Journal: International Journal of Molecular Sciences

Article Title: Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

doi: 10.3390/ijms221910570

Figure Lengend Snippet: Disruption of the HBV X gene derived from a different patient using AdV expressing 8 and 12 gRNAs. ( a ) Sequences of the HBV X gene present in a chromosome of Gx11 cells. Blue bold line, X gene coding region. Three nucleotides differing between the two patients are shown as vertical boxes containing the diverse nucleotides in red. The blue numbers 2, 4, 5, 8, and 9 with bold and red vertical arrows show cut sites by Cas9. Their 20 nt recognition sequences are boxed in red. The black numbers 1, 3, 6, and 7 with thin and broken vertical arrows show the sites not cleaved by Cas9. Their 20 nt recognition sequences are boxed in black over and under the sequences. The PCR primers used for ( b ) are indicated at the top and bottom of this figure. ( b ) Multiple cleavages of the X gene using AdVs expressing 8 and 12 gRNAs. (Upper) Cleavage patterns using Axg8HBV-X. Gx11 cells were infected with Axg8HBV-X together with AxCBCas9 (AxCas9), and 3 days later, total cellular DNA was extracted and PCR was performed using the primer set shown in ( a ). The rates of disruption of the original 0.55 kb DNA using conventional PCR, which detects only deletions but not indels, are shown under the lanes. (Lower) Cleavage patterns using Axg12HBV-X-DR1. The representations are the same as above. Asterisks indicate the region consisting of close bands from 0.47 kb to 0.51 kb shown in ( c ). ( c ) Schematic explanation of the PCR fragments produced by 8 and 12 gRNAs expressed by AdVs. The PCR fragment of 0.55 kb without deletion is shown at the top, the positions of active cut 2, cut 4, cut 5, and cut 8 using Axg8HBV-X are shown in blue characters, and the position of cut 9, an extra cleavage site using Axg12HBV-X-DR1, is indicated in red character. The produced DNA fragments are shown below the top line together with their sizes. Among them, cut 2 and cut 8 were major features at high MOI, and the 0.44 kb fragment produced by them is shown as a bold line. Cut 1, cut 3, cut 6, and cut 7 shown in small green characters on the top line are uncleaved sites, with the exception of cut 1, which showed partial cleavage probably due to a strong off-target effect. The possible 0.54 kb fragment produced by double cleavages of cut 4 and cut 5, the second fragment of gray in the figure, was not detected. The reason for this may be that their cutting sites overlap ((a), fourth sequence row) and one cleavage abolished the 20 nt recognition sequence of the other. All other expected fragments were detected.

Article Snippet: Cassette plasmids of the improved version of the method of Tetraplex-guide Tandem will become available from Addgene, and the AdV expressing Cas9 under the control of CB promoter, AxCBCas9, will be available from RIKEN BioResorce Bank ( https://dna.brc.riken.jp/en/rvd/adenoen , Clone Search, accessed on 28 September 2021).

Techniques: Derivative Assay, Expressing, Infection, Produced, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

doi: 10.3390/ijms221910570

Figure Lengend Snippet: Southern analyses of HBV X gene integrated in Gx11 cell chromosome disrupted by the AdV expressing eight gRNA units. ( a ) The deleted region of the HBV X gene. All cleaved positions and the ends of the produced fragments are common among the upper, middle, and lower parts. (Upper) Cleavage sites of Cas9 and restriction enzymes used for Southern experiments at the HBV X coding region. The cleavage sites of cut 2 and cut 8 are shown as red vertical arrows on the HBV X coding region of the bold horizontal arrow. The 0.55 kb fragment amplified in the PCR deletion assay is shown above the arrow of the HBV X coding region; the range of the Southern probe (BamHI–HindIII fragment) is also shown below the HBV X arrow. neo, neo gene used for the selection of Gx11 cells; GpA, poly(A) sequences of the rabbit β-globin gene. These sequences are present because the integrated DNA fragments are derived from the pCAGGS plasmid. (Middle) The positions of the BamHI–HindIII fragment containing the deletion between cut 2 and cut 8. Because the same enzymes BamHI and HindIII are used for preparation of the Southern probe and for cleavages of total cell DNA, their terminal positions are the same. Expected sizes of the fragments in Southern analysis are shown on the right in kb. (Lower) The positions of the StyI–StyI fragment containing the deletion between cut 2 and cut 8. ( b ) The results of Southern analyses. Gx11 cells were infected with Axg8HBV-X at the indicated MOIs together with AxCas9 at the same MOI.

Article Snippet: Cassette plasmids of the improved version of the method of Tetraplex-guide Tandem will become available from Addgene, and the AdV expressing Cas9 under the control of CB promoter, AxCBCas9, will be available from RIKEN BioResorce Bank ( https://dna.brc.riken.jp/en/rvd/adenoen , Clone Search, accessed on 28 September 2021).

Techniques: Expressing, Produced, Amplification, DNA Deletion Assay, Selection, Derivative Assay, Plasmid Preparation, Infection

Journal: eLife

Article Title: Delta glutamate receptor conductance drives excitation of mouse dorsal raphe neurons

doi: 10.7554/eLife.56054

Figure Lengend Snippet: ( A ) A graphic representation of AAV vectors used in conjunction with AAV-Cas9 to encode eGFP reporter only (top, AAV-empty) and mouse Grid1 guide RNA with eGFP reporter (bottom, AAV- Grid1 ). ( B ) A graphic representation of the third exon of mouse Grid1 , marked with the location of the gRNA (390F) and the overlapping Bgl I restriction site. Filled arrows represent the location of primers used for PCR amplification while the hollow arrow indicates the primer used for sequencing. ( C ) A nucleotide representation of gRNA target site with the translated peptide sequence underneath. The Bgl I recognition site (GCCNNNN^NGGC) is contained by the grey box and overlaps with the predicted Cas9 cleavage site (|). ( D ) Fragment analysis on Bgl I-digested Grid1 PCR products amplified from the dorsal raphe of mice injected with (AAV-Cas9 and AAV-empty) or (AAV-Cas9 and AAV- Grid1 ). ‘% PCR undigested’ refers to the amount of the undigested PCR product relative to the total PCR product as determined by AATI fragment analysis.

Article Snippet: Genetic reagent (adeno-associated virus) , AAV-Cas9 , PMIDs: 25326897 30792150 , NIDA IRP Core Facility, AAV1, pX551 , RRID: Addgene_60957 , Lot# AAV692.

Techniques: Amplification, Sequencing, Injection

Journal: eLife

Article Title: Delta glutamate receptor conductance drives excitation of mouse dorsal raphe neurons

doi: 10.7554/eLife.56054

Figure Lengend Snippet: ( A ) Membrane resistance (R m , ΔV −65 to −120 mV) decreased after stimulation in transduced neurons from mice injected with AAV-Cas9 and AAV-empty (ctrl, p=0.0002, n = 13), but not in transduced neurons from mice injected with AAV-Cas9 and AAV- Grid1 ( Grid1 , p=0.562, n = 16). ( B ) Representative traces (left) and grouped data (right, p<0.0001, n = 15 and 16 and 7) demonstrating the presence of an α1-A R -EPSC in transduced neurons from control mice, but not from Grid1 mice. Neighboring non-transduced neurons from mice injected with AAV-Cas9 and AAV- Grid1 (non) had an α1-A R -EPSC that was indistinguishable from transduced neurons from control mice (p>0.999). ( C ) Current-voltage relationship of the α1-A R -EPSC from control (n = 13) and Grid1 (n = 16) grouped data. Shaded area represents mean ± SEM. ( D ) Targeting GluD1 R reduced the inward current to noradrenaline (NA, I NA, 30 μM) as compared to transduced neurons from control mice and neighboring non-transduced neurons (p=0.004, n = 16 and 16 and 4). Inward I NA in non-transduced neurons from mice injected with AAV-Cas9 and AAV- Grid1 was similar to transduced neurons from control mice (p=0.631). ( E ) Targeting GluD1 R reduced the tonic inward current revealed by bath application of NASPM (100 μM, I NSP ) as compared to transduced neurons from control mice (p=0.009, n = 5 and 11). Line and error bars represent mean ± SEM, * denotes statistical significance, ns denotes not significant. Figure 5—source data 1. Numerical data that were used to generate graphs in .

Article Snippet: Genetic reagent (adeno-associated virus) , AAV-Cas9 , PMIDs: 25326897 30792150 , NIDA IRP Core Facility, AAV1, pX551 , RRID: Addgene_60957 , Lot# AAV692.

Techniques: Injection

Journal: eLife

Article Title: Delta glutamate receptor conductance drives excitation of mouse dorsal raphe neurons

doi: 10.7554/eLife.56054

Figure Lengend Snippet:

Article Snippet: Genetic reagent (adeno-associated virus) , AAV-Cas9 , PMIDs: 25326897 30792150 , NIDA IRP Core Facility, AAV1, pX551 , RRID: Addgene_60957 , Lot# AAV692.

Techniques: Sequencing, Amplification, Clone Assay, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail

doi: 10.1073/pnas.1903230116

Figure Lengend Snippet: Generation of targeted gene knockout quail using adenovirus. (A) Schematic representation of research plan. gRNA of the MLPH gene was selected from exon 2, and the adenoviral CRISPR/Cas9 vector was constructed. Recombinant adenovirus was subsequently produced and injected into the quail blastoderm. Chimeras (G0) were maintained and mated with wild-type (WT) quail to produce G1 quail with a heterozygous genotype of the MLPH gene (MLPH+/−). Male and female G1 MLPH+/− quail were subsequently mated to generate G2 quail with a homozygous genotype of the MLPH gene (MLPH−/−). After production of the recombinant adenovirus, it took a total of 10 wk, 2 wk for egg incubation after virus injection and another 8 wk for sexual maturation, to receive eggs from G0 quail. After G1 quail hatched, the MLPH+/− quail were screened and mated to produce G2 offspring 8 wk later. Fully grown G2 offspring were obtained for further experiments 26 wk after the injection of the adenovirus. (B) Sanger sequencing chromatograms of G1 MLPH+/− quail. Dashed lines indicate the starting point of the mutation, and the deleted or inserted nucleotides are underlined. The PAM sequences are highlighted in gray. Nucleotide sequences are presented in a negative direction.

Article Snippet: We acknowledge the Addgene company and Dr. Andrea Ventura for supplying us with the Adeno Cas9 (#64072) plasmid.

Techniques: Gene Knockout, CRISPR, Plasmid Preparation, Construct, Recombinant, Produced, Injection, Incubation, Sequencing, Mutagenesis

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

doi: 10.1016/j.omtm.2019.01.003

Figure Lengend Snippet: Recombinant HBoV1 Vector Production and Analysis of Packaging Capacity (A) Plasmids for rAAV/HBoV1 production using either the 4-plasmid (constructs 1–4) or 3-plasmid (constructs 1, 2+3, and 4) transfection protocols. In both systems, the plasmids are co-transfected into HEK293T cells, which are then harvested 72 h later. To release the viral particles, cells are subjected to five freeze-thaw cycles, before free plasmid DNA is digested with benzonase. The resulting crude lysate is purified using iodixanol gradient centrifugation, and the vector-containing 40% phase is collected. (B) Purification of scAAV-YFP/HBoV1 via iodixanol gradient centrifugation. Shown is the distribution of benzonase-resistant particles in the different indicated iodixanol fractions. Data are mean (±SD) genome copies per milliliter (n = 3), as determined by TaqMan RT-PCR. (C) Production of scAAV-YFP/HBoV1 using the 3- or 4-plasmid transfection protocols. Data are mean (±SD) genome copies per milliliter (n = 3), as determined by TaqMan real-time PCR. (D) Oversized ssAAV-CRISPR constructs used in this work. Sp Cas9 and gRNA cassettes are expressed from different RNA polymerase II (Pol II) (first column) or Pol III (second column) promoters, respectively. Total genome sizes are shown in the third column. (E) Southern blot analysis of the ssAAV-CRISPR genomes from (D), which were packaged into and isolated from HBoV1 particles and then resolved on an alkaline agarose gel. The number above each lane indicates the size of the packaged genome. AAV vector genomes were detected with a probe against Sp Cas9. (F) Low-molecular-weight (Hirt) extracts of the indicated constructs followed by Southern blot analysis. Brackets indicate monomeric (M) and dimeric (D) AAV replicative forms. (G) Oversized scAAV genomes used in this work. Stuffer sequences from lacZ with the indicated lengths (first column) were inserted to increase the total genome size (second column). (H) Southern blot analysis of the scAAV-YFP genomes from (G), which were packaged into and isolated from HBoV1 particles and then resolved on an alkaline agarose gel. The number above each lane indicates the size of the packaged genome. AAV vector genomes were labeled with a probe against yfp . (I) Low-molecular-weight (Hirt) extracts of the indicated constructs followed by Southern blot analysis. Brackets indicate monomeric (M) and dimeric (D) AAV replicative forms. ss, single-stranded; sc, self-complementary.

Article Snippet: This variant was now replaced with the Sp Cas9 cDNA from the Zhang lab, by PCR-amplifying this cDNA from former Addgene plasmid 49535 (in the meantime replaced by Addgene by plasmid 52961) using primers 46 and 47 and by cloning the NheI/ClaI-digested PCR product into our appropriately cleaved original AAV/ Sp Cas9 plasmid, resulting in pAAV-FZ Sp Cas9.

Techniques: Recombinant, Plasmid Preparation, Construct, Transfection, Purification, Gradient Centrifugation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, CRISPR, Southern Blot, Isolation, Agarose Gel Electrophoresis, Molecular Weight, Labeling