Fig. 2: Chemical synthesis and assembly of hAZFa in yeast.
From: De novo assembly and delivery of synthetic megabase-scale human DNA into mouse early embryos
a, The AZFa locus and UCSC database gene annotations are indicated. The hAZFa was designed to split into four large fragments—SynA, SynG, SynB and SynC—with two HERV15 block deletions. b, The vertical blue lines indicate the position of repetitive elements, including STRs, LTRs, LINEs and SINEs. c, Circos plot showing the assembly of hAZFa. Twelve 28-bp watermark sequences were inserted into the intron region of hAZFa. WM(n), watermark(n). d, The recombination-based combinatorial assembly strategy for Mb-scale synthetic human DNA with highly repetitive sequences. e, Restriction digestion analysis of hAZFa followed by PFGE. Yeast_hAZFa: S. cerevisiae VL6-48 containing hAZFa; Yeast_Control: S. cerevisiae VL6-48 harboring pRS416 as a control. Left, AscI enzyme digestion, with VL6-48 genome as a marker. Right, BsiWI enzyme digestion, with λ PFG ladder as a marker. n = 3 biological replicates. f, Growth assay of Yeast_hAZFa compared to Yeast_Control. g, Transcriptome analysis of Yeast_hAZFa compared to Yeast_Control. A two-sided adjusted P value (q value) threshold of 0.001 (Wald test with Benjamini–Hochberg correction) and a |log2(FC)| > 1.0 were used. h, The length of isoforms on hAZFa compared to yeast endogenous genome. i, Proteomic analysis of Yeast_hAZFa compared to Yeast_Control. Differentially expressed genes were identified using a two-sided P value threshold of 0.001 (Welch’s t-test) with FC > 1.2 or FC < 0.83. j, Genome-wide Hi-C contact map of the Yeast_hAZFa chromosome at a 10-kb resolution (left). The Hi-C interaction heatmap of hAZFa is magnified on the right. FC, fold change; LINE, long interspersed nuclear element; LTR, long terminal repeat; SINE, short interspersed nuclear element; STR, short terminal repeat; PFG, pulsed-field gel.
