Pervasive Genotypic Mosaicism in Founder Mice Derived from Genome Editing through Pronuclear Injection
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{"title"=>"Pervasive genotypic mosaicism in founder mice derived from genome editing through pronuclear injection", "type"=>"journal", "authors"=>[{"first_name"=>"Daniel", "last_name"=>"Oliver", "scopus_author_id"=>"56421423100"}, {"first_name"=>"Shuiqiao", "last_name"=>"Yuan", "scopus_author_id"=>"55848484700"}, {"first_name"=>"Hayden", "last_name"=>"McSwiggin", "scopus_author_id"=>"56590036100"}, {"first_name"=>"Wei", "last_name"=>"Yan", "scopus_author_id"=>"7402221472"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-84936806646", "sgr"=>"84936806646", "pui"=>"605098129", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"26053263", "doi"=>"10.1371/journal.pone.0129457"}, "id"=>"829f56ba-2c61-35f9-8bf1-1e11abbf6076", "abstract"=>"Genome editing technologies, especially the Cas9/CRISPR system, have revolutionized biomedical research over the past several years. Generation of novel alleles has been simplified to unprecedented levels, allowing for rapid expansion of available genetic tool kits for researchers. However, the issue of genotypic mosaicism has become evident, making stringent analyses of the penetrance of genome-edited alleles essential. Here, we report that founder mice, derived from pronuclear injection of ZFNs or a mix of guidance RNAs and Cas9 mRNAs, display consistent genotypic mosaicism for both deletion and insertion alleles. To identify founders with greater possibility of transmitting the mutant allele through the germline, we developed an effective germline genotyping method. The awareness of the inherent genotypic mosaicism issue with genome editing will allow for a more efficient implementation of the technologies, and the germline genotyping method will save valuable time and resources.", "link"=>"http://www.mendeley.com/research/pervasive-genotypic-mosaicism-founder-mice-derived-genome-editing-through-pronuclear-injection", "reader_count"=>31, "reader_count_by_academic_status"=>{"Unspecified"=>3, "Professor > Associate Professor"=>1, "Researcher"=>7, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>10, "Student > Master"=>3, "Student > Bachelor"=>6}, "reader_count_by_user_role"=>{"Unspecified"=>3, "Professor > Associate Professor"=>1, "Researcher"=>7, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>10, "Student > Master"=>3, "Student > Bachelor"=>6}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>15, "Agricultural and Biological Sciences"=>9, "Neuroscience"=>2, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Neuroscience"=>{"Neuroscience"=>2}, "Chemistry"=>{"Chemistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>9}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>15}, "Unspecified"=>{"Unspecified"=>3}}, "reader_count_by_country"=>{"Poland"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2100501"], "description"=>"<p>(a) Schematic representation of DNA sequence structure and genotyping primer design. Primers are indicated as orientation-specific arrows. The wild-type amplicon is 484bp, while the deletion alleles vary in size, depending on the NHEJ. sgRNA binding sites are indicated by dark grey boxes. <i>miR-741</i> is depicted as a light grey box. The approximate deletion in Founder 5 is depicted by the green line, whereas that of Founder 15 is depicted by the red line. (b) Agarose gel images showing tail DNA PCR genotyping for founder mice using the external primer set. The approximate band size of the WT allele amplicon is indicated by the black arrow. The deletion/insertion alleles were variable in size, depending on the founder mice; Founder 5 and 15 had a 132bp and a 30bp deletion, respectively. Founders 5 and 6 represent mosaic males, showing both wild-type allele amplification (upper band) and deletion allele amplification (lower band). (c) PCR germline genotyping results of Founders 5 and 15, showing the presence of the multiple alleles (t = testis, s = sperm). (d) PCR tail genotyping of F1 pups (F1 Pups 16–22) fathered by Founder 5. (e) PCR tail genotyping of F1 pups derived from Founder 15 (F1 Pups 23–29). (f) Representative sequencing results for wild-type miR741 (upper), Founder 5 (lower) and Founder 15 (middle). (g) Representative sequencing results for wild-type miR741 (upper) and F1 Pup 23 fathered by Founder 15.</p>", "links"=>[], "tags"=>["Pervasive Genotypic Mosaicism", "founder", "Pronuclear Injection Genome editing technologies", "Founder Mice Derived", "technology", "germline genotyping method", "genotypic mosaicism issue", "genotypic mosaicism", "allele", "Cas 9 mRNAs", "zfn"], "article_id"=>1440605, "categories"=>["Biological Sciences"], "users"=>["Daniel Oliver", "Shuiqiao Yuan", "Hayden McSwiggin", "Wei Yan"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0129457.g003", "stats"=>{"downloads"=>6, "page_views"=>116, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cas9_CRISPR_mediated_deletion_of_miR_741_shows_mosaicism_in_multiple_forms_/1440605", "title"=>"Cas9/CRISPR-mediated deletion of <i>miR-741</i> shows mosaicism in multiple forms.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-06-08 03:44:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2100499"], "description"=>"<p>(a) Schematic representation of DNA sequence structure and genotyping primer design. Primers are indicated as orientation-specific arrows. The wild-type external amplicon is 376bp, while the deletion alleles vary in size, relative to the wild-type amplicon. The deletion forward (Fwd) primer, paired with the external reverse (Rev) primer, amplifies a 192bp amplicon in wild type. Deletion alleles that cover this region do not amplify. sgRNA binding sites are indicated by the dark grey boxes. The approximate regions of the deletions in Founders 7 and 10 are depicted by the red line. (b) The founder pups (1–17) were genotyped using the external primer set. The WT allele band is indicated by the black arrow. Any band, above or below, represents either an insertion, or deletion, respectively (*). (c) The founder generation was likewise genotyped with the deletion internal primer amplicon. (d) Founders 7 and 10 were germline genotyped, using the external primer set. (e) Founders 7 and 10 were germline genotyped, using deletion primer set (t = testis; s = sperm; L = left; R = right). (f-g) The F1 progeny of Founder 7 (deletion/deletion based on tail lysate genotyping) was likewise genotyped, and the results suggest mosaicism in Founder 7’s germline because some pups received the deletion allele from Founder 7, while others were homozygous wild type. (h) Representative sequencing results for Founder 7 using tail DNA. (i) Representative sequencing results for Founder 7’s F1 progeny using tail DNA. Sequences of the amplicons using external primers for Founder 7 and one of Founder 7’s progeny (F1 Pup 49) displayed a large deletion.</p>", "links"=>[], "tags"=>["Pervasive Genotypic Mosaicism", "founder", "Pronuclear Injection Genome editing technologies", "Founder Mice Derived", "technology", "germline genotyping method", "genotypic mosaicism issue", "genotypic mosaicism", "allele", "Cas 9 mRNAs", "zfn"], "article_id"=>1440603, "categories"=>["Biological Sciences"], "users"=>["Daniel Oliver", "Shuiqiao Yuan", "Hayden McSwiggin", "Wei Yan"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0129457.g002", "stats"=>{"downloads"=>3, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cas9_CRISPR_mediated_deletion_in_Ubqlnl_/1440603", "title"=>"Cas9/CRISPR-mediated deletion in <i>Ubqlnl</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-06-08 03:44:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2100497"], "description"=>"<p>(a) Schematic representation of DNA sequence structure and genotyping primer design. Primers are indicated as orientation-specific arrows. The <i>loxp</i> insertion adds 46bp to the 288bp WT external amplicon size (334bp total, white box). <i>miR-34c</i> is indicated by the light grey box (77bp). The sgRNA target site is indicated by the dark grey box (20bp). The sgRNA was designed to target the wild-type sequence, with minimal effect on the HDR construct, or edited genomic sequence. (b) Agarose gel images showing amplicons using external forward (Fw) and revers (Rv) primers. WT bands are marked with black arrows, whereas the “<i>loxp</i> shift” bands are indicated using red arrows. Note that successful <i>loxp</i> insertion led to band shifts ranging from 288bp to 334bp. Germline genotyping assays were performed using the testes from Founder 3 (3t) and Founder 9 (9t) (t = testis). Founder 9 showed much greater abundance of the <i>loxp</i> shift bands than Founder 3. (c) Tail DNA PCR genotyping for F1 progeny of Founder 3, which was bred with WT females, showed no germline transmission of the <i>loxp</i> allele (Pups 13–21). (d) Tail DNA PCR genotyping results of F1 pups from Founder 9, which was crossed with WT females, showing successful germline transmission of the <i>loxp</i> allele. (e) Sequencing results of Founder 9 tail DNA, showing the successful <i>loxp</i> insertion, flanked by two engineered Kpn1 sites. (f) Sequencing results of tail DNA from Founder 9’s F1 progeny (Pup 22).</p>", "links"=>[], "tags"=>["Pervasive Genotypic Mosaicism", "founder", "Pronuclear Injection Genome editing technologies", "Founder Mice Derived", "technology", "germline genotyping method", "genotypic mosaicism issue", "genotypic mosaicism", "allele", "Cas 9 mRNAs", "zfn"], "article_id"=>1440601, "categories"=>["Biological Sciences"], "users"=>["Daniel Oliver", "Shuiqiao Yuan", "Hayden McSwiggin", "Wei Yan"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0129457.g001", "stats"=>{"downloads"=>2, "page_views"=>100, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cas9_CRISPR_mediated_loxp_sequence_insertion_adjacent_to_miR_34c_/1440601", "title"=>"Cas9/CRISPR-mediated <i>loxp</i> sequence insertion adjacent to <i>miR-34c</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-06-08 03:44:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2100502", "https://ndownloader.figshare.com/files/2100503"], "description"=>"<div><p>Genome editing technologies, especially the Cas9/CRISPR system, have revolutionized biomedical research over the past several years. Generation of novel alleles has been simplified to unprecedented levels, allowing for rapid expansion of available genetic tool kits for researchers. However, the issue of genotypic mosaicism has become evident, making stringent analyses of the penetrance of genome-edited alleles essential. Here, we report that founder mice, derived from pronuclear injection of ZFNs or a mix of guidance RNAs and Cas9 mRNAs, display consistent genotypic mosaicism for both deletion and insertion alleles. To identify founders with greater possibility of transmitting the mutant allele through the germline, we developed an effective germline genotyping method. The awareness of the inherent genotypic mosaicism issue with genome editing will allow for a more efficient implementation of the technologies, and the germline genotyping method will save valuable time and resources.</p></div>", "links"=>[], "tags"=>["Pervasive Genotypic Mosaicism", "founder", "Pronuclear Injection Genome editing technologies", "Founder Mice Derived", "technology", "germline genotyping method", "genotypic mosaicism issue", "genotypic mosaicism", "allele", "Cas 9 mRNAs", "zfn"], "article_id"=>1440607, "categories"=>["Biological Sciences"], "users"=>["Daniel Oliver", "Shuiqiao Yuan", "Hayden McSwiggin", "Wei Yan"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0129457.s001", "https://dx.doi.org/10.1371/journal.pone.0129457.s002"], "stats"=>{"downloads"=>0, "page_views"=>24, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Pervasive_Genotypic_Mosaicism_in_Founder_Mice_Derived_from_Genome_Editing_through_Pronuclear_Injection_/1440607", "title"=>"Pervasive Genotypic Mosaicism in Founder Mice Derived from Genome Editing through Pronuclear Injection", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-06-08 03:44:49"}

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Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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