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Disruption of HPV16-E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells.

Hu Z, Yu L, Zhu D, Ding W, Wang X, Zhang C, Wang L, Jiang X, Shen H, He D, Li K, Xi L, Ma D, Wang H - Biomed Res Int (2014)

Bottom Line: By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells.Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb.Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.

ABSTRACT
High-risk human papillomavirus (HR-HPV) has been recognized as a major causative agent for cervical cancer. Upon HPV infection, early genes E6 and E7 play important roles in maintaining malignant phenotype of cervical cancer cells. By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells. Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb. Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

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Evaluation of gRNA/Cas9-mediated HPV16-E7 DNA cleavage by the SSA assay. (a) Schematic representation of luciferase assay. Inactive luciferase gene is separated by left homology arm (LH), 20 nt gRNA recognition site, corresponding PAM sequence, a stop codon, and right homology arm (RH). When the CRISPR gRNA/Cas9 system causes DSBs at specific recognition sites, an active luciferase will form due to SSA repair pathway. Cas9 plasmid, site-specific gRNA, pSSA Rep-gRNA, and renilla plasmid were cotransfected into HEK293 cells. At 48 h after transfection, the firefly luciferase (b) and renilla luciferase (c) activity were measured by a microplate reader. Cells transfected with GZF3-L3, GZF1-R3, and pSSA Rep3-1 plasmids were used as positive control and cells treated with only Cas9 plasmid were applied as negative control. Cells treated with Con-gRNA (HPV16E6-gRNA-1) which had been proved to be inactive preexperimentally and Cas9 enzyme together were used as a control. RLU represents relative light units (**P < 0.01 compared to the negative test, n = 6, per Student's t-test).
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fig2: Evaluation of gRNA/Cas9-mediated HPV16-E7 DNA cleavage by the SSA assay. (a) Schematic representation of luciferase assay. Inactive luciferase gene is separated by left homology arm (LH), 20 nt gRNA recognition site, corresponding PAM sequence, a stop codon, and right homology arm (RH). When the CRISPR gRNA/Cas9 system causes DSBs at specific recognition sites, an active luciferase will form due to SSA repair pathway. Cas9 plasmid, site-specific gRNA, pSSA Rep-gRNA, and renilla plasmid were cotransfected into HEK293 cells. At 48 h after transfection, the firefly luciferase (b) and renilla luciferase (c) activity were measured by a microplate reader. Cells transfected with GZF3-L3, GZF1-R3, and pSSA Rep3-1 plasmids were used as positive control and cells treated with only Cas9 plasmid were applied as negative control. Cells treated with Con-gRNA (HPV16E6-gRNA-1) which had been proved to be inactive preexperimentally and Cas9 enzyme together were used as a control. RLU represents relative light units (**P < 0.01 compared to the negative test, n = 6, per Student's t-test).

Mentions: Construction of the SSA luciferase reporter pSSA Rep3-1 plasmid has been described previously [11]. The pSSA Rep3-1 and control GZF3-L3 + GZF1-R3 ZFN plasmids were kindly provided by Professor David Segal. Briefly, a CRISPR sgRNA target sequence, its corresponding PAM sequence, and a stop codon were inserted into the direct repeat halves of the firefly luciferase gene pSSA Rep3-1 (Figure 2(a)), which was named as pSSA Rep-gRNA. 400 ng of each Cas9 plasmid, 100 ng of gRNA plasmid, 100 ng of pSSA Rep-gRNA, and 25 ng of pRL-TK-Renilla luciferase (Promega) were cotransfected into HEK293 cells in 24-well plates. At 48 h after transfection, firefly luciferase and Renilla activities were determined according to the protocol of the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was monitored with a microplate luminometer (BioTek). All the experiments were repeated three times.


Disruption of HPV16-E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells.

Hu Z, Yu L, Zhu D, Ding W, Wang X, Zhang C, Wang L, Jiang X, Shen H, He D, Li K, Xi L, Ma D, Wang H - Biomed Res Int (2014)

Evaluation of gRNA/Cas9-mediated HPV16-E7 DNA cleavage by the SSA assay. (a) Schematic representation of luciferase assay. Inactive luciferase gene is separated by left homology arm (LH), 20 nt gRNA recognition site, corresponding PAM sequence, a stop codon, and right homology arm (RH). When the CRISPR gRNA/Cas9 system causes DSBs at specific recognition sites, an active luciferase will form due to SSA repair pathway. Cas9 plasmid, site-specific gRNA, pSSA Rep-gRNA, and renilla plasmid were cotransfected into HEK293 cells. At 48 h after transfection, the firefly luciferase (b) and renilla luciferase (c) activity were measured by a microplate reader. Cells transfected with GZF3-L3, GZF1-R3, and pSSA Rep3-1 plasmids were used as positive control and cells treated with only Cas9 plasmid were applied as negative control. Cells treated with Con-gRNA (HPV16E6-gRNA-1) which had been proved to be inactive preexperimentally and Cas9 enzyme together were used as a control. RLU represents relative light units (**P < 0.01 compared to the negative test, n = 6, per Student's t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4127252&req=5

fig2: Evaluation of gRNA/Cas9-mediated HPV16-E7 DNA cleavage by the SSA assay. (a) Schematic representation of luciferase assay. Inactive luciferase gene is separated by left homology arm (LH), 20 nt gRNA recognition site, corresponding PAM sequence, a stop codon, and right homology arm (RH). When the CRISPR gRNA/Cas9 system causes DSBs at specific recognition sites, an active luciferase will form due to SSA repair pathway. Cas9 plasmid, site-specific gRNA, pSSA Rep-gRNA, and renilla plasmid were cotransfected into HEK293 cells. At 48 h after transfection, the firefly luciferase (b) and renilla luciferase (c) activity were measured by a microplate reader. Cells transfected with GZF3-L3, GZF1-R3, and pSSA Rep3-1 plasmids were used as positive control and cells treated with only Cas9 plasmid were applied as negative control. Cells treated with Con-gRNA (HPV16E6-gRNA-1) which had been proved to be inactive preexperimentally and Cas9 enzyme together were used as a control. RLU represents relative light units (**P < 0.01 compared to the negative test, n = 6, per Student's t-test).
Mentions: Construction of the SSA luciferase reporter pSSA Rep3-1 plasmid has been described previously [11]. The pSSA Rep3-1 and control GZF3-L3 + GZF1-R3 ZFN plasmids were kindly provided by Professor David Segal. Briefly, a CRISPR sgRNA target sequence, its corresponding PAM sequence, and a stop codon were inserted into the direct repeat halves of the firefly luciferase gene pSSA Rep3-1 (Figure 2(a)), which was named as pSSA Rep-gRNA. 400 ng of each Cas9 plasmid, 100 ng of gRNA plasmid, 100 ng of pSSA Rep-gRNA, and 25 ng of pRL-TK-Renilla luciferase (Promega) were cotransfected into HEK293 cells in 24-well plates. At 48 h after transfection, firefly luciferase and Renilla activities were determined according to the protocol of the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was monitored with a microplate luminometer (BioTek). All the experiments were repeated three times.

Bottom Line: By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells.Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb.Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.

ABSTRACT
High-risk human papillomavirus (HR-HPV) has been recognized as a major causative agent for cervical cancer. Upon HPV infection, early genes E6 and E7 play important roles in maintaining malignant phenotype of cervical cancer cells. By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells. Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb. Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

Show MeSH
Related in: MedlinePlus