U6 sgrna cloning vector

BE3 fragments were synthesized by Guangzhou IGE Biotechnology Ltd. The fragments were then cloned into the pCS2 vector with the SP6 promoter and into the pCDNA3.1 ( + ) vector with the CMV promoter. The obtained vectors were named as pCS2-BE3 and pCDNA3.1-BE3. pCMV-hA3A-BE3 vector (#113410) and U6-sgRNA cloning vector (#48962) were purchased from Addgene. pCS2-hA3A-BE3 vector was constructed by ligating linearized pCS2 vector and hA3A-BE3 fragment. DMD-sgRNA, TYR-sgRNA, LMNA-sgRNA, pol-sgRNA, RAG1-sgRNA, RAG2-sgRNA, and IL2RG-sgRNA were designed following the G-N19-NGG rule. Two complementary oligonucleotides of sgRNAs were synthesized and then annealed to double-stranded DNAs. The annealed products were then cloned into the BbsI-digested U6-sgRNA cloning vector and obtained sgRNA-expressing plasmid. The primers used above are listed in Supplementary Data 3.

To construct pCMV-Cdx2 and pCMV-Gata6 vector, Cdx2 and Gata6 genes were amplified by RT-PCR, respectively, followed by cloning into pEGFP-N2 vector (Clontech) between EcoRI and NotI. To construct pCMV-dCAS9-VP64 vector, hCAS9 vector (#41815, Addgene, a gift from George Church) was mutated (D10A + H840A) and named dCAS9, and VP64 activation domain module was fused with the dCAS9 gene as previously described41 (link). U6-sgRNA cloning vector was also a gift from George Church (#41819, Addgene). sgRNAs were designed by GN19NGG rule and were constructed as previously described10 (link). The primer sequences for sgRNAs that guide dCas9 to Cdx2 promoter (sgCdx2P) and Gata6 promoter (sgGata6P) loci were designed as shown in Supplementary Table 1.
To generate a Cdx2-reproter system, sgCdx2 was designed as: caccGGCGGCGGCACAGCAATCCC and aaacGGGATTGCTGTGCCGCCGCC. U6-sgCdx2 was amplified and cloned into hCAS9 vector between MfeI and SpeI. Left homology arm (HA-L) and right homology arm (HA-R) around the Cdx2 stop codon site amplified from mouse ESC genome by PCR were cloned into upstream and downstream of 2A-tdTomato, as described in Supplementary Fig. 1a.