TABLE 1.
sgRNAs, siRNAs, and primers used in this study
| Purpose | Name | Sequence (5′–3′) |
|---|---|---|
| Gene knockouta | CD46-sgRNA1 | GGACACTCGCGGCGGCCGGG |
| CD46-sgRNA2 | GCAAATGGGACTTACGAGTT | |
| DSG2-sgRNA1 | GCAGGGCGTACGCGCGTCCC | |
| DSG2-sgRNA2 | GGGATTACAGAGCCACCTTT | |
| Gene knockdown | siCD46-1 sense | GGAGCCACCAACAUUUGAATT |
| siCD46-1 antisense | UUCAAAUGUUGGUGGCUCCTT | |
| siCD46-2 sense | GCCUGUUAUAGAGAAACAUTT | |
| siCD46-2 antisense | AUGUUUCUCUAUAACAGGCTT | |
| siDSG2-1 sense | CCUCCAGUGUUCUACCUAATT | |
| siDSG2-1 antisense | UUAGGUAGAACACUGGAGGTT | |
| siDSG2-2 sense | CCAAUUGCCAAGAUACAUUTT | |
| siDSG2-2 antisense | AAUGUAUCUUGGCAAUUGGTT | |
| siCAR sense | GCCAGAAGUUUGAGUAUCATT | |
| siCAR antisense | UGAUACUCAAACUUCUGGCTT | |
| Scramble sense | UUCUCCGAAGGUGUCACGUTT | |
| Scramble antisense | ACGUGACACGUUCGGAGAATT | |
| Genome quantification | Q-Ad55 F | TGTTTTCCACTGAATGGCATAGG |
| Q-Ad55 R | GGAAACTTCGCCATAGATTGGC | |
| Gene expression | Q-CD46 F | TGTTTGAATGCGATAAGGGT |
| Q-CD46 R | TGAGACTGGAGGCTTGTAAG | |
| Q-DSG2 F | CTACACCCATTCCCATCAAGGTC | |
| Q-DSG2 R | GCTTGATCTATCCATGCTCTCG | |
| Q-βactin F | GACCCTGAAGTACCCCATTGA | |
| Q-βactin R | CTCAAACATGATCTGGGTCATCT | |
| CD46 F1 | GCCTCCAAGTCTTGACAGTAGATTA | |
| CD46 R1 | GCTTCTGTACACTGCCAGACATG | |
| CD46 R2 | CTATTGGCGTTACTATGGGAACA | |
| Transgenic mouse genotypingb | DSG2 F1 | GTTATCAGTAAGGGAGCTGCAGTG |
| DSG2 R1 | CTGCTTACATAGTCTAACTCGCGAC | |
| DSG2 R2 | GGCAGTTTACCGTAAATACTCCAC | |
| CAG F2 | CTGGTTATTGTGCTGTCTCATCAT | |
| DSG2 R3 | CTGGTAACATTCAGTTCTCCAGTATC | |
| CD46 R3 | GTGCATCTGATAACCAAACTCGTA |
a
The target sequences of sgRNAs for hCD46 and hDSG2 gene are shown. The sense and antisense DNAs corresponding to these targets were synthesized.
b
For mouse genotyping, two sets of primers for both hCD46 cassette and hDSG2 cassette were designed. F1-R1/R2 pairs were used to distinguish homozygous, heterozygous, and nontransgenic mice. CAG F2-R3 pairs were used to confirm the insertion of the cassette in mouse genome and validate the deletion of loxP-poly(A)-loxP sequence.