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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

Types of mutations in TALE repeat arrays identified by DNA sequence analysis. Clones with a 1.76 kb DNA fragment of PCR insert as revealed by the RE digestion method were subjected to DNA sequencing examination. The samples of P3 baculovirus genome (n = 239) were analyzed. The correct TALE repeat arrays in left and right TALEN arms are listed on the top.
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fig3: Types of mutations in TALE repeat arrays identified by DNA sequence analysis. Clones with a 1.76 kb DNA fragment of PCR insert as revealed by the RE digestion method were subjected to DNA sequencing examination. The samples of P3 baculovirus genome (n = 239) were analyzed. The correct TALE repeat arrays in left and right TALEN arms are listed on the top.

Mentions: The changes in DNA sequences of the TALE repeat arrays of these samples included deletion, addition, substitution, and translocation. Deletion and addition caused changes in the number of RVD-containing TALE repeat units, while substitution mutation resulted in codon change. DNA strand exchange between left and right TALEN arms led to translocation of a portion of a TALE DNA binding domain. The results obtained from genomic DNA isolated from P3 viruses including BV-TALEN(L), BV-TALEN(R) and BV-TALEN(L-R) are shown in Figure 3. In these samples, more than 60% of genetic mutations were attributed to deletion, which could involve 1 to 4 RVD-containing repeat units (Supplementary Table S2). As expected, translocation was observed in BV-TALEN(L-R) with a reduction in the frequency of intra-array recombination within the left or right TALE DNA binding domain (Supplementary Table S2). Apart from missense mutations, other nucleotide sequence changes related point mutation, including frameshift and nonsense mutations, were detectable but at a negligible frequency. DNA sequence alignment revealed no evidence of silent mutation in the RVD-containing repeat arrays. When looking at effects of MOIs of viral stocks used to generate baculoviruses, we noticed that a higher MOI could increase the incidence of genetic mutation, although it did not cause obvious change in mutation type (Supplementary Table S3).


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)

Types of mutations in TALE repeat arrays identified by DNA sequence analysis. Clones with a 1.76 kb DNA fragment of PCR insert as revealed by the RE digestion method were subjected to DNA sequencing examination. The samples of P3 baculovirus genome (n = 239) were analyzed. The correct TALE repeat arrays in left and right TALEN arms are listed on the top.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Types of mutations in TALE repeat arrays identified by DNA sequence analysis. Clones with a 1.76 kb DNA fragment of PCR insert as revealed by the RE digestion method were subjected to DNA sequencing examination. The samples of P3 baculovirus genome (n = 239) were analyzed. The correct TALE repeat arrays in left and right TALEN arms are listed on the top.
Mentions: The changes in DNA sequences of the TALE repeat arrays of these samples included deletion, addition, substitution, and translocation. Deletion and addition caused changes in the number of RVD-containing TALE repeat units, while substitution mutation resulted in codon change. DNA strand exchange between left and right TALEN arms led to translocation of a portion of a TALE DNA binding domain. The results obtained from genomic DNA isolated from P3 viruses including BV-TALEN(L), BV-TALEN(R) and BV-TALEN(L-R) are shown in Figure 3. In these samples, more than 60% of genetic mutations were attributed to deletion, which could involve 1 to 4 RVD-containing repeat units (Supplementary Table S2). As expected, translocation was observed in BV-TALEN(L-R) with a reduction in the frequency of intra-array recombination within the left or right TALE DNA binding domain (Supplementary Table S2). Apart from missense mutations, other nucleotide sequence changes related point mutation, including frameshift and nonsense mutations, were detectable but at a negligible frequency. DNA sequence alignment revealed no evidence of silent mutation in the RVD-containing repeat arrays. When looking at effects of MOIs of viral stocks used to generate baculoviruses, we noticed that a higher MOI could increase the incidence of genetic mutation, although it did not cause obvious change in mutation type (Supplementary Table S3).

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