Journal: Biotechnology and Bioengineering

Article Title: Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

doi: 10.1002/bit.27200

Figure Lengend Snippet: Assessment of the diversity of Myr and PPYP flanking sequences and CRISPR‐derived mutations by DNA deep sequencing. (a) Schematic illustration of the pipeline established to identify CRISPR‐derived indel mutations in type‐C endogenous retrovirus (ERV) sequences from targeted DNA amplicon sequencing. Type‐C ERV specific primers were used to amplify approximately 300 bp surrounding the Myr or PPYP CRISPR target sites of the gag genes from untreated and CRISPR‐treated cells, and amplicons were analyzed by Illumina sequencing. Untreated reads were clustered as based on 97% sequence similarity to establish weighted profiles. Profiles were used to distinguish between natural ERV variations and indel mutations in CRISPR‐treated cells. (b, c) Clusters of Myr (panel B) or PPYP (panel C) deep sequencing reads of untreated parental CHO‐K1 cells. Clusters consisting of group 1, group 2 and group 3 type‐C ERV sequences are indicated in blue, purple and red lettering, respectively, according to the phylogenetic groups depicted in Figure . Clustered sequences expected to be targeted by CRISPR‐Cas9, as they contain the Myr2 sgRNA or PPYP6 sgRNA recognition sites and an adjacent PAM sequence, are shown in bold. The cluster representing the expressed group 1 type‐C ERV sequence is highlighted in yellow. (d) Number of distinct mutations and their corresponding read frequencies in seven clones (C02, D12, G09, A02, E10, K03, K14) isolated from Myr2 or PPYP6 sgRNA‐treated polyclonal populations, as indicated. Mutations of the expressed group 1 ERV, as previously detected in the mRNA in each clone, are indicated with a bold frame. Gray shaded boxes represent mutations occurring at a frequency higher than 0.4% (left‐hand side axis), thus implying the occurrence of the same mutation in more than one ERV locus, where the distinct ERV loci are separated by dotted lines. The estimated total number of mutated ERV loci of each clone is indicated by the right‐hand side axis. (e) Frequency of Myr2 or PPYP6 sgRNA‐induced repair junctions compatible with C‐NHEJ, alt‐EJ or HR DSB repair mechanisms. Repair junctions incompatible with these three main DSB repair mechanisms are grouped as Unknown. A total of 67 DNA repair junctions (n Myr = 45, n PPYP = 22) obtained from both Sanger cDNA and Illumina deep DNA sequencing were analyzed. (f, g) Proportion of the various mutations detected in each of the ERV sequence clusters shown in panels B and C, respectively. Clusters containing the Myr2 or PPYP6 sgRNA recognition sites including an adjacent PAM site are shown in bold letters as in panels B and C, while clusters with sgRNA possessing mismatches at position 13 or 15 in the sgRNA recognition site mismatches are shown in normal letters. The cluster representing the expressed group 1 type‐C ERV sequence is highlighted in yellow, as for panels B and C [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The mammalian codon‐optimized Streptococcus pyogenes Cas9 (SpCas9) nuclease expression plasmid JDS246 (Addgene plasmid #43861) was used to introduce site‐specific DSBs (Fu et al., ).

Techniques: CRISPR, Derivative Assay, Sequencing, Amplification, Illumina Sequencing, Clone Assay, Isolation, Mutagenesis, DNA Sequencing

Journal: Cell

Article Title: TMEM41B Is a Pan-flavivirus Host Factor

doi: 10.1016/j.cell.2020.12.005

Figure Lengend Snippet: Genome-wide CRISPR-Cas9 Screens for Zika and Yellow Fever Viruses Identify TMEM41B and VMP1 as Required Host Factors (A) Bubble plot of genes significantly enriched in a genome-wide CRISPR KO screen in HAP1 cells challenged with ZIKV (top) and YFV (bottom). Colors indicate association with cellular pathways or protein complexes and domains. Red lines denote Z = ± 2. (B) Heatmap of Z scores for genes in the autophagy pathway ordered sequentially by functional role: L, lipid mobilization; 1, initiation; 2, nucleation; 3, elongation; 4, sequestration; 5, tethering/fusion. Rows represent replicate screens. (C) Scatterplot of gene-wise log 2 fold change (LFC) from this study (ZIKV) versus Moretti et al. (2018) autophagy screen. (D) HAP1 WT and (n = 3) individual KO clones for VTT domain-containing proteins infected with ZIKV. (E) WT and TMEM41B KO HAP1 cells overexpressing individual VTT domain proteins infected with ZIKV. (F) Same as (E) but in VMP1 KO HAP1 cells. (G) HAP1 WT and (n = 3–5) individual KO clones for autophagy genes infected with ZIKV. (H–K) Same as (D–G) but infected with YFV Asibi. Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen-positive cells. Dots in (D), (G), (H), and (K) represent the average of n = 3 replicates from individual single-cell clones. Error bars in (E), (F), (I), and (J) depict a single KO clone with standard deviation (SD) of n = 3 replicates. See also B–S1I.

Article Snippet: CRISPR/Cas9 plasmids were generated from the pDDC6 vector, which encodes the human codon-optimized Streptococcus pyogenes Cas9 ( hSpCas9 ; a gift from Peter Duchek) (Addgene plasmid: #59985; http://n2t.net/addgene:59985 ; RRID: Addgene_59985).

Techniques: Genome Wide, CRISPR, Functional Assay, Clone Assay, Infection, Flow Cytometry, Standard Deviation

Journal: Cell

Article Title: TMEM41B Is a Pan-flavivirus Host Factor

doi: 10.1016/j.cell.2020.12.005

Figure Lengend Snippet:

Article Snippet: CRISPR/Cas9 plasmids were generated from the pDDC6 vector, which encodes the human codon-optimized Streptococcus pyogenes Cas9 ( hSpCas9 ; a gift from Peter Duchek) (Addgene plasmid: #59985; http://n2t.net/addgene:59985 ; RRID: Addgene_59985).

Techniques: Subcloning, Western Blot, Recombinant, Infection, Transfection, Protease Inhibitor, Staining, Bicinchoninic Acid Protein Assay, Immunoprecipitation, SYBR Green Assay, Sequencing, Derivative Assay, Plasmid Preparation, Software

Journal: Human Molecular Genetics

Article Title: PD-linked CHCHD2 mutations impair CHCHD10 and MICOS complex leading to mitochondria dysfunction

doi: 10.1093/hmg/ddy413

Figure Lengend Snippet: Generation of human isogenic lines harboring PD-related CHCHD2 mutants. ( A ) PD-associated CHCHD2 mutants T61I, Q126X and R145Q on functional domains of human CHCHD2 protein. CHCH domain. ( B ) Schematic overview depicting human CHCHD2 locus and Cas9 targeting strategy with designed guide RNA recognized exon 3 sequences in human CHCHD2 locus. ( C ) Sanger sequencing chromatogram showing the heterozygous point mutation leading to Q126X+/−. Representative results from Q10 were shown. ( D ) Sanger sequencing chromatogram on the reverse DNA strand showing the heterozygous point mutation leading to R145Q+/− (R4), the homozygous point mutant leading to R145Q−/− (R17) at the targeting locus of H9 (+/+) hESCs. * the blocking nonsense mutation to enhance the specificity of Cas9 system.

Article Snippet: Capped and polyadenylated Cas9 mRNA was obtained from pT7-Cas9 vector (Origene) by mMESSAGE Mmachine T7 ULTRA kit (Thermo Fisher Scientific).

Techniques: Functional Assay, Sequencing, Mutagenesis, Blocking Assay

Journal: Nucleic Acids Research

Article Title: PLK1 targets CtIP to promote microhomology-mediated end joining

doi: 10.1093/nar/gky810

Figure Lengend Snippet: CDK1, Aurora A and PLK1 kinase activity is required for MMEJ. ( A ) DR-EGFP-HR, ( B ) EGFP-MMEJ reporter and modified DNA DSB induction method by RNA-guided endonuclease Cas9. ( C ) Drug exposure and DSB repair assay. U2OS cells carrying the indicated DSB repair reporter substrate were treated with nocodazole (330 nM) or mock treated with DMSO for 16 h followed by Dox in the presence of nocodazole to induce a DSB. After 24 h Dox induction, the cells were trypsinized and FACS was performed. ( D and E ) After 16 h nocodazole treatment, the cell-cycle profile was determined by FACS and the mean percentage of cells in the indicated cell-cycle phases was determined. ( F ) HR repair efficiency in unperturbed and synchronized cells with or without the indicated drugs was determined by FACS. A CDK1 inhibitor (10 μM RO3306), PLK1 inhibitor (10 μM BI2536), Aurora A inhibitor (2 μM MLN8054) or DMSO was added to the culture before Dox induction. ( G ) MMEJ repair efficiency determined by the same method and strategy as the HR assay. ( H ) EGFP-MMEJ assay was performed in U2OS cells with overexpressed Flag-PLK1 WT or Flag-PLK1 kinase dead (K82M/D176N) mutant. Western blotting shows the expression of Flag-PLK1 variants. ( I and J ) U2OS cells overexpressing Flag-PLK1-WT or a PLK1-AS (analog-sensitive) mutant were treated with or without the indicated amounts of 3MB-PP1 and then analyzed by EGFP-MMEJ assay. Western blotting shows the expression of Flag-PLK1 variants. The data represent the means of three independent experiments, with error bars as SD and P values as noted: ** P ≤ 0.01; n.s. not significant.

Article Snippet: Doxycycline-inducible lentiviral expression vectors containing the humanized Streptococcus pyogenes Cas9 plasmid were obtained from Addgene (#50661).

Techniques: Activity Assay, Modification, Mutagenesis, Western Blot, Expressing

Journal: Plants

Article Title: A Gemini Virus-Derived Autonomously Replicating System for HDR-Mediated Genome Editing of the EPSP Synthase Gene in Indica Rice

doi: 10.3390/plants14030477

Figure Lengend Snippet: ( A ) rcoSpCas9 expression cassette and first round of plant transformation: LB/RB: T-DNA left and right border; hptII- Hygromycin coding sequence (CDS); 35S_P: Promoter of Cauliflower mosaic virus 35S gene; ZmUbq_T- Maize ubiquitin gene terminator, ZmUbq_P- Zea maiz Ubiquitin gene promoter rcoSpCas9- Rice codon-optimized Streptococcus pyrogens Cas9 CDS. ( B ) Development of stable lines and molecular confirmation of rcoSpCas9 expressing MTU1010 transgenic rice (i) and (ii): Regeneration and rooting of putative transgenic lines; (iii): 1–10 putative rcoSpCas9 transgenic lines confirmed through PCR; B: blank lane; P: pMDC99:rcoSpCas9 plasmid as positive control; L: 1 kb DNA ladder (GeneRuler) (iv): Southern blot analysis of PCR positive control lines; L: 1 kb DNA ladder (GeneRuler); W: wild type MTU1010 DNA as negative control; B: blank lane; E1–E20: PCR positive rcoSpCas9 lines (v): Coomassie-stained SDS-PAGE gel showing equal loading of total protein extracted from E1, E2, E20 and W lines. The gel serves as a loading control to confirm uniform protein quantities across lanes. (vi) Western blot analysis of the same gel, probed with an anti-Cas9 antibody, confirming the expression of rcoSpCas9 protein in the transgenic lines (E1, E2, and E20). The position of the Cas9 protein (~expected 160 kDa) and W: wild type MTU1010 total soluble protein as negative control, E1, E2, and E20: Southern positive rcoSpCas9 expressed lines; M- Protein ladder.

Article Snippet: A rice codon-optimized Streptococcus pyogenes Cas9 ( rcoSpCas9 ) was synthesized from GenScript ( https://www.genscript.com ).

Techniques: Expressing, Transformation Assay, Sequencing, Virus, Ubiquitin Proteomics, Transgenic Assay, Plasmid Preparation, Positive Control, Southern Blot, Negative Control, Staining, SDS Page, Control, Western Blot

Journal: Nature biomedical engineering

Article Title: Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions

doi: 10.1038/s41551-016-0003

Figure Lengend Snippet: Construction of WT1-2A-eGFP knockin ES03 hESC line using Cas9 nuclease. (A) Schematic diagram of the knockin strategy at the stop codon of the WT1 locus. Vertical arrows indicate sgRNA1 and sgRNA2 targeting sites. Red and blue horizontal arrows are PCR primers for assaying WT1 locus targeting and homozygosity, respectively. (B) Representative PCR genotyping of hESC clones after puromycin selection is shown, and the expected PCR product for correctly targeted WT1 locus is ~3 kbp (red arrows) with an efficiency of 21/44. A homozygosity assay was performed on the knockin clones, and those without ~200 bp PCR products were homozygous (blue arrows). (C) PCR genotyping of hESC clones after TAT-Cre mediated excision of the PGK-Puro cassette. Clones with the PCR products of ~1 kbp are PGK-Puro free, and those with ~3 kbp contain PGK-Puro. (D) Live cell flow analysis of GFP+ cells at day 0, day 10 and day 12 during CHIR treatment of WT1-2A-eGFP knockin ES03. (E) Phase contrast images and corresponding eGFP fluorescent images of WT1-2A-eGFP hPSC-derived epicardial cells after excision of the PGK-Puro cassette. Scale bars, 100 μm.

Article Snippet: Human codon-optimized Streptococcus pyogenes wild-type Cas9 (pCas9-2A-eGFP #) was obtained from Addgene (plasmid #44719) and chimeric guide RNA expression cassette was cloned into this Cas9-2A-eGFP plasmid with two BbsI restriction sites for rapid sgRNA cloning.

Techniques: Knock-In, Clone Assay, Selection, Derivative Assay

Journal: International journal of biological macromolecules

Article Title: Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression.

doi: 10.1016/j.ijbiomac.2022.04.114

Figure Lengend Snippet: Fig. 2. Finding the most efficient aptamer/ABP pair for Cas9 mRNA delivery. A. Aptamers and ABPs tested for Cas9 mRNA delivery by VLPs. PP7, BoxB, or com replaced MS2 in the Cas9-expressing construct and PCP, λ N22 peptide, or Com replaced MCP in the packaging plasmid. Red asterisk indicates stop codon. UTR: Human HBB 3′ untranslated region sequence for improving Cas9 mRNA stability and translatability. B. Comparing com/Com and MS2/MCP aptamer/ABP pairs for SaCas9 mRNA delivery. *** indicates p < 0.0001 (two-tailed t-test). C. Comparing com/Com, BoxB/N22 and PP7/PCP aptamer/ABP pairs for SaCas9 mRNA delivery. *** indicates p < 0.001 for com/Com VLPs versus BoxB/N22 or PP7/PCP VLPs (Tukey's post hoc analysis following ANOVA). For (B and C), 0.25 μg pFCK-HBB(n)-g1 plasmid DNA (expressing HBB-sgRNA targeting the sickle mutant seqeunce in GFP reporter cells) was transfected into 2.5 × 104 GFP-reporter cells 12 h before transducing 500 μl indicated Cas9 mRNA VLPs. Our typical VLP-containing supernatants contained 100–150 ng/ml p24. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: pRSV-Rev (Addgene #12253), a plasmid conferring expression of HIV-1-derived Rev. (coding for sequence-specific RNA-binding protein) under the transcriptional control of the Rous Sarcoma Virus (RSV) promoter [37]; pMD2.G (Addgene #12259), a plasmid expressing the spike G glycoprotein of the vesicular stomatitis virus (VSV-G) for pseudotyping; pMDLg/pRRE (Addgene #12251), a third generation packaging plasmid [37]; psPAX2-D64V (Addgene #63586) [38], a second generation packaging plasmid with an inactivating D64V substitution in the integrase to prevent integration of the LV genomic RNA [39]; pLHsgRNA1 (Addgene #75388), a lentiviral transfer plasmid for expressing Streptococcus pyogenes Cas9 (SpCas9) sgRNA [40]; were purchased from Addgene.

Techniques: Expressing, Construct, Plasmid Preparation, Sequencing, Two Tailed Test, Mutagenesis, Transfection

Journal: International journal of biological macromolecules

Article Title: Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression.

doi: 10.1016/j.ijbiomac.2022.04.114

Figure Lengend Snippet: Fig. 3. Co-packaging Cas9 mRNA and sgRNA-expressing LV genomic RNA in VLPs. A. Illustration of co-packaging mRNA and LV genomic RNA in VLPs. Envelope proteins are not shown. Only one modified and one unmodified Gag precursor in the immature virion are shown. B. Determining the best ratio of unmodified and modified packaging plasmid for most efficient co-packaging. Indicated ratios of unmodified (Um) packaging plasmid (psPAX2-D64V) and Com-modified (M) packaging plasmid (psPAX2-D64V-NC-Com) were used to transfect HEK293T cells by PEI to make VLPs, which contained Cas9 mRNA and LV genomic RNA for expressing sickle sgRNA. The particle-containing supernatants (500 μl) were transduced into GFP-reporter cells and GFP-positive cells were analyzed by flow cytometry. Each data point indicates one technical replicate from the same batch of VLP particles. C. com/Com outperformed MS2/MCP for mRNA and sgRNA expressing cassette co-packaging. Experiments were done similarly to those in (B) and means of three biological replicates were shown. *** indicates p < 0.001 in Bonferroni post hoc tests following ANOVA. D. Determining the best ratio of Cas9-expressing DNA and sgRNA-expressing lentiviral transfer plasmid DNA for most efficient co-packaging. Experiments were done similarly to those in (B), with a psPAX2-D64V and psPAX2-D64V-NC-Com ratio of 2:1. Data points indicate biological replicates. ** indicates p < 0.01 in Tukey's post hoc test following ANOVA. (B–D): 500 μl unconcentrated VLP-containing supernatant was added to 2.5 × 104 GFP- reporter cells.

Article Snippet: pRSV-Rev (Addgene #12253), a plasmid conferring expression of HIV-1-derived Rev. (coding for sequence-specific RNA-binding protein) under the transcriptional control of the Rous Sarcoma Virus (RSV) promoter [37]; pMD2.G (Addgene #12259), a plasmid expressing the spike G glycoprotein of the vesicular stomatitis virus (VSV-G) for pseudotyping; pMDLg/pRRE (Addgene #12251), a third generation packaging plasmid [37]; psPAX2-D64V (Addgene #63586) [38], a second generation packaging plasmid with an inactivating D64V substitution in the integrase to prevent integration of the LV genomic RNA [39]; pLHsgRNA1 (Addgene #75388), a lentiviral transfer plasmid for expressing Streptococcus pyogenes Cas9 (SpCas9) sgRNA [40]; were purchased from Addgene.

Techniques: Expressing, Modification, Plasmid Preparation, Flow Cytometry

Journal: International journal of biological macromolecules

Article Title: Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression.

doi: 10.1016/j.ijbiomac.2022.04.114

Figure Lengend Snippet: Fig. 4. Characterization of Cas9 mRNA and sgRNA all-in-one VLPs. A. Genome editing activities of singly packaged particles (Cas9 mRNA VLPs or sgRNA IDLVs) versus co-packaged VLPs. For single packaging, either the Cas9-expressing DNA (pBlue-SaCas9–3 aptamers) or the sickle sgRNA-expressing lentiviral transfer plasmid DNA [pFCK-HBB (n)-g1] was replaced by pCDNA3 DNA to control transfection conditions. B. Genome editing activities of the RNP VLPs and co-packaged RNA VLPs. Five hundred microliter of supernatant were added to 2.5 × 104 GFP-reporter cells. For (A, B), *** indicates p < 0.0001 in two-tailed unpaired t-tests. Three preparations of VLPs were analyzed in each experiment. C. Deep sequencing analysis of insertions and deletions in the target region. The PAM (on the opposite strand) region is highlighted and the target sequence is underlined. The red “G” indicates substitutions. DNA was amplified from GFP-reporter cells treated with 50 μl all-in-one VLP-containing supernatant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: pRSV-Rev (Addgene #12253), a plasmid conferring expression of HIV-1-derived Rev. (coding for sequence-specific RNA-binding protein) under the transcriptional control of the Rous Sarcoma Virus (RSV) promoter [37]; pMD2.G (Addgene #12259), a plasmid expressing the spike G glycoprotein of the vesicular stomatitis virus (VSV-G) for pseudotyping; pMDLg/pRRE (Addgene #12251), a third generation packaging plasmid [37]; psPAX2-D64V (Addgene #63586) [38], a second generation packaging plasmid with an inactivating D64V substitution in the integrase to prevent integration of the LV genomic RNA [39]; pLHsgRNA1 (Addgene #75388), a lentiviral transfer plasmid for expressing Streptococcus pyogenes Cas9 (SpCas9) sgRNA [40]; were purchased from Addgene.

Techniques: Expressing, Plasmid Preparation, Control, Transfection, Two Tailed Test, Sequencing, Amplification