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CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA.

Miura H, Gurumurthy CB, Sato T, Sato M, Ohtsuka M - Sci Rep (2015)

Bottom Line: We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis.Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression.We also provide a strategy to establish conditional knockdown models with this method.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.

ABSTRACT
Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.

No MeSH data available.


Targeted insertion of ssDNA encoding anti-Otx2 amiRNA by CRISPR/Cas9 system (Exp. 4).(a) Schematics of targeted integration of amiR-Otx2_546 into intron 6 of eEF-2 gene (upper panel) and Otx2 genomic region showing amiR-Otx2_546 binding site (lower panel). The exons of Otx2 are shown as boxes and the boxes with homeodomain region are shaded black. Red arrows indicate the location of primer set (PP119/PP120) used for detection of fetuses with targeted insertion. (b) The resultant fetuses photographed at E14.5. (c) Genotyping of fetuses by PCR using primer set shown in (a). The embryo numbers in (b) and (c) correspond with each other. Expected fragment sizes: wild-type = 301-bp (black arrow), targeted insertion = 487-bp (blue arrow). (d) Targeted insertion efficiency. Fetuses containing functional amiRNA sequence were considered as ‘embryos with targeted insertions’ even though insertions were not fully accurate.
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f3: Targeted insertion of ssDNA encoding anti-Otx2 amiRNA by CRISPR/Cas9 system (Exp. 4).(a) Schematics of targeted integration of amiR-Otx2_546 into intron 6 of eEF-2 gene (upper panel) and Otx2 genomic region showing amiR-Otx2_546 binding site (lower panel). The exons of Otx2 are shown as boxes and the boxes with homeodomain region are shaded black. Red arrows indicate the location of primer set (PP119/PP120) used for detection of fetuses with targeted insertion. (b) The resultant fetuses photographed at E14.5. (c) Genotyping of fetuses by PCR using primer set shown in (a). The embryo numbers in (b) and (c) correspond with each other. Expected fragment sizes: wild-type = 301-bp (black arrow), targeted insertion = 487-bp (blue arrow). (d) Targeted insertion efficiency. Fetuses containing functional amiRNA sequence were considered as ‘embryos with targeted insertions’ even though insertions were not fully accurate.

Mentions: Based on the results of eGFP knockdown, the TS2 site that showed a better rate of insertion of intact sequences was used as the target site for insertion of amiRNA sequences against Otx2 (Fig. 3a and Supplementary Fig. S5a). Two different amiRNA target sequences against Otx2 were tested: amiR-Otx2_518 (Exp. 3) and amiR-Otx2_546 (Exp. 4). The ssDNAs for these amiRNA sequences were 296-bases long that were injected at 14 to 20 ng/μl concentration into C57BL/6 zygotes, together with 10 ng/μl of Cas9 mRNA and 10 ng/μl of sgRNA. Injected zygotes were transferred to oviducts of pseudo-pregnant mice. The fetuses were recovered at E14.5 and examined for knockdown phenotypes. We observed putative Otx2 knockdown phenotypes in two of the embryos derived from Exp. 4 that included amiR-Otx2_546 in the injection mix (Fig. 3b). One embryo (Exp. 4 sample #3) exhibited clear reduction in head, eye, and body size. This phenotype partially resembled Otx2 conditional knockout mouse reported by Fossat et al. (2006)23. The anophthalmia (lack of both eyes) phenotype was observed in the embryo #6 in Exp. 4. This is similar to the Otx2 hypomorphic (Otx2AA/AA) phenotype reported by Bernard et al. (2014)22. However, no embryos showing putative Otx2 knockdown phenotypes were obtained from amiR-Otx2_518 injected fetuses (Supplementary Fig. S5b).


CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA.

Miura H, Gurumurthy CB, Sato T, Sato M, Ohtsuka M - Sci Rep (2015)

Targeted insertion of ssDNA encoding anti-Otx2 amiRNA by CRISPR/Cas9 system (Exp. 4).(a) Schematics of targeted integration of amiR-Otx2_546 into intron 6 of eEF-2 gene (upper panel) and Otx2 genomic region showing amiR-Otx2_546 binding site (lower panel). The exons of Otx2 are shown as boxes and the boxes with homeodomain region are shaded black. Red arrows indicate the location of primer set (PP119/PP120) used for detection of fetuses with targeted insertion. (b) The resultant fetuses photographed at E14.5. (c) Genotyping of fetuses by PCR using primer set shown in (a). The embryo numbers in (b) and (c) correspond with each other. Expected fragment sizes: wild-type = 301-bp (black arrow), targeted insertion = 487-bp (blue arrow). (d) Targeted insertion efficiency. Fetuses containing functional amiRNA sequence were considered as ‘embryos with targeted insertions’ even though insertions were not fully accurate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525291&req=5

f3: Targeted insertion of ssDNA encoding anti-Otx2 amiRNA by CRISPR/Cas9 system (Exp. 4).(a) Schematics of targeted integration of amiR-Otx2_546 into intron 6 of eEF-2 gene (upper panel) and Otx2 genomic region showing amiR-Otx2_546 binding site (lower panel). The exons of Otx2 are shown as boxes and the boxes with homeodomain region are shaded black. Red arrows indicate the location of primer set (PP119/PP120) used for detection of fetuses with targeted insertion. (b) The resultant fetuses photographed at E14.5. (c) Genotyping of fetuses by PCR using primer set shown in (a). The embryo numbers in (b) and (c) correspond with each other. Expected fragment sizes: wild-type = 301-bp (black arrow), targeted insertion = 487-bp (blue arrow). (d) Targeted insertion efficiency. Fetuses containing functional amiRNA sequence were considered as ‘embryos with targeted insertions’ even though insertions were not fully accurate.
Mentions: Based on the results of eGFP knockdown, the TS2 site that showed a better rate of insertion of intact sequences was used as the target site for insertion of amiRNA sequences against Otx2 (Fig. 3a and Supplementary Fig. S5a). Two different amiRNA target sequences against Otx2 were tested: amiR-Otx2_518 (Exp. 3) and amiR-Otx2_546 (Exp. 4). The ssDNAs for these amiRNA sequences were 296-bases long that were injected at 14 to 20 ng/μl concentration into C57BL/6 zygotes, together with 10 ng/μl of Cas9 mRNA and 10 ng/μl of sgRNA. Injected zygotes were transferred to oviducts of pseudo-pregnant mice. The fetuses were recovered at E14.5 and examined for knockdown phenotypes. We observed putative Otx2 knockdown phenotypes in two of the embryos derived from Exp. 4 that included amiR-Otx2_546 in the injection mix (Fig. 3b). One embryo (Exp. 4 sample #3) exhibited clear reduction in head, eye, and body size. This phenotype partially resembled Otx2 conditional knockout mouse reported by Fossat et al. (2006)23. The anophthalmia (lack of both eyes) phenotype was observed in the embryo #6 in Exp. 4. This is similar to the Otx2 hypomorphic (Otx2AA/AA) phenotype reported by Bernard et al. (2014)22. However, no embryos showing putative Otx2 knockdown phenotypes were obtained from amiR-Otx2_518 injected fetuses (Supplementary Fig. S5b).

Bottom Line: We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis.Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression.We also provide a strategy to establish conditional knockdown models with this method.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.

ABSTRACT
Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.

No MeSH data available.