Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (6): 181-188.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1251
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ZHANG Zu-lin1,2(), LIU Fang-fang1,2, ZHOU Qing-niao1,2, ZHAO Rui-qiang1,2, HE Shu-jia1,2, LIN Wen-zhen1,2()
Received:
2022-10-11
Online:
2023-06-26
Published:
2023-07-07
Contact:
LIN Wen-zhen
E-mail:1343381135@qq.com;linwenzhen@msn.com
ZHANG Zu-lin, LIU Fang-fang, ZHOU Qing-niao, ZHAO Rui-qiang, HE Shu-jia, LIN Wen-zhen. Construction and Identification of Huh7 Hepatoma Cell Line with ACE2 Gene Knockout Based on CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2023, 39(6): 181-188.
名称Primer name | 引物序列Primer sequence(5'-3') |
---|---|
U6-F | ATGGACTATCATATGCTTACCGTA |
CMV-R | TCGTTGGGCGGTCAGC |
验证-F Verification-F | GACGACTTCCTGACAGCTCATCAT |
验证-R Verification-R | AGTGTCACCTGACCCAAGGAAAT |
Table 1 Sequencing primers and PCR sequences
名称Primer name | 引物序列Primer sequence(5'-3') |
---|---|
U6-F | ATGGACTATCATATGCTTACCGTA |
CMV-R | TCGTTGGGCGGTCAGC |
验证-F Verification-F | GACGACTTCCTGACAGCTCATCAT |
验证-R Verification-R | AGTGTCACCTGACCCAAGGAAAT |
Fig. 1 Identified ACE2 domain A: ACE2 domain identified by NCBI CD-search, with major domains marked in red boxes. B: The Esembl database searches for the protein domain encoded by the largest transcript of ACE2. Exons 9 and 10 and their domain are marked in red boxes
sgRNA | 序列Sequence(5'-3') |
---|---|
sgRNA1F | CACCGATCGGGTGACAGAAGACCAA |
sgRNA1R | AAACTTGGTCTTCTGTCACCCGATC |
sgRNA2F | CACCGTCGTGAGTGCTTGTTTGAGC |
sgRNA2R | AAACGCTCAAACAAGCACTCACGAC |
Table 2 Sequences of two sgRNAs targeting ACE2 gene
sgRNA | 序列Sequence(5'-3') |
---|---|
sgRNA1F | CACCGATCGGGTGACAGAAGACCAA |
sgRNA1R | AAACTTGGTCTTCTGTCACCCGATC |
sgRNA2F | CACCGTCGTGAGTGCTTGTTTGAGC |
sgRNA2R | AAACGCTCAAACAAGCACTCACGAC |
Fig. 2 Specificity score and off-target analysis of two sgRNA oligonucleotide fragments A: sgRNA1 specific score and off-target analysis; B: sgRNA2 specific score and off-target analysis
Fig. 5 Agarose gel electrophoresis to verify the effect of gene editing M: DL10000 marker. Lane 1-5 are Huh7 wild-type; PXNC(control group); PX1 #, PX2 #, PX3#(knockout group)
Fig. 6 Results of gene editing A: PXNC(control group)gene fragment. B: PX2#(knockout group), gene fragment deletion after sgRNA acts on specific exons, which is connected to form repaired DNA fragment by non-homologous recombination. C: Blast comparison of PX2# monoclonal cell line and PXNC sequencing results showed that some exons and introns were missing, and the edited PX2# gene sequence was shown in red. Compared with PXNC, the PX2# gene with fragment knockout was fragmented
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