Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene

Figures

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  Figure 1. Binding sites of E1A-targeting gRNAs The E1A gene serving as the target for CRISPR-Cas9-mediated cleavage of the viral DNA is shown in detail. The gRNAs binding to E1A are indicated by arrows numbered 1–10. The orientation of the arrows indicates binding to the plus and minus strand, respectively. The binding sites within the two most important E1A isoforms (243R and 289R) are depicted.

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  Figure 2. Structure of the recombinant adenovirus vector constructs and gRNA sequences incorporated into them (A) Schematic of HAdV-5-based vectors with inserted individual gRNA sequences. All adenoviral vectors are based on the HAdV-5-derived vector pAd/PL-DEST (Thermo Fisher Scientific) and lack the E1 and E3 regions. Cas9 and gRNA expression cassettes were inserted into the deleted E1 region. The expression of spCas9-HF1 is driven by a tetracycline repressor-controlled CMV promoter comprising two binding sites for the repressor (2×TetO2). The expression of the individual targeting or non-targeting gRNAs is under control of a constitutive human U6 (hU6) promoter. The structure and sequence of the gRNAs are exemplarily shown for E1A gRNA 9 bound to its target site. The control vector containing only the Cas9 expression cassette is also depicted. (B) Target sequences for the individual gRNAs and their positions within the HAdV-5 genome (AY339865.1).

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  Figure 3. Detection of CRISPR-Cas9-mediated gene editing in T7 endonuclease mismatch assays and surrogate reporter assays (A) HeLa cells were transduced with the adenoviral CRISPR-Cas9 expression vectors containing either Cas9 alone or Cas9 in combination with the individual targeting gRNAs or a non-targeting (NT) gRNA at an MOI of 100, followed by infection with HAdV-5 at an MOI of 0.01 24 h later. Four days post infection, DNA was isolated, and the target region comprising all target sites was amplified by PCR. The amplicon DNA was heat denatured, re-annealed to form heteroduplex DNA, subjected to T7 endonuclease I treatment, and analyzed by agarose gel electrophoresis. The uncleaved DNA band is indicated with an arrow. Cleavage products indicating heteroduplex DNA formed because of insertion of mutations by CRISPR-Cas9 at the specific target sites are indicated with asterisks. (B) Schematic of the EGFP surrogate reporter vectors and of the methodology. A fusion protein of RFP (red) and two out-of-frame EGFP copies (black), each in a different reading frame, are expressed from a CMV promoter. The E1A region comprising the individual target sites was inserted into the linker region…

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  Figure 4. E1A-targeting gRNAs significantly decrease the numbers of infectious viral particles and HAdV-5 genome copy numbers (A) HeLa cells were transduced with adenoviral vectors containing either Cas9 alone or Cas9 in combination with targeting gRNAs 8 or 9 or with an NT gRNA at an MOI of 100. 24 h after transduction, cells were infected with HAdV-5 at an MOI of 0.01. Numbers of infectious virus particles were determined on day 2 post infection and were expressed as infectious units (IFUs) per milliliter. Data represent the means (n = 3) ± SD of triplicate infections of a representative experiment of 3. ∗p < 0.05. (B) HeLa cells were transduced and infected as in (A) and grown for a prolonged period of time. Numbers of infectious virus particles were determined at time points 0, 2, 4, and 6 days post infection. Data represent the means (n = 3) ± SD of triplicate infections of a representative experiment of 3. ∗∗∗∗p < 0.0001. (C) Same experimental setup as in (A) with the difference that HAdV-5 genome copy numbers were determined by qPCR. Data represent the means (n = 3) ± SD of triplicate infections of a representative experiment of 3. ∗p < 0.05.

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  Figure 5. Low doses of CDV increase inhibition of HAdV-5 replication by CRISPR-Cas9 (A and B) HeLa cells were transduced with the adenoviral CRISPR-Cas9 expression vectors containing either Cas9 alone or Cas9 in combination with E1A gRNA 9 or an NT gRNA at an MOI of 100, followed by infection with HAdV-5 at an MOI of 0.01 24 h later. Concomitant with infection, the cultures were treated with or without CDV at a concentration of 30 μM (A) and 10 μM (B), respectively. Numbers of infectious HAdV-5 particles were determined on days 0, 2, 4, and 6 post infection and expressed as IFUs per milliliter. Data represent the means (n = 3) ± SD of triplicate infections of a representative experiment of 3. ∗∗∗∗p < 0.0001.

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  Figure 6. Targeting E1A with 4 different gRNAs potently inhibits HAdV-5 replication (A) Schematic of the HAdV-5-based CRISPR-Cas9 vectors for multiplex gRNA expression. The sequences of gRNAs 1, 7, 8, and 9 were expressed from individual constitutive RNA polymerase III promoters (mU6, h7SK, hH1, and hU6). An analogous vector containing 4 identical NT gRNAs instead of the targeting gRNAs was constructed as well. SpCas9-HF1 was expressed from a doxycycline-regulatable CMV promoter harboring two TetO2 binding sites for the tetracycline repressor. (B) HeLa cells were transduced with the adenoviral vector containing Cas9 in combination with gRNAs 1, 7, 8, and 9 or with a control vector carrying 4 NTNT gRNAs at an MOI of 100, followed by infection of the cells with HAdV-5 at an MOI of 0.01 24 h later. Numbers of infectious virus particles were determined at the indicated time points and expressed as IFUs per milliliter. Data represent the means (n = 3) ± SD of triplicate infections of a representative experiment of 3. ∗p < 0.05, ∗∗∗p < 0.001. (C) Experimental settings were as in (A) with the difference that HeLa cells were infected with HAdV-5 6 h prior to transduction with the vectors. Numbers…

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  Figure 7. Concatemerized gRNAs separated by NT amiRNA spacers allow potent inhibition of HAdV-5 replication (A) Schematic of the adenoviral vector for multiplex expression of concatemerized gRNAs and processing of the primary transcripts within the cell. SpCas9-HF1 expression is under control of a doxycycline-regulatable CMV promoter harboring two TetO2 binding sites for the tetracycline repressor, and multiplexed gRNAs separated by NT amiRNA are expressed from a common hU6 promoter. A negative control vector carrying 4 NT gRNAs was constructed in an analogous manner. The individual gRNAs are released from the primary transcript by DROSHA-mediated cleavage at the base of each interjacent amiRNA hairpin. The gRNA intermediates are further trimmed by Cas9 to give rise to the mature gRNAs. (B) HeLa cells were transduced with the adenoviral vectors containing Cas9 in combination with the concatemerized targeting gRNAs 1, 7, 8, and 9 or with a control vector bearing Cas9 in combination with 4 NT gRNAs at an MOI of 100. 24 h after transduction, the cells were infected with HAdV-5 at an MOI of 0.01. Numbers of infectious virus particles were determined at the indicated time points and expressed…

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  Figure 8. Targeting of E1A with 4 different gRNAs primarily leads to deletions, inversions, and duplications in E1A HeLa cells were transduced with adenoviral vectors harboring Cas9 in combination with the 4 gRNAs 1, 7, 8, and 9 expressed from separate promoters at an MOI of 100 or were mock transduced. 24 h after transduction, the cells were infected with HAdV-5 at an MOI of 0.01. Two days post infection, DNA was isolated from the cells, a large region comprising all E1A target sites was amplified by PCR, and the amplicon DNA was subjected to loop sequencing. (A) Total percentage of deletions/insertions/inversions in the target region. (B) Alignment of reads around the most heavily affected region comprising the target site for gRNA 7. Deletions (del; dashed lines), inversions (inv; in red and lowercase), and duplications (dupl; in red and lowercase) are shown. The cleavage position for gRNA 7 is indicated with a vertical dashed line. The frequency of the mutations (percentage of reads mapping to the target region) are given on the right of the alignments. (C) Types of deletions, inversions, and duplications detectable in the target region. Deletions are indicated with dashed lines, and…