Extended Data Fig. 5: Genomically rewriting mouse Ace2 with human ACE2.
From: Mouse genome rewriting and tailoring of three important disease loci
(a) Schematic workflow. (b) Restriction enzyme digestion verification of the 116 kb-hACE2 payload. Digestion products were separated using low-melting-point agarose gel by pulse field gel electrophoresis (see methods). (c) 180 kb-hACE2 payload assembly. Scissors mark in vitro CRISPR-Cas9 digestion sites. mHA, mouse homology arm. YAV, yeast assembly vector. (d) Sequencing coverage of two human BACs and the two hACE2 payloads mapped to hg38. Black bars represent single nucleotide polymorphisms (SNPs). (e) Marker cassette 1 insertion to the downstream of mouse Ace2. Integration was confirmed by junction PCR. HA-L, left homology arm, HA-R, right homology arm. L, left junction assay with primers oWZ1502 and oWZ920. R, right junction assay with primers oWZ211 and oWZ1503. F, full MC1 amplification with primers oWZ1502 and oWZ1503. (f) Genotyping PCR analysis of 116 kb- hACE2 and 180 kb-hACE2 mSwAP-In clones. Double headed arrows mark PCR amplicons from either mAce2 (assays 1–6) or hACE2 payloads (assays 7–16). (g) Sequencing coverage of Cas9 in 116 kb- hACE2 and 180 kb-hACE2 mESCs.
