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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 Cre-activatable anti-eGFP amiRNA by CRISPR/Cas9 system (Exp. 5).(a) Schematics of targeted integration of reverse orientated ‘amiR-eGFP123/419’ and mutant loxP sites (JT15 and JTZ17) into the intron 6 of eEF-2 gene and subsequent Cre-loxP recombination to switch the amiRNA cassette to the right orientation. Functional amiRNAs do not get produced from the targeted ‘amiRNA-off’ allele because of the opposite orientation of amiRNA with respect to the eEF-2 gene. After Cre recombination, the allele gets converted to ‘amiRNA-on’ that produces functional amiRNA. Red arrows indicate the primers (PP119, PP120 and M412) used for genotyping. (b) Genotyping of fetuses by PCR using primer set (PP119/PP120). Expected fragment sizes: wild-type = 301-bp (black arrow), targeted insertion = 705-bp (blue arrow). (c) Targeted insertion efficiency. (d) Dotplot of embryonic feeder cells, derived from Exp. 5 samples #4 (upper) and #6 (lower), nine days after transfection with (left) or without (right) the iCre plasmid. The cells showing weak eGFP fluorescence are partitioned within the box in each plot. (e) Genotyping of embryonic feeder cells used in the experiment (d) by PCR with primer sets shown in (a).
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f4: Targeted insertion of ssDNA encoding Cre-activatable anti-eGFP amiRNA by CRISPR/Cas9 system (Exp. 5).(a) Schematics of targeted integration of reverse orientated ‘amiR-eGFP123/419’ and mutant loxP sites (JT15 and JTZ17) into the intron 6 of eEF-2 gene and subsequent Cre-loxP recombination to switch the amiRNA cassette to the right orientation. Functional amiRNAs do not get produced from the targeted ‘amiRNA-off’ allele because of the opposite orientation of amiRNA with respect to the eEF-2 gene. After Cre recombination, the allele gets converted to ‘amiRNA-on’ that produces functional amiRNA. Red arrows indicate the primers (PP119, PP120 and M412) used for genotyping. (b) Genotyping of fetuses by PCR using primer set (PP119/PP120). Expected fragment sizes: wild-type = 301-bp (black arrow), targeted insertion = 705-bp (blue arrow). (c) Targeted insertion efficiency. (d) Dotplot of embryonic feeder cells, derived from Exp. 5 samples #4 (upper) and #6 (lower), nine days after transfection with (left) or without (right) the iCre plasmid. The cells showing weak eGFP fluorescence are partitioned within the box in each plot. (e) Genotyping of embryonic feeder cells used in the experiment (d) by PCR with primer sets shown in (a).

Mentions: The constitutive expression of certain amiRNA sequences that leads to embryonic lethality can eventually result in unavailability of a model for further studies2425. A conditional expression strategy offers the best solution in such cases to generate a viable model. Hence, we next tested applicability of Cre-loxP system, a widely used conditional activation system, in our knockdown strategy. For this purpose, mutant loxP sequences JT15 and JTZ17 were included to flank the amiRNA sequence, as shown in Fig. 4a, and the cassette was placed in the opposite direction to eEF-2 gene orientation. Because of the opposite orientation, the functional amiRNA will not be produced, and the allele will be in the ‘off’ state (i.e., ‘amiRNA-off’ allele). After Cre administration, Cre-loxP-mediated recombination occurred between inversely-oriented JT15 and JTZ1726 that resulted in inversion of amiRNA region to convert the allele to ‘on’ state (‘amiRNA-on’ allele), which will allow for expression of functional amiRNA driven by the eEF-2 promoter. Because JT15 and JTZ17 contain mutations within their inverted-repeat regions, the inversion step will be unidirectional and the ‘amiRNA-on’ state will get locked after Cre recombination27.


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 Cre-activatable anti-eGFP amiRNA by CRISPR/Cas9 system (Exp. 5).(a) Schematics of targeted integration of reverse orientated ‘amiR-eGFP123/419’ and mutant loxP sites (JT15 and JTZ17) into the intron 6 of eEF-2 gene and subsequent Cre-loxP recombination to switch the amiRNA cassette to the right orientation. Functional amiRNAs do not get produced from the targeted ‘amiRNA-off’ allele because of the opposite orientation of amiRNA with respect to the eEF-2 gene. After Cre recombination, the allele gets converted to ‘amiRNA-on’ that produces functional amiRNA. Red arrows indicate the primers (PP119, PP120 and M412) used for genotyping. (b) Genotyping of fetuses by PCR using primer set (PP119/PP120). Expected fragment sizes: wild-type = 301-bp (black arrow), targeted insertion = 705-bp (blue arrow). (c) Targeted insertion efficiency. (d) Dotplot of embryonic feeder cells, derived from Exp. 5 samples #4 (upper) and #6 (lower), nine days after transfection with (left) or without (right) the iCre plasmid. The cells showing weak eGFP fluorescence are partitioned within the box in each plot. (e) Genotyping of embryonic feeder cells used in the experiment (d) by PCR with primer sets shown in (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Targeted insertion of ssDNA encoding Cre-activatable anti-eGFP amiRNA by CRISPR/Cas9 system (Exp. 5).(a) Schematics of targeted integration of reverse orientated ‘amiR-eGFP123/419’ and mutant loxP sites (JT15 and JTZ17) into the intron 6 of eEF-2 gene and subsequent Cre-loxP recombination to switch the amiRNA cassette to the right orientation. Functional amiRNAs do not get produced from the targeted ‘amiRNA-off’ allele because of the opposite orientation of amiRNA with respect to the eEF-2 gene. After Cre recombination, the allele gets converted to ‘amiRNA-on’ that produces functional amiRNA. Red arrows indicate the primers (PP119, PP120 and M412) used for genotyping. (b) Genotyping of fetuses by PCR using primer set (PP119/PP120). Expected fragment sizes: wild-type = 301-bp (black arrow), targeted insertion = 705-bp (blue arrow). (c) Targeted insertion efficiency. (d) Dotplot of embryonic feeder cells, derived from Exp. 5 samples #4 (upper) and #6 (lower), nine days after transfection with (left) or without (right) the iCre plasmid. The cells showing weak eGFP fluorescence are partitioned within the box in each plot. (e) Genotyping of embryonic feeder cells used in the experiment (d) by PCR with primer sets shown in (a).
Mentions: The constitutive expression of certain amiRNA sequences that leads to embryonic lethality can eventually result in unavailability of a model for further studies2425. A conditional expression strategy offers the best solution in such cases to generate a viable model. Hence, we next tested applicability of Cre-loxP system, a widely used conditional activation system, in our knockdown strategy. For this purpose, mutant loxP sequences JT15 and JTZ17 were included to flank the amiRNA sequence, as shown in Fig. 4a, and the cassette was placed in the opposite direction to eEF-2 gene orientation. Because of the opposite orientation, the functional amiRNA will not be produced, and the allele will be in the ‘off’ state (i.e., ‘amiRNA-off’ allele). After Cre administration, Cre-loxP-mediated recombination occurred between inversely-oriented JT15 and JTZ1726 that resulted in inversion of amiRNA region to convert the allele to ‘on’ state (‘amiRNA-on’ allele), which will allow for expression of functional amiRNA driven by the eEF-2 promoter. Because JT15 and JTZ17 contain mutations within their inverted-repeat regions, the inversion step will be unidirectional and the ‘amiRNA-on’ state will get locked after Cre recombination27.

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.