Supplementary Figure 3: Analyses of the sgRNA coverage and base-mutation efficiency in cell clones and SC embryos obtained from 4B2N1 cells with an sgRNA library. | Nature Cell Biology

Supplementary Figure 3: Analyses of the sgRNA coverage and base-mutation efficiency in cell clones and SC embryos obtained from 4B2N1 cells with an sgRNA library.

From: CRISPR–Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development

Supplementary Figure 3

a, E12.5 SC embryos from 4B2N1 cells carrying Dnd1 sgRNA library. b, Identification of sgRNA in E12.5 SC embryos by PCR. All tested SC embryos carried sgRNA transgenes. Data represent 3 independent experiments. c, Determination of the inserted sgRNA by Sanger sequencing of RCR products in b. Each sgRNA carried a specific sequence that can be recognized as a ‘barcode’ of the SC embryo. The sgRNA transcription is driven by U6 promoter. d, Top, the number of identified sgRNAs in cell clones and SC embryos from 4B2N1-DP1 cells. Bottom, the number of the sgRNAs that induced homozygous base mutations in cell clones and SC embryos determined by Sanger sequencing. e, Deep-sequencing of sgRNAs in 4B2N1-DP2 cells carrying an sgRNA library consisting of 30 sgRNAs. The horizontal axis represents the sgRNA number. n = 30 sgRNAs. f, Distribution of sgRNAs in 4B2N1-DP2 cells according to their read counts. g, SgRNA distribution in 4B2N1-DP2 cells. Out of 142 cell clones, 76.8%carried only one sgRNA. h, Similar sgRNA distribution patterns revealed by cell-clone and deep-sequencing of 4B2N1-DP2 cells. 109 cell clones with only one sgRNA covering 26 sgRNAs were analyzed. The horizontal axis represents the sgRNA number. The left vertical axis represents the number of cell clones with a specific sgRNA. The right vertical axis represents the frequency of the corresponding sgRNA through deep-sequencing. i, Summary of the mutations in tested cell clones through Sanger sequencing of sgRNA-targeted sites in cell clones with only one sgRNA from 4B2N1-DP2 cells. Other represents the cell clones without sgRNA or with 2 or more sgRNAs. j, The whole-exome sequencing data of three 4B2N1-DP2 cell clones (4B2N1-C1, C2 and C3) from showed a low rate of mosaicism at the target sites of Dnd1. The target sequences are shown with yellow shadow. The PAM sites and substituted nucleotides are shown in green and red, respectively. The expected editing windows are shown with dotted orthogon. The columns on the right indicate frequencies of mutant alleles.

Back to article page