Journal of Animal Reproduction and Biotechnology 2023; 38(3): 99-108
Published online September 30, 2023
https://doi.org/10.12750/JARB.38.3.99
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Ga-Yeon Kim and Man-Jong Kang*
Department of Animal Science, College of Agriculture and Life Science, Chonnam National University, Gwangju 61186, Korea
Correspondence to: Man-Jong Kang
E-mail: mjkang@jnu.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Efficient gene editing technology is needed for successful knock-in. Homologous recombination (HR) is a major double-strand break repair pathway that can be utilized for accurately inserting foreign genes into the genome. HR occurs during the S/G2 phase, and the DNA mismatch repair (MMR) pathway is inextricably linked to HR to maintain HR fidelity. This study was conducted to investigate the effect of inhibiting MMR-related genes using CdCl2, an MMR-related gene inhibitor, on HR efficiency in HC11 cells.
Methods: The mRNA and protein expression levels of MMR-related genes (Msh2, Msh3, Msh6, Mlh1, Pms2), the HR-related gene Rad51, and the NHEJ-related gene DNA Ligase IV were assessed in HC11 cells treated with 10 μM of CdCl2 for 48 hours. In addition, HC11 cells were transfected with a CRISPR/sgRNA expression vector and a knock-in vector targeting Exon3 of the mouse-beta casein locus, and treated with 10 μM cadmium for 48 hours. The knock-in efficiency was monitored through PCR.
Results: The treatment of HC11 cells with a high-dose of CdCl2 decreased the mRNA expression of the HR-related gene Rad51 in HC11 cells. In addition, the inhibition of MMR-related genes through CdCl2 treatment did not lead to an increase in knock-in efficiency.
Conclusions: The inhibition of MMR-related gene expression through high-dose CdCl2 treatment reduces the expression of the HR-related gene Rad51, which is active during recombination. Therefore, it was determined that CdCl2 is an inappropriate compound for improving HR efficiency.
Keywords: cadmium chloride, CRISPR-Cas9 mediated knock-in efficiency, DNA mismatch repair, homologous recombination, non-homologous end joining
Gene targeting is a useful technique for inserting foreign genes into a specific locus in the genome. The major pathways of double-strand break (DSB) repair are homologous recombination (HR) and non-homologous end joining (NHEJ). The HR pathway is utilized to accurately insert foreign genes into the genome, but it occurs only during the S/G2 phase, and its efficiency for DSB repair is lower than that of NHEJ (Vasquez et al., 2001; Mao et al., 2008). Thus, studies have been performed to improve the efficiency of HR for accurate gene targeting. The HR-related gene
The DNA mismatch repair (MMR) is known to be inextricably linked to HR (Spies and Fishel, 2015), and is reported as a post-replication repair pathway that occurs during replication or recombination (Li, 2008). The MMR-related MutS α (MSH2-MSH6) and MutS β (MSH2-MSH3) proteins bind to a mismatched DNA base pair, and MutL α (MLH1-PMS2) is an endonuclease that binds to MutS-heteroduplex complexes (Lahue et al., 1989; Habraken et al., 1996). The MMR maintains HR fidelity and prevents recombination between divergent sequences through heteroduplex rejection in the presence of extensive heteroduplexes during the recombination process (Goldfarb and Alani, 2005; Kunkel and Erie, 2015; Chakaraborty and Alani, 2016).
CdCl2 treatment inhibits ATP hydrolysis of MutS α (MSH2-MSH6) in eukaryotic cells and specific binding to mismatched DNA (Clark and Kunkel, 2004). Meanwhile, it has been reported that knock-out of
To address the conflicting reports, we aimed to determine the change in knock-in efficiency when MMR is suppressed through high-dose CdCl2 treatment (10 μM), an MMR inhibitor, in HC11 cells. Furthermore, to assess the mechanism of HR inhibition by CdCl2 treatment, we assessed the expression of MMR-related genes, the HR-related gene
The knock-in vector used in this study expresses hEPO protein using the GST fusion system at the mouse beta-casein Exon3 locus. The knock-in vector, which consists of a 5’ homology arm (1.024 kb), GST-PreScission protease-hEPO, SV40polyA, CMV-EGFP, and a 3’ homology arm (1.81 kb), was originally developed in our laboratory. The vector was modified in this study; CMV-EGFP, a positive selection marker, was replaced with PGK neo, a negative selection marker, and the 3’ homology arm of 1.81 kb was modified to a 3’ homology arm of 1.015 kb. As a result, the final knock-in vector was constructed as PBSK_1.024kb mBC5’arm_GST_hEPO_SV40polyA_PGK neo_1.015kb mBC3’arm.
Mouse mammary epithelial cells (HC11) were cultured in the growth medium consisting of Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone), 1% penicillin/streptomycin (Hyclone), 0.1% gentamicin (Sigma, St. Louis, MO, USA), 0.1% EGF (Gibco, Thermo Fisher, USA), and 0.05% insulin (Sigma). HC11 cells were seeded at a density of 2.4 × 105 cells per well into 6-well plates (SPL, Gyeonggi-do, Korea) and incubated at 37℃ with 5% CO2. The medium was replaced with 1 mL of fresh medium before transfection. The knock-in vector (3.75 μg) and pGuide it Zs Green1_sgRNA expression vector (1.875 μg) were transfected into HC11 cells using the Xfect transfection reagent (Takara, Tokyo, Japan). After 24 hours of transfection, the cells were treated with 10 μM CdCl2 and incubated for 48 hours.
Total RNA from HC11 cells was isolated using an RNeasy mini kit (QIAGEN, Hilden, Germany). A total of 3 μg of RNA was used as a template for cDNA synthesis, which was performed using random hexamers and M-MLV reverse transcriptase (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. Quantitative PCR was performed to measure the mRNA expression of DNA MMR-related genes (
Table 1 . Primers used for GST_hEPO screening PCR analysis and quantitative PCR analysis of specific genes
Gene | Primer name | Sequence (5’ to 3’) |
---|---|---|
Primers used for GST_hEPO screening PCR analysis | ||
Mouse β-casein 5’ arm | m C5 CV S5 β | ATAAGAACTGCAGTGGGATTCTTC |
m C3 GST AS9 β | AGCATTGAAATCTCTGCACGCTC | |
m C5 CV S6 β | GTCTACCAATTCACTCTAGAAGTG | |
m C3 GST AS7 β | ACGTATGATGGCCATAGACTGTG | |
Primers used for quantitative real-time PCR analysis of MMR-related gene, | ||
m | CGACCTGGAAGTCCAACTA | |
m | ATCTGCTGCATCTGCTTG | |
MMR-related gene ( | m | TGAAGTTGGACATGGCAGCA |
m | TAATGCGGCCAGAGACTGAG | |
m | CCATCGCCTATGCAACTCTA | |
m | TCACAGACTGGTGGATAGTG | |
m | ATACTCAGGCATGCAACAGC | |
m | TCAAAAGTTGCGGTGCCTCT | |
m | AGATTAGTGAGCGGTGCCAT | |
m | ACTGAGGATTCACACAGCCC | |
m | ACTTCCAGGACGCCACAAAA | |
m | AACTGCCTGTCTGTTGCACT | |
HR-related gene | AATTCCGAACTGGGAAGACAC | |
TCACCTCCACCACGGTCAAT | ||
NHEJ-related gene | qPCR | TCCACAGGAAGGCTCTCTCA |
DNA | qPCR | TCTCACCGTCAAGGATGCAC |
Nuclear and cytoplasmic proteins from HC11 cells were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Total protein from HC11 cells was extracted using the PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. The nuclear and cytoplasmic protein extracts (15 μg) and total protein (30 μg) derived from the cells were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes (Bio-Rad Co., Hercules, CA, USA). The membranes were then blocked for 2 hours at room temperature in 5% skim milk in TBST (Tris-buffered saline with 0.1% Tween 20), and blotted with a rabbit polyclonal anti-MSH2 antibody (ab-70270, Abcam, Cambridge, UK; dilution 1:2,000), mouse monoclonal anti-RAD51 (sc398587) and anti-DNA Ligase
The knock-in efficiency of the hEPO knock-in vector in HC11 cells was confirmed through first and second PCR experiments. The genomic DNA isolated from cells in each group was used as a DNA template for PCR. The first PCR was performed using the mβC5 CV S5 primer for regions outside the 5’ arm and the mβC3 GST9 (Table 1) for the GST region using the amplification reagent SolgTM 2X Multiplex (Solgent, Daejeon, South Korea). The PCR consisted 25 cycles of amplification at 95℃ for 20 seconds, annealing at 62℃ for 40 seconds, and strand extension at 72℃ for 2 minutes. Subsequently, a nested PCR was performed using the mβC5 CV primer and the mβC3 GST AS7 primer using the amplification regent KOD FX Neo (Toyobo, Osaka, Japan) through the following steps: 25 cycles of denaturation at 95℃ for 20 seconds, annealing at 64℃ for 30 seconds, and strand extension at 72℃ for 2 minutes; followed by a final extension at 72℃ for 5 min. The PCR fragments were confirmed through electrophoresis on a 0.8% agarose gel. For comparative quantification, each DNA band was normalized to the mβCE7 region.
Densitometric quantification of DNA or protein bands was performed by analyzing the data using UN-SCAN-IT gel Analysis Software (Silk Scientific Inc., Orem, UT, USA). Subsequently, a statistical analysis of the mRNA levels and densitometric quantification of DNA and protein bands was performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). The data were analyzed in two ways. First, the data were analyzed with one-way ANOVA, followed by the Dunnett test, to compare all columns with reference to the control column. Second, a
The diagram illustrating the knock-in vector used for expressing the human erythropoietin (
To determine the effect of high-dose CdCl2 treatment (10 μM) on the mRNA expression of
In order to determine the protein levels of Rad51, DNA Ligase
To investigate the efficiency of CRISPR-Cas9-mediated knock-in, HC11 cells were exposed to high-dose CdCl2 treatment and transfected with the GST-hEPO knock-in and the CRISPR-sgRNA expression vectors. The knock-in efficiency was analyzed after 48 h of CdCl2 treatment (Fig. 4). The CRISPR-Cas9-mediated knock-in efficiency in the HC11 cells treated with a high-dose of CdCl2 decreased by about 13% (
This study investigated whether the HR efficiency at the mouse beta-casein locus using CRISPR/Cas9 system was associated with the regulation of DNA mismatch repair (MMR) through high-dose CdCl2 treatment in HC11 cells.
It has been reported that the efficiency of DSB-promoted recombination for divergence substrates is higher in
Therefore, to evaluate the expression of DNA MMR-related genes, the HR-related gene
Interestingly, the mRNA expression of the
In this study, we analyzed the effects of high-dose CdCl2 treatment on the expression of the HR-related gene
In protein expression analysis, the nuclear levels of the Msh2 protein significantly decreased in HC11 cells treated with high-dose CdCl2 at 24 h; Msh2 levels slightly decreased at 48 h, but there was no significant difference. These results indicate that treatment with 10 μM CdCl2 does not completely inhibit the nuclear expression of Msh2. In addition, the nuclear levels of Rad51 significantly decreased at 48 h compared to non-CdCl2-treated group. Chidambaram et al. (2017) reported that CdCl2 treatment reduces histone deacetylase 2 (Hdac2) expression, and, in this study, the protein expression of Hdac2 decreased with increasing duration of CdCl2 treatment. There was no significant difference in the cytoplasmic protein levels of Msh2 and DNA Ligase
In this study, we performed CRISPR-Cas9-mediated gene targeting at the mouse beta-casein locus to determine the effect of high-dose CdCl2 treatment on HR efficiency in HC11 cells. The results showed that high-dose CdCl2 treatment slightly decreased the knock-in efficiency in HC11 cells compared to the CdCl2-untreated group. Additionally, we examined the mRNA expression of MMR-related genes, the HR-related gene
In this study, high concentrations of cadmium were used to compensate for the problem of MMR-related gene inhibition. However, despite the suppression of MMR-related genes, the knock-in efficiency did not improve. Although it was believed that the inhibition of MMR-related gene expression through high-dose CdCl2 would improve the HR mechanism, this was not the case.
Therefore, the high-dose CdCl2 treatment can be used as a DNA MMR inhibitor that can suppress DNA MMR genes. However, it was deemed inappropriate as a method for improving the efficiency of HR due to the decrease in expression of the HR-related gene
This study aimed to determine the potential changes in the efficiency of CRISPR-Cas9-mediated knock-in induced by treatment with high-dose CdCl2, an MMR inhibitor. During homologous recombination, the expression of MMR-related genes was suppressed in HC11 cells treated with cadmium, and the mRNA and protein expression of the HR-related gene
None.
Conceptualization, G-Y.K. and M-J.K.; investigation, G-Y.K. and M-J.K.; data curation, G-Y.K. and M-J.K.; writing—original draft preparation, G-Y.K. and M-J.K.; writing—review and editing, G-Y.K. and M-J.K.; supervision, M-J.K.
This work was supported by a National Research Foundation of Korea (NRF) grant awarded by the Korea government (MSIT) (No.2021R1F1A1052105).
Not applicable.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
Journal of Animal Reproduction and Biotechnology 2023; 38(3): 99-108
Published online September 30, 2023 https://doi.org/10.12750/JARB.38.3.99
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Ga-Yeon Kim and Man-Jong Kang*
Department of Animal Science, College of Agriculture and Life Science, Chonnam National University, Gwangju 61186, Korea
Correspondence to:Man-Jong Kang
E-mail: mjkang@jnu.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Efficient gene editing technology is needed for successful knock-in. Homologous recombination (HR) is a major double-strand break repair pathway that can be utilized for accurately inserting foreign genes into the genome. HR occurs during the S/G2 phase, and the DNA mismatch repair (MMR) pathway is inextricably linked to HR to maintain HR fidelity. This study was conducted to investigate the effect of inhibiting MMR-related genes using CdCl2, an MMR-related gene inhibitor, on HR efficiency in HC11 cells.
Methods: The mRNA and protein expression levels of MMR-related genes (Msh2, Msh3, Msh6, Mlh1, Pms2), the HR-related gene Rad51, and the NHEJ-related gene DNA Ligase IV were assessed in HC11 cells treated with 10 μM of CdCl2 for 48 hours. In addition, HC11 cells were transfected with a CRISPR/sgRNA expression vector and a knock-in vector targeting Exon3 of the mouse-beta casein locus, and treated with 10 μM cadmium for 48 hours. The knock-in efficiency was monitored through PCR.
Results: The treatment of HC11 cells with a high-dose of CdCl2 decreased the mRNA expression of the HR-related gene Rad51 in HC11 cells. In addition, the inhibition of MMR-related genes through CdCl2 treatment did not lead to an increase in knock-in efficiency.
Conclusions: The inhibition of MMR-related gene expression through high-dose CdCl2 treatment reduces the expression of the HR-related gene Rad51, which is active during recombination. Therefore, it was determined that CdCl2 is an inappropriate compound for improving HR efficiency.
Keywords: cadmium chloride, CRISPR-Cas9 mediated knock-in efficiency, DNA mismatch repair, homologous recombination, non-homologous end joining
Gene targeting is a useful technique for inserting foreign genes into a specific locus in the genome. The major pathways of double-strand break (DSB) repair are homologous recombination (HR) and non-homologous end joining (NHEJ). The HR pathway is utilized to accurately insert foreign genes into the genome, but it occurs only during the S/G2 phase, and its efficiency for DSB repair is lower than that of NHEJ (Vasquez et al., 2001; Mao et al., 2008). Thus, studies have been performed to improve the efficiency of HR for accurate gene targeting. The HR-related gene
The DNA mismatch repair (MMR) is known to be inextricably linked to HR (Spies and Fishel, 2015), and is reported as a post-replication repair pathway that occurs during replication or recombination (Li, 2008). The MMR-related MutS α (MSH2-MSH6) and MutS β (MSH2-MSH3) proteins bind to a mismatched DNA base pair, and MutL α (MLH1-PMS2) is an endonuclease that binds to MutS-heteroduplex complexes (Lahue et al., 1989; Habraken et al., 1996). The MMR maintains HR fidelity and prevents recombination between divergent sequences through heteroduplex rejection in the presence of extensive heteroduplexes during the recombination process (Goldfarb and Alani, 2005; Kunkel and Erie, 2015; Chakaraborty and Alani, 2016).
CdCl2 treatment inhibits ATP hydrolysis of MutS α (MSH2-MSH6) in eukaryotic cells and specific binding to mismatched DNA (Clark and Kunkel, 2004). Meanwhile, it has been reported that knock-out of
To address the conflicting reports, we aimed to determine the change in knock-in efficiency when MMR is suppressed through high-dose CdCl2 treatment (10 μM), an MMR inhibitor, in HC11 cells. Furthermore, to assess the mechanism of HR inhibition by CdCl2 treatment, we assessed the expression of MMR-related genes, the HR-related gene
The knock-in vector used in this study expresses hEPO protein using the GST fusion system at the mouse beta-casein Exon3 locus. The knock-in vector, which consists of a 5’ homology arm (1.024 kb), GST-PreScission protease-hEPO, SV40polyA, CMV-EGFP, and a 3’ homology arm (1.81 kb), was originally developed in our laboratory. The vector was modified in this study; CMV-EGFP, a positive selection marker, was replaced with PGK neo, a negative selection marker, and the 3’ homology arm of 1.81 kb was modified to a 3’ homology arm of 1.015 kb. As a result, the final knock-in vector was constructed as PBSK_1.024kb mBC5’arm_GST_hEPO_SV40polyA_PGK neo_1.015kb mBC3’arm.
Mouse mammary epithelial cells (HC11) were cultured in the growth medium consisting of Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone), 1% penicillin/streptomycin (Hyclone), 0.1% gentamicin (Sigma, St. Louis, MO, USA), 0.1% EGF (Gibco, Thermo Fisher, USA), and 0.05% insulin (Sigma). HC11 cells were seeded at a density of 2.4 × 105 cells per well into 6-well plates (SPL, Gyeonggi-do, Korea) and incubated at 37℃ with 5% CO2. The medium was replaced with 1 mL of fresh medium before transfection. The knock-in vector (3.75 μg) and pGuide it Zs Green1_sgRNA expression vector (1.875 μg) were transfected into HC11 cells using the Xfect transfection reagent (Takara, Tokyo, Japan). After 24 hours of transfection, the cells were treated with 10 μM CdCl2 and incubated for 48 hours.
Total RNA from HC11 cells was isolated using an RNeasy mini kit (QIAGEN, Hilden, Germany). A total of 3 μg of RNA was used as a template for cDNA synthesis, which was performed using random hexamers and M-MLV reverse transcriptase (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. Quantitative PCR was performed to measure the mRNA expression of DNA MMR-related genes (
Table 1. Primers used for GST_hEPO screening PCR analysis and quantitative PCR analysis of specific genes.
Gene | Primer name | Sequence (5’ to 3’) |
---|---|---|
Primers used for GST_hEPO screening PCR analysis | ||
Mouse β-casein 5’ arm | m C5 CV S5 β | ATAAGAACTGCAGTGGGATTCTTC |
m C3 GST AS9 β | AGCATTGAAATCTCTGCACGCTC | |
m C5 CV S6 β | GTCTACCAATTCACTCTAGAAGTG | |
m C3 GST AS7 β | ACGTATGATGGCCATAGACTGTG | |
Primers used for quantitative real-time PCR analysis of MMR-related gene, | ||
m | CGACCTGGAAGTCCAACTA | |
m | ATCTGCTGCATCTGCTTG | |
MMR-related gene ( | m | TGAAGTTGGACATGGCAGCA |
m | TAATGCGGCCAGAGACTGAG | |
m | CCATCGCCTATGCAACTCTA | |
m | TCACAGACTGGTGGATAGTG | |
m | ATACTCAGGCATGCAACAGC | |
m | TCAAAAGTTGCGGTGCCTCT | |
m | AGATTAGTGAGCGGTGCCAT | |
m | ACTGAGGATTCACACAGCCC | |
m | ACTTCCAGGACGCCACAAAA | |
m | AACTGCCTGTCTGTTGCACT | |
HR-related gene | AATTCCGAACTGGGAAGACAC | |
TCACCTCCACCACGGTCAAT | ||
NHEJ-related gene | qPCR | TCCACAGGAAGGCTCTCTCA |
DNA | qPCR | TCTCACCGTCAAGGATGCAC |
Nuclear and cytoplasmic proteins from HC11 cells were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Total protein from HC11 cells was extracted using the PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. The nuclear and cytoplasmic protein extracts (15 μg) and total protein (30 μg) derived from the cells were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes (Bio-Rad Co., Hercules, CA, USA). The membranes were then blocked for 2 hours at room temperature in 5% skim milk in TBST (Tris-buffered saline with 0.1% Tween 20), and blotted with a rabbit polyclonal anti-MSH2 antibody (ab-70270, Abcam, Cambridge, UK; dilution 1:2,000), mouse monoclonal anti-RAD51 (sc398587) and anti-DNA Ligase
The knock-in efficiency of the hEPO knock-in vector in HC11 cells was confirmed through first and second PCR experiments. The genomic DNA isolated from cells in each group was used as a DNA template for PCR. The first PCR was performed using the mβC5 CV S5 primer for regions outside the 5’ arm and the mβC3 GST9 (Table 1) for the GST region using the amplification reagent SolgTM 2X Multiplex (Solgent, Daejeon, South Korea). The PCR consisted 25 cycles of amplification at 95℃ for 20 seconds, annealing at 62℃ for 40 seconds, and strand extension at 72℃ for 2 minutes. Subsequently, a nested PCR was performed using the mβC5 CV primer and the mβC3 GST AS7 primer using the amplification regent KOD FX Neo (Toyobo, Osaka, Japan) through the following steps: 25 cycles of denaturation at 95℃ for 20 seconds, annealing at 64℃ for 30 seconds, and strand extension at 72℃ for 2 minutes; followed by a final extension at 72℃ for 5 min. The PCR fragments were confirmed through electrophoresis on a 0.8% agarose gel. For comparative quantification, each DNA band was normalized to the mβCE7 region.
Densitometric quantification of DNA or protein bands was performed by analyzing the data using UN-SCAN-IT gel Analysis Software (Silk Scientific Inc., Orem, UT, USA). Subsequently, a statistical analysis of the mRNA levels and densitometric quantification of DNA and protein bands was performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). The data were analyzed in two ways. First, the data were analyzed with one-way ANOVA, followed by the Dunnett test, to compare all columns with reference to the control column. Second, a
The diagram illustrating the knock-in vector used for expressing the human erythropoietin (
To determine the effect of high-dose CdCl2 treatment (10 μM) on the mRNA expression of
In order to determine the protein levels of Rad51, DNA Ligase
To investigate the efficiency of CRISPR-Cas9-mediated knock-in, HC11 cells were exposed to high-dose CdCl2 treatment and transfected with the GST-hEPO knock-in and the CRISPR-sgRNA expression vectors. The knock-in efficiency was analyzed after 48 h of CdCl2 treatment (Fig. 4). The CRISPR-Cas9-mediated knock-in efficiency in the HC11 cells treated with a high-dose of CdCl2 decreased by about 13% (
This study investigated whether the HR efficiency at the mouse beta-casein locus using CRISPR/Cas9 system was associated with the regulation of DNA mismatch repair (MMR) through high-dose CdCl2 treatment in HC11 cells.
It has been reported that the efficiency of DSB-promoted recombination for divergence substrates is higher in
Therefore, to evaluate the expression of DNA MMR-related genes, the HR-related gene
Interestingly, the mRNA expression of the
In this study, we analyzed the effects of high-dose CdCl2 treatment on the expression of the HR-related gene
In protein expression analysis, the nuclear levels of the Msh2 protein significantly decreased in HC11 cells treated with high-dose CdCl2 at 24 h; Msh2 levels slightly decreased at 48 h, but there was no significant difference. These results indicate that treatment with 10 μM CdCl2 does not completely inhibit the nuclear expression of Msh2. In addition, the nuclear levels of Rad51 significantly decreased at 48 h compared to non-CdCl2-treated group. Chidambaram et al. (2017) reported that CdCl2 treatment reduces histone deacetylase 2 (Hdac2) expression, and, in this study, the protein expression of Hdac2 decreased with increasing duration of CdCl2 treatment. There was no significant difference in the cytoplasmic protein levels of Msh2 and DNA Ligase
In this study, we performed CRISPR-Cas9-mediated gene targeting at the mouse beta-casein locus to determine the effect of high-dose CdCl2 treatment on HR efficiency in HC11 cells. The results showed that high-dose CdCl2 treatment slightly decreased the knock-in efficiency in HC11 cells compared to the CdCl2-untreated group. Additionally, we examined the mRNA expression of MMR-related genes, the HR-related gene
In this study, high concentrations of cadmium were used to compensate for the problem of MMR-related gene inhibition. However, despite the suppression of MMR-related genes, the knock-in efficiency did not improve. Although it was believed that the inhibition of MMR-related gene expression through high-dose CdCl2 would improve the HR mechanism, this was not the case.
Therefore, the high-dose CdCl2 treatment can be used as a DNA MMR inhibitor that can suppress DNA MMR genes. However, it was deemed inappropriate as a method for improving the efficiency of HR due to the decrease in expression of the HR-related gene
This study aimed to determine the potential changes in the efficiency of CRISPR-Cas9-mediated knock-in induced by treatment with high-dose CdCl2, an MMR inhibitor. During homologous recombination, the expression of MMR-related genes was suppressed in HC11 cells treated with cadmium, and the mRNA and protein expression of the HR-related gene
None.
Conceptualization, G-Y.K. and M-J.K.; investigation, G-Y.K. and M-J.K.; data curation, G-Y.K. and M-J.K.; writing—original draft preparation, G-Y.K. and M-J.K.; writing—review and editing, G-Y.K. and M-J.K.; supervision, M-J.K.
This work was supported by a National Research Foundation of Korea (NRF) grant awarded by the Korea government (MSIT) (No.2021R1F1A1052105).
Not applicable.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
Table 1 . Primers used for GST_hEPO screening PCR analysis and quantitative PCR analysis of specific genes.
Gene | Primer name | Sequence (5’ to 3’) |
---|---|---|
Primers used for GST_hEPO screening PCR analysis | ||
Mouse β-casein 5’ arm | m C5 CV S5 β | ATAAGAACTGCAGTGGGATTCTTC |
m C3 GST AS9 β | AGCATTGAAATCTCTGCACGCTC | |
m C5 CV S6 β | GTCTACCAATTCACTCTAGAAGTG | |
m C3 GST AS7 β | ACGTATGATGGCCATAGACTGTG | |
Primers used for quantitative real-time PCR analysis of MMR-related gene, | ||
m | CGACCTGGAAGTCCAACTA | |
m | ATCTGCTGCATCTGCTTG | |
MMR-related gene ( | m | TGAAGTTGGACATGGCAGCA |
m | TAATGCGGCCAGAGACTGAG | |
m | CCATCGCCTATGCAACTCTA | |
m | TCACAGACTGGTGGATAGTG | |
m | ATACTCAGGCATGCAACAGC | |
m | TCAAAAGTTGCGGTGCCTCT | |
m | AGATTAGTGAGCGGTGCCAT | |
m | ACTGAGGATTCACACAGCCC | |
m | ACTTCCAGGACGCCACAAAA | |
m | AACTGCCTGTCTGTTGCACT | |
HR-related gene | AATTCCGAACTGGGAAGACAC | |
TCACCTCCACCACGGTCAAT | ||
NHEJ-related gene | qPCR | TCCACAGGAAGGCTCTCTCA |
DNA | qPCR | TCTCACCGTCAAGGATGCAC |
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pISSN: 2671-4639
eISSN: 2671-4663