(A) The locations of primer sets for PCR are presented on
DNA with respect to the target sequences of sgRNA. The target sequences
are 20 nts in length, and ‘NGG’ nucleotides acting as PAMs
are numbered as ‘+1’, ‘+2’, and
‘+3’. One primer in A2 amplicon (red arrows) locates on
the target sequences, and A2R-1 primer is on next to the target
sequences. A1 (blue arrows) and A3 primers (green arrows) locate in both
flanking regions of the target sequences. A4F/R (yellow arrows)
primers amplify large region including A1, A2, and A3 amplicons.
(B) Genomic DNA from clones targetted by the
CRISPR/spCas9 system, was amplified by qPCR using the three
primer sets, A1, A2, and A3. PCR product amounts were compared using IVR
amplicon for intervening region between the LCR and
ε-globin gene as an internal control and
then normalized compared with amounts in wild-type (Wt) MEL/ch11
cells. (C) In HS3ΔKL clones, PCR products amplified
by A1F/R and A2F/R-1 primers were visualized on an agarose
gel, M is the DNA marker. (D) DNA sequences in the LCR HS3
are presented for Wt cells and clones with GATA-1, TAL1, or KLF1 motif
deletions. The binding motifs of transcription factors are marked by
colored bases as described in Figure
1. Black dashes represent deleted nucleotides and yellow
bases are inserted nucleotides. (E) Genomic DNA of clones
was amplified and quantitated as described above. PCR products amplified
by A4F/R primers were visualized in an agarose gel.