Figure 4

Screening single-cell clones harboring homozygous frameshift mutant from pooled mammalian cells with SWS. (a, b) Analysis of sequencing graph of a single clone from sgRNA1- treated cells (clones 1–9). In the sequencing graph, a PSAT1-CF ([A/G]G[A/C][C/A]G[A/C]AG) 6-bp downstream of CUIS were used for searching (a). Two corresponding PSAT1-CF′ (PSAT1-CF′1 GGCCGCAG; PSAT1-CF′2 AGAAGAAG) were identified in PSAT1 wild-type sequence and the distance between each PSAT1-CF′ and CUIS were 4 bp (n1) and 10 bp (n2) (b). The deduced number of indels in one allele was n–n1=6–4=2 bp; the one in the other allele was was n–n2=6–10=−4 bp. (c) The actual indels of each allele in clones 1–9 determined by cloning and sequencing strategy. PCR products from clones 1–9 were cloned into a blunt-end ligation vector (pZero-blunt) and the resulting single colonies were sequenced. Compared with the wild-type allele, one mutant allele had 2-bp insertion and the other had 4-bp deletion. (d, e) Analysis of sequencing graph of a single clone from sgRNA3-treated cells (clones 3–4). In the sequencing graph, a PSAT1-CF (AA[T/G][A/T]T[T/C]G[G/T][G/T][A/C]) 12- bp downstream of CUIS were used for searching (c). Two corresponding PSAT1-CF′ (PSAT1-CF′1 AAGTTTGGGA; PSAT1-CF′2 AATATCGTTC) were found in PSAT1 wild-type sequence and the distances between each PSAT1-CF′ and CUIS were 11 bp (n1) and 26 bp (n2) (d). The deduced number of indels in one allele was n–n1=12–11=1 bp; the one in the other allele was n–n2=12–26=−14 bp. (f) The actual indels of each allele in clones 3–4 determined by cloning and sequencing strategy. PCR products from clones 3–4 were cloned into pZero-blunt and the resulting single colonies were sequenced. Compared with the wild-type allele, one mutant allele had 1-bp insertion and the other one had 14-bp deletion.