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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-eGFP amiRNA by CRISPR/Cas9 system (Exp. 2).(a) Schematics of targeted integration of amiR-eGFP123/419 sequences into the intron 6 of eEF-2 gene (upper panel) and location of amiR-eGFP123 and -eGFP419 target sites on eGFP cDNA (lower panel). From the correctly targeted eEF-2 locus, amiRNAs get transcribed and subsequently bind to the target sites (amiR-eGFP123 = pink line and amiR-eGFP419 = purple line) on eGFP mRNA, leading to eGFP knockdown. Red arrows indicate the location of primer set (PP119/PP120) used for detection of fetuses with targeted insertion. (b) eGFP fluorescence among E13.5 day fetuses observed under a fluorescent stereomicroscope showing successful knockdown in some fetuses (see text for details). (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 = 625-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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f2: Targeted insertion of ssDNA encoding anti-eGFP amiRNA by CRISPR/Cas9 system (Exp. 2).(a) Schematics of targeted integration of amiR-eGFP123/419 sequences into the intron 6 of eEF-2 gene (upper panel) and location of amiR-eGFP123 and -eGFP419 target sites on eGFP cDNA (lower panel). From the correctly targeted eEF-2 locus, amiRNAs get transcribed and subsequently bind to the target sites (amiR-eGFP123 = pink line and amiR-eGFP419 = purple line) on eGFP mRNA, leading to eGFP knockdown. Red arrows indicate the location of primer set (PP119/PP120) used for detection of fetuses with targeted insertion. (b) eGFP fluorescence among E13.5 day fetuses observed under a fluorescent stereomicroscope showing successful knockdown in some fetuses (see text for details). (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 = 625-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: We aimed to insert the amiRNA sequences at a genomic site that would readily allow transgene expression without inhibitory positional effects that occur in certain chromosomal locations. Introns of eukaryotic translation elongation factor 2 (eEF-2) gene were selected as candidates because eEF-2 is expressed at high levels ubiquitously and consistently both during developmental and adult stages15. The corresponding intronic regions of mouse and human eEF-2 genes were aligned and compared, which revealed that parts of mouse introns 1 and 6 were among the evolutionally non-conserved regions. We reasoned that such non-conserved regions are less likely to contain regulatory sequences and therefore not have biological functions. The two less conserved intronic regions in eEF-2 gene were named Target Site 1 and 2 (TS1 and TS2) (Fig. 2a, Supplementary Fig. S1a). The sgRNAs were designed against TS1 and TS2, as described in Methods section, to insert amiRNA cassettes into these sites. As a first model to test this, we inserted amiRNA to knockdown eGFP gene, using our previously generated eGFP Tg mouse model that shows highly stable eGFP expression1617. The 434-base ssDNA copies for TS1 and TS2, containing amiR-eGFP123/419, were prepared and used as repair DNAs (20 ng/μl) in microinjections that contained Cas9 mRNA (10 ng/μl) and respective CRISPR sgRNAs (10 ng/μl). These injection experiments for TS1 and TS2 were designated as Exp. 1 and Exp. 2, respectively. The zygotes used for injection were obtained by in vitro fertilization of eggs collected from wild-type C57BL/6 mice using sperm collected from homozygous eGFP Tg mice. Injected zygotes were transferred to oviducts of pseudo-pregnant mice and allowed to develop until embryonic day 13.5 (E13.5), when they were collected and examined for eGFP fluorescence.


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-eGFP amiRNA by CRISPR/Cas9 system (Exp. 2).(a) Schematics of targeted integration of amiR-eGFP123/419 sequences into the intron 6 of eEF-2 gene (upper panel) and location of amiR-eGFP123 and -eGFP419 target sites on eGFP cDNA (lower panel). From the correctly targeted eEF-2 locus, amiRNAs get transcribed and subsequently bind to the target sites (amiR-eGFP123 = pink line and amiR-eGFP419 = purple line) on eGFP mRNA, leading to eGFP knockdown. Red arrows indicate the location of primer set (PP119/PP120) used for detection of fetuses with targeted insertion. (b) eGFP fluorescence among E13.5 day fetuses observed under a fluorescent stereomicroscope showing successful knockdown in some fetuses (see text for details). (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 = 625-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

f2: Targeted insertion of ssDNA encoding anti-eGFP amiRNA by CRISPR/Cas9 system (Exp. 2).(a) Schematics of targeted integration of amiR-eGFP123/419 sequences into the intron 6 of eEF-2 gene (upper panel) and location of amiR-eGFP123 and -eGFP419 target sites on eGFP cDNA (lower panel). From the correctly targeted eEF-2 locus, amiRNAs get transcribed and subsequently bind to the target sites (amiR-eGFP123 = pink line and amiR-eGFP419 = purple line) on eGFP mRNA, leading to eGFP knockdown. Red arrows indicate the location of primer set (PP119/PP120) used for detection of fetuses with targeted insertion. (b) eGFP fluorescence among E13.5 day fetuses observed under a fluorescent stereomicroscope showing successful knockdown in some fetuses (see text for details). (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 = 625-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: We aimed to insert the amiRNA sequences at a genomic site that would readily allow transgene expression without inhibitory positional effects that occur in certain chromosomal locations. Introns of eukaryotic translation elongation factor 2 (eEF-2) gene were selected as candidates because eEF-2 is expressed at high levels ubiquitously and consistently both during developmental and adult stages15. The corresponding intronic regions of mouse and human eEF-2 genes were aligned and compared, which revealed that parts of mouse introns 1 and 6 were among the evolutionally non-conserved regions. We reasoned that such non-conserved regions are less likely to contain regulatory sequences and therefore not have biological functions. The two less conserved intronic regions in eEF-2 gene were named Target Site 1 and 2 (TS1 and TS2) (Fig. 2a, Supplementary Fig. S1a). The sgRNAs were designed against TS1 and TS2, as described in Methods section, to insert amiRNA cassettes into these sites. As a first model to test this, we inserted amiRNA to knockdown eGFP gene, using our previously generated eGFP Tg mouse model that shows highly stable eGFP expression1617. The 434-base ssDNA copies for TS1 and TS2, containing amiR-eGFP123/419, were prepared and used as repair DNAs (20 ng/μl) in microinjections that contained Cas9 mRNA (10 ng/μl) and respective CRISPR sgRNAs (10 ng/μl). These injection experiments for TS1 and TS2 were designated as Exp. 1 and Exp. 2, respectively. The zygotes used for injection were obtained by in vitro fertilization of eggs collected from wild-type C57BL/6 mice using sperm collected from homozygous eGFP Tg mice. Injected zygotes were transferred to oviducts of pseudo-pregnant mice and allowed to develop until embryonic day 13.5 (E13.5), when they were collected and examined for eGFP fluorescence.

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.