (D) Bacteriophage plaque formation assay in E. coli . To assess the efficiency of plating (EOP), phages were serially diluted (10 ×) and spotted onto lawns of E. coli expressing AsCas12a, dAsCas12a or GoCas12m. Effective defense against phage infection resulted in reduction of plaque-forming units. Data are presented as mean (n = 3). S and A indicate targeted sense and antisense DNA strands, respectively.

Article Snippet: Expression and purification of Cas12 effectors and their RNP complexes For Cas12m and Cas12a protein purification, E. coli DH10B strain was transformed with expression vectors (pMBP-AsCas12a (gift from Jennifer Doudna, Addgene plasmid #113430), pGB060 (produced from pMBP-AsCas12a by introducing D908A modification, using Phusion SiteDirected Mutagenesis Kit (Thermo Fisher Scientific), pTK200, pTK203; Supplementary Table S4 ).

Techniques: CRISPR, Activity Assay, Plasmid Preparation, Transformation Assay, Mutagenesis, Fluorescence, Expressing, Binding Assay, Control, Plaque Formation Assay, Infection

Journal: Nucleic acids research

Article Title: Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.

doi: 10.1093/nar/gkae016

Figure Lengend Snippet: Figure 4. GoABE base editing activity in E. coli and human cells. (A) Schematic representation of the experimental w orkflo w to detect base editing activity in E. coli resulting in chloramphenicol resistance (CmR) gene restoration. (B) Plasmid DNA transf ormation assa y in E. coli . To assess base editing efficiency, each E. coli transformant sample was serially diluted (10 ×) and grown overnight at 37 ◦C on the Cm supplemented media. Recovery of the colonies indicates successful targeted base editing. As a positive control (Ctrl), E. coli were transformed with a plasmid encoding the intact CmR gene. NT and T indicate non-targeting and targeting crRNA constructs, respectively. (C) Sanger sequencing of plasmids obtained from reco v ered E. coli colonies. (D) Experimental w orkflo w of the enhanced GFP (eGFP) reco v ery assa y perf ormed in HEK293T cells. (E) Flo w cytometry results of counting eGFP-positive HEK293T cells after 24, 48 and 72 h. Data are presented as mean ± SD ( n = 3). (F) Base editing at endogenous sites in HEK293T cells. Selected DNA target sites are labeled according to Richter et al. ( 67 ). Gray areas indicate non-A nucleotides at specific positions across all targets. Data are presented as mean ± SD ( n = 3).

Article Snippet: Expression and purification of Cas12 effectors and their RNP complexes For Cas12m and Cas12a protein purification, E. coli DH10B strain was transformed with expression vectors (pMBP-AsCas12a (gift from Jennifer Doudna, Addgene plasmid #113430), pGB060 (produced from pMBP-AsCas12a by introducing D908A modification, using Phusion SiteDirected Mutagenesis Kit (Thermo Fisher Scientific), pTK200, pTK203; Supplementary Table S4 ).

Techniques: Activity Assay, Plasmid Preparation, Positive Control, Transformation Assay, Construct, Sequencing, Cytometry, Labeling

Journal: EMBO Reports

Article Title: PARK15 / FBXO7 is dispensable for PINK1 /Parkin mitophagy in iNeurons and HeLa cell systems

doi: 10.15252/embr.202256399

Figure Lengend Snippet:

Article Snippet: SpCas9 and AsCas12a/AsCpf1 expression plasmids pET‐NLS‐Cas9‐6xHis (Addgene plasmid # 62934) and modified pDEST‐his‐AsCpf1‐EC, generated by deleting the MBP sequence from plasmid pDEST‐hisMBP‐AsCpf1‐EC (Addgene plasmid # 79007), were transformed into RosettaTM(DE3)pLysS Competent Cells (Novagen), respectively, for expression.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Membrane, Electron Microscopy, Staining, Western Blot, Protease Inhibitor, Peptide Fractionation, Bicinchoninic Acid Protein Assay, Software, Mass Spectrometry, Transfection, Imaging

Journal: Molecular Therapy. Nucleic Acids

Article Title: A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020

doi: 10.1016/j.omtn.2021.02.012

Figure Lengend Snippet: Lb2Cas12a efficiently cleaved mammalian genomes at both integrated and endogenous targets (A) A phylogenetic tree generated by Phylo.io based on an alignment of Cas12a orthologs of the indicated species. , (B) A representation of the indicated Cas12a orthologs with domains indicated. WED: crRNA binding and processing domains, split by REC and PI domains into WED-I, II, and III (green). REC: DNA binding domains REC1 and REC2 (gray). PI: PAM-interacting domain (orange). RuvC: DNA cleavage domains, split by BH and NUC domains into RuvC-I, II, and III (blue). BH: bridge helix (light green). Nuc: DNA processing domain (red). Number at right indicates the number of amino acids of each ortholog. (C) A diagram illustrating the double-stranded-break-induced gain-of-expression assay used in subsequent figures. A construct with an in-frame (+1) crRNA target sequence (orange) preceding an out-of-frame (+3) luciferase gene (light green) is stably integrated into the genomes of HEK293T cells. Upon cleavage by a Cas12a/crRNA complex, non-homologous end-joining (NHEJ) repair places approximately one-third of the downstream luciferase genes in frame (dark green), enabling their expression. Luciferase activity reflects the efficiency of Cas12a-mediated cleavage. (D) A list of target sequences used in subsequent panels with their preceding PAMs (blue). (E) The editing efficiency of AsCas12a, LbCas12a, and Lb2Cas12a, measured with the assay shown in panel (C), were compared using the six indicated lentivirally integrated targets. The means of three independent replicates are shown, with error bars indicating ± standard error of the mean (SEM). The significance of differences with Lb2Cas12a are indicated above bars representing AsCas12a and LbCas12 (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; values of p > 0.05 are indicated in the graph), as determined by two-way ANOVA, followed by Tukey’s multiple comparison tests. (F) Lb2Cas12a edited an endogenous locus of the DNMT1 gene (DNMT1-3) with high efficiency. A T7E1 assay was used to compare the editing efficiencies of the Cas12a orthologs. Percent edited, indicated beneath the figure, is calculated as in Guschin et al. and indicates the average of three independent biological replicates. Neg: negative-control cells transfected with vector alone. (G) Expression of AsCas12a, LbCas12a, and Lb2Cas12a in HEK293T cells used in (F). A western blot, representative of three independent biological replicates with similar results, is shown.

Article Snippet: Wild-type AsCas12a (pcDNA3.1-hAsCpf1), LbCas12a (pcDNA3.1-hLbCpf1), and Lb2Cas12a (pcDNA3.1-hLb2Cpf1) and pX330 (pX330-U6-chimeric_BB-CBh-hSpCas9) plasmids were gifts from Dr. Feng Zhang (Addgene plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Cas12a variants in E. coli for protein production.

Techniques: Generated, Binding Assay, Expressing, Construct, Sequencing, Luciferase, Stable Transfection, Non-Homologous End Joining, Activity Assay, Comparison, Negative Control, Transfection, Plasmid Preparation, Western Blot