Journal: Viruses

Article Title: CRISPR/Cas9-Driven Engineering of AcMNPV Using Dual gRNA for Optimized Recombinant Protein Production

doi: 10.3390/v17081041

Figure Lengend Snippet: CRISPR dual-sgRNA knockout of Ac15 - Ac16 in Bac-eGFP/HRPc. ( A ) Schematic representation of the Ac15 - Ac16 genomic locus before and after CRISPR editing. The Ac15 and Ac16 are represented in green and purple, respectively. After deletion of the Ac15 -Ac 16 fragment, the resulting pseudogene consists of the 5′ portion of Ac15 (green) and the 3′ portion of Ac16 (purple). Gene-specific primers used for PCR are indicated by black arrows. DNA excision is marked with scissors and a triangle (▲). The hybridization positions of primers and sgRNA targets are shown in parentheses (+/−), using the ATG start codon of each ORF as the reference point. PAM sequences (red nucleotides) are highlighted. ( B ) Identification of edited clones by PCR using specific primers (Fw- Ac15 and Rv- Ac16 ). The lane labeled “ Ac-eGFP/HRPc ” corresponds to the PCR product amplified from the unedited parental virus (2033 bp). The lane labeled “ Ac-eGFP / HRPc ΔAc15-Ac16 ” corresponds to the PCR product amplified from the edited virus (749 bp). Marker: Trans 2K Plus (TransGen Biotech, Beijin, China). ( C ) Sanger sequencing chromatogram of the edited virus. Only the flanking regions of the knockout are shown. ( D ) Amino acid sequence of the truncated aberrant protein originating from the fusion of Ac15 and Ac16 . The conserved N-terminal region from Ac15 is shown in green, followed by a frameshift-derived sequence (i.e., a sequence resulting from a mutation that alters the original reading frame) leading to a premature stop codon (*). Not at scale.

Article Snippet: All were synthesized using the Engen ® sgRNA Synthesis Kit, S. pyogenes (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s protocols.

Techniques: CRISPR, Knock-Out, Hybridization, Clone Assay, Labeling, Amplification, Virus, Marker, Sequencing, Derivative Assay, Mutagenesis

Journal: Viruses

Article Title: CRISPR/Cas9-Driven Engineering of AcMNPV Using Dual gRNA for Optimized Recombinant Protein Production

doi: 10.3390/v17081041

Figure Lengend Snippet: CRISPR dual-sgRNA knockout of Ac129 - Ac131 in Bac-eGFP/HRPc. ( A ) Schematic representation of Ac129 - Ac131 genomic locus before and after CRISPR editing. The non-edited Ac128 is shown as a dark grey arrow, while Ac129 , Ac130 , and Ac131 are represented in green, light gray, and purple, respectively. After deletion of the Ac129-Ac131 fragment, the resulting pseudogene consists of the 5′ portion of Ac129 (green) and the 3′ portion of Ac131 (purple). Ac130 (light grey) is completely removed. Gene-specific primers used for PCR are indicated by black arrows. DNA excision is marked with scissors and a triangle (▲). The hybridization positions of primers and sgRNA targets are shown in parentheses (+/−), using the ATG start codon of each ORF as the reference point. PAM sequences (red nucleotides) are highlighted. ( B ) Identification of edited clones by PCR using specific primers (Fw- Ac129 and Rv- Ac131 ). The lane labeled “ Ac-eGFP/HRPc ” corresponds to the PCR product amplified from the unedited parental virus (1733 bp). The lane labeled “ Ac-eGFP/HRPc ΔAc129-Ac131 ” corresponds to the PCR product amplified from the edited virus (569 bp). Marker: Trans 2K Plus (TransGen Biotech). ( C ) Sanger sequencing chromatogram of the edited virus. Only the flanking regions of the knockout are shown. A single nucleotide insertion (T) at the fusion is indicated with a square. ( D ) Amino acid sequence of the truncated aberrant protein originating from the junction of Ac129 and Ac131 . The conserved N-terminal region from Ac129 is shown in green, followed by a frameshift-derived sequence that leads to a premature stop codon (*). Not at scale.

Article Snippet: All were synthesized using the Engen ® sgRNA Synthesis Kit, S. pyogenes (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s protocols.

Techniques: CRISPR, Knock-Out, Hybridization, Clone Assay, Labeling, Amplification, Virus, Marker, Sequencing, Derivative Assay

Journal: Viruses

Article Title: CRISPR/Cas9-Driven Engineering of AcMNPV Using Dual gRNA for Optimized Recombinant Protein Production

doi: 10.3390/v17081041

Figure Lengend Snippet: CRISPR dual-sgRNA knockout of Ac136 - Ac138 in Bac-eGFP/HRPc. ( A ) Schematic representation of the Ac136-Ac138 genomic locus before and after CRISPR editing. The non-edited HR5 is shown as a dark grey arrow, while Ac136 , Ac137 , and Ac138 are represented in green, light gray, and purple, respectively. After deletion of the Ac136 -Ac 138 fragment, the resulting pseudogene consists of the 5′ portion of Ac136 (green) and the 3′ portion of Ac138 (purple). Ac137 (light grey) is completely removed. Gene-specific primers used for PCR are shown as black arrows. DNA excision is marked with scissors and a triangle (▲). The hybridization positions of primers and sgRNA targets are shown in parentheses (+/−), using the ATG start codon of each ORF as the reference point. PAM sequences (red nucleotides) are highlighted. ( B ) Identification of edited clones by PCR using specific primers (Fw- Ac136 and Rv- Ac138 ). The lane labeled “ Ac-eGFP/HRPc ” corresponds to the PCR product amplified from the unedited parental virus (3134 bp). The lane labeled “ Ac-eGFP/HRPc ΔAc129-Ac131 ” corresponds to the PCR product amplified from the edited virus (379 bp). Marker: Trans 2K Plus (TransGen Biotech). ( C ) Sanger sequencing chromatogram of the edited virus. Only the flanking regions of the knockout are shown. A single nucleotide insertion (c) at the junction is indicated with a square. ( D ) Amino acid sequence of the truncated aberrant protein originating from the fusion of Ac13 6 and Ac138 . 1. The conserved N-terminal region from Ac129 is shown in green, followed by a frameshift-derived sequence that leads to a premature stop codon (*). 2. The conserved N-terminal region from Ac138 is shown in green, followed by a frameshift-derived sequence that leads to a premature stop codon (*). Not at scale.

Article Snippet: All were synthesized using the Engen ® sgRNA Synthesis Kit, S. pyogenes (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s protocols.

Techniques: CRISPR, Knock-Out, Hybridization, Clone Assay, Labeling, Amplification, Virus, Marker, Sequencing, Derivative Assay

Journal: Viruses

Article Title: CRISPR/Cas9-Driven Engineering of AcMNPV Using Dual gRNA for Optimized Recombinant Protein Production

doi: 10.3390/v17081041

Figure Lengend Snippet: CRISPR dual-sgRNA knockout of Ac148 -Ac 150 in Bac-eGFP/HRPc. ( A ) Schematic representation of Ac148 -Ac 150 genomic locus before and after CRISPR editing. The edited Ac148 , Ac149 , and Ac150 are represented in green, light gray and purple, respectively. After deletion of the Ac148-Ac150 fragment, the resulting pseudogene is composed of the 5′ portion of Ac148 (green) and the 3′ portion of Ac150 (purple). Ac149 (light grey) is completely removed. Gene-specific primers used for PCR are indicated by black arrows. DNA excision is marked with scissors and a triangle (▲). The hybridization positions of primers and sgRNA targets are shown in parentheses, using the ATG start codon of each ORF as the reference point. PAM sequences (red nucleotides) are highlighted. ( B ) Identification of edited clones by PCR using specific primers (Fw- Ac148 and Rv- Ac150 ). The lane labeled “ Ac-eGFP/HRPc ” corresponds to the PCR product amplified from the unedited parental virus (886 bp). The lane labeled “ Ac-eGFP/HRPc ΔAc12-Ac131 ” corresponds to the PCR product amplified from the edited mutant virus (367 bp). Marker: Trans 2K Plus (TransGen Biotech). ( C ) Sanger sequencing chromatogram of the edited virus. Only the flanking regions of the knockout are shown. Not at scale.

Article Snippet: All were synthesized using the Engen ® sgRNA Synthesis Kit, S. pyogenes (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s protocols.

Techniques: CRISPR, Knock-Out, Hybridization, Clone Assay, Labeling, Amplification, Virus, Mutagenesis, Marker, Sequencing