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Fig. 1 | Genome Biology

Fig. 1

From: A versatile reporter system for CRISPR-mediated chromosomal rearrangements

Fig. 1

An inverted GFP reporter (iGFP) to visualize CRISPR/Cas9-mediated DNA inversion. a Schematic of iGFP. Red arrowheads indicate the Cas9 cutting sites recognized by the sgiGFP.1 and sgiGFP.2. Inversion of the GFP cassette will lead to GFP expression from the CMV promoter. PAM sequences are underlined. Red and blue color indicate sequences flanking the predicted fusion site (indicated by ‘|’). The blue sequence in the inverted plasmid will be reverse-complementary of the original sequence. b 293 T cells were co-transfected with 0.5 μg iGFP and 0.5 μg of two px330 plasmids (sgiGFP.1 + 2) and imaged 24 h later. c A PCR reaction detected inversion (primers p1 + p2) from total cellular DNA. The arrowhead indicates the expected inversion band. d Deep-sequencing identified perfect fusion and indels (insertions or deletions) at the DNA fusion sites. Purple bars in representative IGV images (two biological replicates) indicate insertions. Position indicates basepair position in the reference sequence. e Quantification of indels. VarFreq is the average of two replicates. 22 % of the reads mapped perfectly with predicted reference sequence, corresponding to precise ligation of the DNA breaks. f Two sgRNAs also induced deletion between CRISPR/Cas9 cutting sites. A PCR reaction detected deletion of the iGFP reporter (primers p1 + p3). The top bands are full length PCR products. An arrowhead indicates the expected deletion band

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