sgrna cloning vector  (Addgene inc)

Journal: BIO-PROTOCOL

Article Title: Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1

doi: 10.21769/bioprotoc.5108

Figure Lengend Snippet: Figure 4. AsCpf1-mediated mutation formation in hPSCs. A. Representative fluorescent micrographs of hPSCs electroporated with the corresponding constructs on the left. B. Disruption of tdTomato expression in H1-tdT cells by CAGGS promoter driven AsCpf1 with a tdT crRNA. A 4-nucleotide deletion was identified in cells that lost tdTomato expression but not in control cells. Scale bar: 100 μm. G. Knockin of hPSC and multiplex genome editing

Article Snippet: Anneal the tdT-targeting crRNA oligos and ligate to CAGGS-AsCpf1-2A-GFP-U6-crRNA-cloning vector (Addgene, #159281) based on the procedures described in section B, giving rise to the CAGGS-AsCpf12A-GFP-U6-tdT-crRNA plasmid (Addgene, #194724).

Techniques: Mutagenesis, Construct, Disruption, Expressing, Control, Knock-In, Multiplex Assay

Journal: BIO-PROTOCOL

Article Title: Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1

doi: 10.21769/bioprotoc.5108

Figure Lengend Snippet: Figure 7. AsCpf1-mediated targeting in the INS locus in hPSCs enables monitoring of INS expression. A. The design of an INS targeting crRNA. The PAM sequence is highlighted in magenta, and the guide sequence is highlighted in yellow. B. The targeting strategy for generating INS luciferase and tdTomato reporter INS- luciferase-tdT allele by knocking the 2A-luciferase-2A-tdt cassette to the 3′ end of the INS coding sequence. C. Southern blots with internal probe (left panel) and external probe (right panel) to identify clones with a single copy of the 2A-luciferase-2A-tdt cassette inserted into the INS locus. Correct clones were labeled in green. The double bands in the control samples for the external probe (right panels) correspond to two wild-type INS alleles of the H1-OCT4-GFP cells. D. Expression of tdTomato in cells during the differentiation of the INS-luciferase- tdT reporter hPSCs toward pancreatic β-like cells. E. Representative confocal micrographs of INS-luciferase- tdT hPSC differentiated β-like cells with anti-tdTomato staining (red) and anti-C-peptide staining (green). F.

Article Snippet: Anneal the tdT-targeting crRNA oligos and ligate to CAGGS-AsCpf1-2A-GFP-U6-crRNA-cloning vector (Addgene, #159281) based on the procedures described in section B, giving rise to the CAGGS-AsCpf12A-GFP-U6-tdT-crRNA plasmid (Addgene, #194724).

Techniques: Expressing, Sequencing, Luciferase, Clone Assay, Labeling, Control, Staining