c Number of exonic SNVs (SNVs are located on exons and splicing sites) or other SNVs detected in highly modified clones, as compared to the sequence of the parental HEK293T. NC, negative control, HEK293T-BE4max stable cell clone 19 from method_2, clone 21 from method_3 clone 19-1, 19-16, 19-21 from second transfection by method_2. b Heatmap of target “C” editing efficiency for converting TAG to TAA. wt-no allele editing hz (heterozygous) – partial allele editing hm(homozygous) - all allele editing. In b, c, e, and f, dots and triangle represent individual biological replicates ( n = 3 independent experiments) and bars represent mean values ± s.d.Ī Allele editing of all target sites in each clone by Sanger sequencing and EditR. Frequency (%) of C-to-T conversion in two stable HEK293T cell lines (FNLS-BE3-NG ( e) and evoAPOBEC1-BE4max-NG ( f)) transduced with gBlock-PC and gBlock-YC1 separately. APOBAEC1, rat APOBEC1 evoAPOBAEC1, evolved rat APOBEC1. F Flag tag, NLS Nuclear localization signal, Cas9n-NG, Cas9D10A with recognizing NG PAM. d Schematic diagram of dox-inducible cytidine deaminase piggyBac construct. Frequency (%) of C-to-T conversion was obtained by Sanger sequencing and editR analysis. Co-transfected with gblock-PC ( b) and gBlock-YC1 ( c) with evoAPOBEC-BE4max-NG into HEK293T cells separately. gBlock-PC carries published five sgRNAs targeting at five endogenous loci (HEK2, HEK3, HEK4, EMX1, RNF2) and gBlock-YC1 carries 5 sgRNAs targeting TAG of five genes loci (ORC3-1, ORC3-2, PTPA, PMSD13, NOP2-1). Schematic diagram of gBlock-PC and gBlock-YC1. 9 Wyss Institute for Biologically Inspired Engineering, Boston, MA, 02115, USA. 8 Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA. 7 CAS Key Laboratory of Quantitative Engineering Biology, Center for Genome Engineering and Therapy, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.1 Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA. 6 Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215, USA. 5 Wyss Institute for Biologically Inspired Engineering, Boston, MA, 02115, USA. 4 Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA.
3 CAS Key Laboratory of Quantitative Engineering Biology, Center for Genome Engineering and Therapy, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.2 Wyss Institute for Biologically Inspired Engineering, Boston, MA, 02115, USA.
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