Journal: BioTechniques

Article Title: CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning.

doi: 10.2144/000114261

Figure Lengend Snippet: Figure 2. Cas9/sgRNA digestion specificity. (A) Cas9/T3gRNA digestion of plasmids A1, B1, and C1. The Cas9/T3gRNA digestion was 1 h for A1, and 72 h for B1 and C1. When Cas9/T3gRNA digestion was combined with restriction digestion with PvuI, the expected bands were produced for each plasmid (red arrows). The red X denotes a band not cleaved by Cas9/T3gRNA. (B) Cas9/sgRNA digestion requires the presence of the target sequence corresponding to the guide sequence of the sgRNA. The three positive clones (G5–G7) and two negative clones (Q1 and Q2) obtained from the CRISPR/Gibson cloning were digested with the Cas9/T3gRNA and PvuI. A positive clone has the insert, but the target sequence has been deleted, while a negative clone does not have the insert but still has the target sequence. (C) Cas9/LAgRNA only cleaves a vector containing a target sequence with a perfect match to the guide sequence. The target sequence of vector D has a perfect match (m, match) with the guide sequence of LAgRNA, while vector E has a target sequence with several mismatches (mm) with the guide sequence. (D) Sequence alignment of the target sequences of plasmids A1, B1 and C1 with the 19 bp T3gRNA guide sequence. (E) Sequence alignment of the target sequences of vectors D and E that are matched (m) and mismatched (mm), respectively, with the guide se quence of LAgRNA. The PAMs including the 5´-NRG are also shown underlined. The number is the length of the corresponding sequence shown in (D) & (E).

Article Snippet: 58 | No. 4 | 2015 and the resulting clone was named pX330-LAsg. b) PCR amplification of DNA templates for synthesis of sgRNAs by in vitro transcription with T7 RNA polymerase: The template for the sgRNA (T3gRNA) targeting the T3 promoter sequence in the cloning vector A1 (pLACAGRFPTetOn; 21683 bp) was PCR amplified from pX330 using the forward primer T3gRNAF, which contained 3 parts: a T7 promoter sequence, the 19-bp guide sequence for the T3 promoter of vector A1 (position 12651–12669), and the first 20 bases of the tracrRNA sequence from the pX330 vector (16) (Addgene plasmid 42230) (Table 1).

Techniques: Produced, Plasmid Preparation, Sequencing, Clone Assay, CRISPR, Cloning

Journal: BioTechniques

Article Title: CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning.

doi: 10.2144/000114261

Figure Lengend Snippet: Figure 3. Gibson cloning with the Cas9/T3gRNA-linearized plasmid A1. (A) Restriction enzyme characterization of plasmid DNA extracted from four clones from the Gibson cloning and four clones from QC cloning. All clones shown were double digested with NheI/PspXI. A PspXI site is present in the insert but not in the vector. Clones Q1–Q4 from QC cloning are negative. Clones G5–G8 from Gibson cloning are positive, as indicated by the presence of the largest band of 9152 bp and the bottom doublet bands of 3562 bp/3334 bp. (B) Sequencing electropherograms show that the insert is correctly cloned into the vector at the 5´ end. The shaded portion is the homologous sequence in the forward PCR primer. (C) Sequencing electropherograms showing that the insert is correctly cloned into the vector at the 3´ end. The shaded portion is the homologous sequence used for the cloning.

Article Snippet: 58 | No. 4 | 2015 and the resulting clone was named pX330-LAsg. b) PCR amplification of DNA templates for synthesis of sgRNAs by in vitro transcription with T7 RNA polymerase: The template for the sgRNA (T3gRNA) targeting the T3 promoter sequence in the cloning vector A1 (pLACAGRFPTetOn; 21683 bp) was PCR amplified from pX330 using the forward primer T3gRNAF, which contained 3 parts: a T7 promoter sequence, the 19-bp guide sequence for the T3 promoter of vector A1 (position 12651–12669), and the first 20 bases of the tracrRNA sequence from the pX330 vector (16) (Addgene plasmid 42230) (Table 1).

Techniques: Cloning, Plasmid Preparation, Clone Assay, Sequencing