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Genetic rearrangements of variable di-residue (RVD)-containing repeat arrays in a baculoviral TALEN system.

Lau CH, Zhu H, Tay JC, Li Z, Tay FC, Chen C, Tan WK, Du S, Sia VK, Phang RZ, Tang SY, Yang C, Chi Z, Liang CC, Ning E, Wang S - Mol Ther Methods Clin Dev (2014)

Bottom Line: The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms.Based on these observations, we have developed a protocol using a low MOI to produce baculoviral vectors expressing TALEN left and right arms separately.Cotransduction of the viruses produced by this optimal protocol provided an improved TALEN cleavage efficiency and enabled effective site-specific transgene integration in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.

ABSTRACT
Virus-derived gene transfer vectors have been successfully employed to express the transcription activator-like effector nucleases (TALENs) in mammalian cells. Since the DNA-binding domains of TALENs consist of the variable di-residue (RVD)-containing tandem repeat modules and virus genome with repeated sequences is susceptible to genetic recombination, we investigated several factors that might affect TALEN cleavage efficiency of baculoviral vectors. Using a TALEN system designed to target the AAVS1 locus, we observed increased sequence instability of the TALE repeat arrays when a higher multiplicity of infection (MOI) of recombinant viruses was used to produce the baculoviral vectors. We also detected more deleterious mutations in the TALE DNA-binding domains when both left and right TALEN arms were placed into a single expression cassette as compared to the viruses containing one arm only. The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms. Based on these observations, we have developed a protocol using a low MOI to produce baculoviral vectors expressing TALEN left and right arms separately. Cotransduction of the viruses produced by this optimal protocol provided an improved TALEN cleavage efficiency and enabled effective site-specific transgene integration in human cells.

No MeSH data available.


Related in: MedlinePlus

Quantification of the TALEN cleavage efficiency using single strand annealing (SSA) assay. (a) Schematic representation of SSA assay designed to quantify the TALEN cleavage efficiency at the targeted AAVS1 site. An AAVS1 SSA assay testing vector is constructed by cloning annealed oligonucleotides bearing the targeted AAVS1 site into BsaI-treated pGL4-SSA vector through their sticky ends. TALEN-induced double strand break (DSB) at the AAVS1 site of the testing vector is repaired via SSA to generate a functional luciferase reporter vector. (b) Comparisons of TALEN cleavage efficiency of TALEN baculoviral vectors prepared with different ways. U87 cells were transfected with the AAVS1 SSA vector, followed by transduction of BV-TALENs at an MOI of 50. BV-TALEN (L-R): Transduction with viruses containing an expression cassette with both left and right TALEN arms. BV-TALEN (L/R): Cotransduction with two types of viruses carrying left and right TALEN arms, respectively. P2 or P3 viruses used for cell transduction and the initial MOI used to generate these BV-TALENs (in brackets) are indicated. Luciferase activity assay was performed 48 hours after transduction, which is directly proportional to the cleavage efficiency mediated by the TALENs. Values present mean ± SD (n = 5). *, ***: p < 0.05 and p < 0.001 versus BV-TALEN(L-R) by Student’s t-test. +++: p < 0.001 between P2 BV- TALEN(L/R) and P3 BV-TALEN(L/R) by Student’s t-test. The background readings for the AAVS1 SSA vector were 291,027 ± 121,415.
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fig5: Quantification of the TALEN cleavage efficiency using single strand annealing (SSA) assay. (a) Schematic representation of SSA assay designed to quantify the TALEN cleavage efficiency at the targeted AAVS1 site. An AAVS1 SSA assay testing vector is constructed by cloning annealed oligonucleotides bearing the targeted AAVS1 site into BsaI-treated pGL4-SSA vector through their sticky ends. TALEN-induced double strand break (DSB) at the AAVS1 site of the testing vector is repaired via SSA to generate a functional luciferase reporter vector. (b) Comparisons of TALEN cleavage efficiency of TALEN baculoviral vectors prepared with different ways. U87 cells were transfected with the AAVS1 SSA vector, followed by transduction of BV-TALENs at an MOI of 50. BV-TALEN (L-R): Transduction with viruses containing an expression cassette with both left and right TALEN arms. BV-TALEN (L/R): Cotransduction with two types of viruses carrying left and right TALEN arms, respectively. P2 or P3 viruses used for cell transduction and the initial MOI used to generate these BV-TALENs (in brackets) are indicated. Luciferase activity assay was performed 48 hours after transduction, which is directly proportional to the cleavage efficiency mediated by the TALENs. Values present mean ± SD (n = 5). *, ***: p < 0.05 and p < 0.001 versus BV-TALEN(L-R) by Student’s t-test. +++: p < 0.001 between P2 BV- TALEN(L/R) and P3 BV-TALEN(L/R) by Student’s t-test. The background readings for the AAVS1 SSA vector were 291,027 ± 121,415.

Mentions: To evaluate the DNA disruption efficiency by BV-TALEN vectors, we constructed a luciferase reporter vector bearing the targeted AAVS1 site and performed a single strand annealing assay (Figure 5a) in human U87 cells. When P3 viruses generated with an initial MOI from 0.025 to 0.2 pfu per insect cell were used, their DNA disruption efficiency decreased with increase in MOI used for virus generation, regardless of whether the cells were transduced with BV-TALEN(L-R) or cotransduced with BV-TALEN(L) and BV-TALEN(R) (Figure 5b). These results are consistent with the above findings that the viruses generated with a higher MOI bear more incorrect DNA binding sequences, thus being less effective in cleavage of a target site. Likewise, cotransduction of U87 cells with BV-TALEN(L) and BV-TALEN(R) provided a more robust TALEN activity than transduction with BV-TALEN(L-R) generated at the same MOI (Figure 5b). Being consistent with the observations that P2 viruses carry less functionally defective RVD-containing repeat units than P3 viruses (Table 1), the highest TALEN activity was achieved when the cells were cotransduced with two P2 viruses, BV-TALEN(L) and BV-TALEN(R), exhibiting an activity to 156% of that provided by two P3 viruses generated with the same MOI. These results support the notion that placing two TALEN arms into two viral vectors, generating viruses with a low MOI, and using a low passage number of working viruses could be an optimal protocol to reduce the sequence instability of a TALE repeat array in a baculoviral vector.


Genetic rearrangements of variable di-residue (RVD)-containing repeat arrays in a baculoviral TALEN system.

Lau CH, Zhu H, Tay JC, Li Z, Tay FC, Chen C, Tan WK, Du S, Sia VK, Phang RZ, Tang SY, Yang C, Chi Z, Liang CC, Ning E, Wang S - Mol Ther Methods Clin Dev (2014)

Quantification of the TALEN cleavage efficiency using single strand annealing (SSA) assay. (a) Schematic representation of SSA assay designed to quantify the TALEN cleavage efficiency at the targeted AAVS1 site. An AAVS1 SSA assay testing vector is constructed by cloning annealed oligonucleotides bearing the targeted AAVS1 site into BsaI-treated pGL4-SSA vector through their sticky ends. TALEN-induced double strand break (DSB) at the AAVS1 site of the testing vector is repaired via SSA to generate a functional luciferase reporter vector. (b) Comparisons of TALEN cleavage efficiency of TALEN baculoviral vectors prepared with different ways. U87 cells were transfected with the AAVS1 SSA vector, followed by transduction of BV-TALENs at an MOI of 50. BV-TALEN (L-R): Transduction with viruses containing an expression cassette with both left and right TALEN arms. BV-TALEN (L/R): Cotransduction with two types of viruses carrying left and right TALEN arms, respectively. P2 or P3 viruses used for cell transduction and the initial MOI used to generate these BV-TALENs (in brackets) are indicated. Luciferase activity assay was performed 48 hours after transduction, which is directly proportional to the cleavage efficiency mediated by the TALENs. Values present mean ± SD (n = 5). *, ***: p < 0.05 and p < 0.001 versus BV-TALEN(L-R) by Student’s t-test. +++: p < 0.001 between P2 BV- TALEN(L/R) and P3 BV-TALEN(L/R) by Student’s t-test. The background readings for the AAVS1 SSA vector were 291,027 ± 121,415.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4362386&req=5

fig5: Quantification of the TALEN cleavage efficiency using single strand annealing (SSA) assay. (a) Schematic representation of SSA assay designed to quantify the TALEN cleavage efficiency at the targeted AAVS1 site. An AAVS1 SSA assay testing vector is constructed by cloning annealed oligonucleotides bearing the targeted AAVS1 site into BsaI-treated pGL4-SSA vector through their sticky ends. TALEN-induced double strand break (DSB) at the AAVS1 site of the testing vector is repaired via SSA to generate a functional luciferase reporter vector. (b) Comparisons of TALEN cleavage efficiency of TALEN baculoviral vectors prepared with different ways. U87 cells were transfected with the AAVS1 SSA vector, followed by transduction of BV-TALENs at an MOI of 50. BV-TALEN (L-R): Transduction with viruses containing an expression cassette with both left and right TALEN arms. BV-TALEN (L/R): Cotransduction with two types of viruses carrying left and right TALEN arms, respectively. P2 or P3 viruses used for cell transduction and the initial MOI used to generate these BV-TALENs (in brackets) are indicated. Luciferase activity assay was performed 48 hours after transduction, which is directly proportional to the cleavage efficiency mediated by the TALENs. Values present mean ± SD (n = 5). *, ***: p < 0.05 and p < 0.001 versus BV-TALEN(L-R) by Student’s t-test. +++: p < 0.001 between P2 BV- TALEN(L/R) and P3 BV-TALEN(L/R) by Student’s t-test. The background readings for the AAVS1 SSA vector were 291,027 ± 121,415.
Mentions: To evaluate the DNA disruption efficiency by BV-TALEN vectors, we constructed a luciferase reporter vector bearing the targeted AAVS1 site and performed a single strand annealing assay (Figure 5a) in human U87 cells. When P3 viruses generated with an initial MOI from 0.025 to 0.2 pfu per insect cell were used, their DNA disruption efficiency decreased with increase in MOI used for virus generation, regardless of whether the cells were transduced with BV-TALEN(L-R) or cotransduced with BV-TALEN(L) and BV-TALEN(R) (Figure 5b). These results are consistent with the above findings that the viruses generated with a higher MOI bear more incorrect DNA binding sequences, thus being less effective in cleavage of a target site. Likewise, cotransduction of U87 cells with BV-TALEN(L) and BV-TALEN(R) provided a more robust TALEN activity than transduction with BV-TALEN(L-R) generated at the same MOI (Figure 5b). Being consistent with the observations that P2 viruses carry less functionally defective RVD-containing repeat units than P3 viruses (Table 1), the highest TALEN activity was achieved when the cells were cotransduced with two P2 viruses, BV-TALEN(L) and BV-TALEN(R), exhibiting an activity to 156% of that provided by two P3 viruses generated with the same MOI. These results support the notion that placing two TALEN arms into two viral vectors, generating viruses with a low MOI, and using a low passage number of working viruses could be an optimal protocol to reduce the sequence instability of a TALE repeat array in a baculoviral vector.

Bottom Line: The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms.Based on these observations, we have developed a protocol using a low MOI to produce baculoviral vectors expressing TALEN left and right arms separately.Cotransduction of the viruses produced by this optimal protocol provided an improved TALEN cleavage efficiency and enabled effective site-specific transgene integration in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.

ABSTRACT
Virus-derived gene transfer vectors have been successfully employed to express the transcription activator-like effector nucleases (TALENs) in mammalian cells. Since the DNA-binding domains of TALENs consist of the variable di-residue (RVD)-containing tandem repeat modules and virus genome with repeated sequences is susceptible to genetic recombination, we investigated several factors that might affect TALEN cleavage efficiency of baculoviral vectors. Using a TALEN system designed to target the AAVS1 locus, we observed increased sequence instability of the TALE repeat arrays when a higher multiplicity of infection (MOI) of recombinant viruses was used to produce the baculoviral vectors. We also detected more deleterious mutations in the TALE DNA-binding domains when both left and right TALEN arms were placed into a single expression cassette as compared to the viruses containing one arm only. The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms. Based on these observations, we have developed a protocol using a low MOI to produce baculoviral vectors expressing TALEN left and right arms separately. Cotransduction of the viruses produced by this optimal protocol provided an improved TALEN cleavage efficiency and enabled effective site-specific transgene integration in human cells.

No MeSH data available.


Related in: MedlinePlus