Journal: Nature Communications

Article Title: Engineered reproductively isolated species drive reversible population replacement

doi: 10.1038/s41467-021-23531-z

Figure Lengend Snippet: a Homozygous dCas9-VPR flies were crossed bidirectionally to homozygous sgRNA flies. Shaded gray background indicates expected lethal crosses. b Homozygous dCas9-VPR individuals were crossed bidirectionally to heterozygous spCas9/sgRNA individuals. Surviving individuals were repeatedly inbred to generate SPECIES. c , d Plots depicting % progeny survival from the crosses depicted in A and B. N = 3 biologically independent replicates for each gRNA combination (eight total combinations). Middle lines indicate mean. Gray dots indicate homozygous sgRNA and heterozygous spCas9/sgRNA flies crossed to wildtype flies. Light and dark blue dots indicate homozygous sgRNA and heterozygous spCas9/sgRNA flies crossed to homozygous dCas9-VPR mothers and fathers, respectively. Purple stars indicate the presence of maternally deposited Cas9. Source data are provided as a Source Data file.

Article Snippet: The resulting plasmid was then digested with PacI, and the following components were cloned into generate the final dCas9-expressing vectors: the Ubiquitin-63E promoter fragment amplified with primers 986.C9 and 986.C10 from D. melanogaster genomic DNA and a dCas9-VPR fragment amplified from Addgene plasmid #78898 with primers 986.C11 and 986.C12 to generate plasmid OA-986B (Addgene #124999); the bottleneck promoter fragment amplified with primers 986.C13 and 986.C14 from D. melanogaster genomic DNA and a dCas9-VPR fragment amplified from Addgene plasmid #78898 with primers 986.C15 and 986.C12 to generate plasmid OA-986C (Addgene #125000); the Ubiquitin-63E promoter fragment amplified with primers 986.C9 and 986.C16 from D. melanogaster genomic DNA and a dCas9-VP64 fragment amplified from Addgene plasmid #78897 with primers 986.C17 and 986.C18 to generate plasmid OA-986D (Addgene #125001); and the bottleneck promoter fragment amplified with primers 986.C13 and 986.C19 from D. melanogaster genomic DNA and a dCas9-VP64 fragment amplified from Addgene plasmid #78897 with primers 986.C20 and 986.C18 to generate plasmid OA-986E (Addgene #125002).

Techniques:

Journal: Nature Communications

Article Title: Engineered reproductively isolated species drive reversible population replacement

doi: 10.1038/s41467-021-23531-z

Figure Lengend Snippet: a Crosses used to determine embryo and adult viability. (Cross #1) Homozygous dCas9-VPR;sgRNA “SPECIES” females crossed to homozygous dCas9-VPR;sgRNA SPECIES males. (Cross #2) Homozygous dCas9-VPR;sgRNA SPECIES females crossed to wildtype (WT) males. (Cross #3) WT females crossed to homozygous dCas9-VPR;sgRNA SPECIES males. Gray shaded background indicates expected lethal crosses. (Cross #4) WT females crossed to WT males. Each cross type was performed for all eight generated SPECIES (Fig. S3). b Schematic detailing the methods of determining embryo and adult survival compared to WT. c % embryo and adult survival was calculated and plotted. The number below each x -axis group indicates cross number (#1–4). N = 3 biologically independent replicates for each cross number. ✝ indicates that embryos did not survive past L1/L2 stages. Unpaired two-tailed t -tests were performed for each SPECIES compared to WT (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). Middle lines indicate mean, while error bars represent standard deviation. The color of the dots represent each species as indicated in figure key. d Embryo (left graph) and adult (right graph) Cohen’s d effect sizes compared to WT. N = 6 observations per cross number per SPECIES (effect size based on mean comparison between three replicates of each experimental cross (#1–3) to control cross (#4)). Error bars represent 95% confidence interval. The color of the dots represent each species as indicated in figure key. Source data are provided as a Source Data file.

Article Snippet: The resulting plasmid was then digested with PacI, and the following components were cloned into generate the final dCas9-expressing vectors: the Ubiquitin-63E promoter fragment amplified with primers 986.C9 and 986.C10 from D. melanogaster genomic DNA and a dCas9-VPR fragment amplified from Addgene plasmid #78898 with primers 986.C11 and 986.C12 to generate plasmid OA-986B (Addgene #124999); the bottleneck promoter fragment amplified with primers 986.C13 and 986.C14 from D. melanogaster genomic DNA and a dCas9-VPR fragment amplified from Addgene plasmid #78898 with primers 986.C15 and 986.C12 to generate plasmid OA-986C (Addgene #125000); the Ubiquitin-63E promoter fragment amplified with primers 986.C9 and 986.C16 from D. melanogaster genomic DNA and a dCas9-VP64 fragment amplified from Addgene plasmid #78897 with primers 986.C17 and 986.C18 to generate plasmid OA-986D (Addgene #125001); and the bottleneck promoter fragment amplified with primers 986.C13 and 986.C19 from D. melanogaster genomic DNA and a dCas9-VP64 fragment amplified from Addgene plasmid #78897 with primers 986.C20 and 986.C18 to generate plasmid OA-986E (Addgene #125002).

Techniques: Generated, Two Tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Cancer therapy with a CRISPR-assistant telomerase-activating gene expression system

doi: 10.1101/291302

Figure Lengend Snippet: PCR am plification assay of telomerase elongation products of 293T, HepG2, MEC-5 and HL7702 cell lines. M, DL2000 DNA marker; Lane 1, cells co-transfected with sTMEP & TsgRNA & dCas9-VP64; Lane 2, cells transfected with sTMEP; Lane 3, cells transfected with bTMEP; Lane 4, cells transfected with lipofectin; Lane 5, PCR negative control.

Article Snippet: The plasmid C1-HO was constructed by replacing the EGFP gene with the HO genes using the cutting sites of NheI and KpnI enzymes (ThermoFisher Scientific) with primers HO NheI-F and HO KpnI-R. A linear TsgRNA-dCas9-VP64 fragment were amplified from plasmid TsgRNA-dCas9-VP64 with primers A TsgRNA-F, A TsgRNA-R, A dCas9-F and A dCas9-R, then cloned into plasmid pAAV-MCS vector (Stratagene) using the cutting sites of SalI and BglII enzymes (ThermoFisher Scientific) to generate plasmid pAAV-TsgRNA-dCas9-VP64.

Techniques: Marker, Transfection, Negative Control

Journal: bioRxiv

Article Title: Cancer therapy with a CRISPR-assistant telomerase-activating gene expression system

doi: 10.1101/291302

Figure Lengend Snippet: (A) The microscope pictures and representative acridine orange staining pictures of HepG2 cell lines. (B) The microscope pictures and representative acridine orange staining pictures of Hepa1-6 cell lines. (C) The microscope pictures and representative acridine orange staining pictures of MRC-5 cell lines. (D) The microscope pictures and representative acridine orange staining pictures of HL7702 cell lines. rAAV-TSD refers to the equivalent mixture of three viruses including rAAV-HOsite-TMCP & rAAV-TsgRNA-dCas9-VP64 & rAAV-HO.

Article Snippet: The plasmid C1-HO was constructed by replacing the EGFP gene with the HO genes using the cutting sites of NheI and KpnI enzymes (ThermoFisher Scientific) with primers HO NheI-F and HO KpnI-R. A linear TsgRNA-dCas9-VP64 fragment were amplified from plasmid TsgRNA-dCas9-VP64 with primers A TsgRNA-F, A TsgRNA-R, A dCas9-F and A dCas9-R, then cloned into plasmid pAAV-MCS vector (Stratagene) using the cutting sites of SalI and BglII enzymes (ThermoFisher Scientific) to generate plasmid pAAV-TsgRNA-dCas9-VP64.

Techniques: Microscopy, Staining

Journal: bioRxiv

Article Title: Cancer therapy with a CRISPR-assistant telomerase-activating gene expression system

doi: 10.1101/291302

Figure Lengend Snippet: (A) Mice subcutaneously transplanted with Hepa1-6 cells mixed with rAAV-TSD and rAAV-MCS, respectively. (B) Tumor size of mice subcutaneously transplanted with the Hepa1-6 cells mixed with rAAV-TSD and rAAV-MCS, respectively. (C) Tumor-burdened mice intravenously injected with rAAV-TSD and rAAV-MCS, respectively. (D) Tumor size of tumor-burdened mice intravenously injected with rAAV-TSD and rAAV-MCS, respectively. (E) Viral DNA presence in various tissues of tumor-burdened mice intravenously injected with rAAV-TSD and rAAV-MCS, respectively. (F) Cas9 mRNA presence in various tissues of tumor-burdened nude mice intravenously injected with rAAV-TSD and rAAV-MCS, respectively. rAAV-TSD refers to a equivalent mixture of three rAAVs including rAAV-HOsite-TMCP, rAAV-TsgRNA-dCas9-VP64, and rAAV-HO. L, left side; R, right side. * p ≤ 0.05 , ** p ≤ 0.01 .

Article Snippet: The plasmid C1-HO was constructed by replacing the EGFP gene with the HO genes using the cutting sites of NheI and KpnI enzymes (ThermoFisher Scientific) with primers HO NheI-F and HO KpnI-R. A linear TsgRNA-dCas9-VP64 fragment were amplified from plasmid TsgRNA-dCas9-VP64 with primers A TsgRNA-F, A TsgRNA-R, A dCas9-F and A dCas9-R, then cloned into plasmid pAAV-MCS vector (Stratagene) using the cutting sites of SalI and BglII enzymes (ThermoFisher Scientific) to generate plasmid pAAV-TsgRNA-dCas9-VP64.

Techniques: Injection

Journal: bioRxiv

Article Title: Cancer therapy with a CRISPR-assistant telomerase-activating gene expression system

doi: 10.1101/291302

Figure Lengend Snippet: (A) Mice subcutaneously transplanted with the Hepa1-6 cells mixed with rAAV-MCS. (B) Mice subcutaneously transplanted with the Hepa1-6 cells mixed with rAAV-TSD. rAAV-TSD refers to the equivalent mixture of three rAVVs, including rAAV-HOsite-TMCP, rAAV-TsgRNA-dCas9-VP64, and rAAV-HO.

Article Snippet: The plasmid C1-HO was constructed by replacing the EGFP gene with the HO genes using the cutting sites of NheI and KpnI enzymes (ThermoFisher Scientific) with primers HO NheI-F and HO KpnI-R. A linear TsgRNA-dCas9-VP64 fragment were amplified from plasmid TsgRNA-dCas9-VP64 with primers A TsgRNA-F, A TsgRNA-R, A dCas9-F and A dCas9-R, then cloned into plasmid pAAV-MCS vector (Stratagene) using the cutting sites of SalI and BglII enzymes (ThermoFisher Scientific) to generate plasmid pAAV-TsgRNA-dCas9-VP64.

Techniques:

Journal: bioRxiv

Article Title: Cancer therapy with a CRISPR-assistant telomerase-activating gene expression system

doi: 10.1101/291302

Figure Lengend Snippet: (A) Mice intravenously injected with rAAV-MCS. (B) Mice intravenously injected with rAAV-TSD. rAAV-TSD refers to the equivalent mixture of three rAVVs, including rAAV-HOsite-TMCP, rAAV-TsgRNA-dCas9-VP64, and rAAV-HO.

Article Snippet: The plasmid C1-HO was constructed by replacing the EGFP gene with the HO genes using the cutting sites of NheI and KpnI enzymes (ThermoFisher Scientific) with primers HO NheI-F and HO KpnI-R. A linear TsgRNA-dCas9-VP64 fragment were amplified from plasmid TsgRNA-dCas9-VP64 with primers A TsgRNA-F, A TsgRNA-R, A dCas9-F and A dCas9-R, then cloned into plasmid pAAV-MCS vector (Stratagene) using the cutting sites of SalI and BglII enzymes (ThermoFisher Scientific) to generate plasmid pAAV-TsgRNA-dCas9-VP64.

Techniques: Injection

Journal: bioRxiv

Article Title: Genome and epigenome engineering CRISPR toolkit for probing in vivo cis -regulatory interactions in the chicken embryo

doi: 10.1101/135525

Figure Lengend Snippet: (A) Schematic of dCas9-VP64-MCP SAM complex. (B) One sgRNA targeting the Sox10 promoter co-electroporated with dCas9-VP64 and MCP-VP64 causes a premature activation of Sox10 transcription on the experimental (left, magenta) as compared to the control side (right, blue), 50% embryos, n=12, *p<0.05 ). Results confirmed in three independent experiments; representative embryos are shown, error bars represent the standard deviation.

Article Snippet: The MCP-VP64 construct was generated by removing the IRES-H2B-RFP cassette from pCI_H2B-RFP vector ( ) and inserting the MCP and VP64 fragments (amplified from Addgene #61423 and #47319, respectively) using 2-fragment Infusion cloning.

Techniques: Activation Assay, Control, Standard Deviation