An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique
Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately...
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| Veröffentlicht in: | Molecular biology reports 2021-02, Vol.48 (2), p.1951-1957 |
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| creator | Gasanov, Eugene V. Jędrychowska, Justyna Pastor, Michal Wiweger, Malgorzata Methner, Axel Korzh, Vladimir P. |
| description | Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the
kcng4b
,
gdap1
, and
ghitm
genes in the zebrafish
Danio rerio
using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of
D. rerio
. |
| doi_str_mv | 10.1007/s11033-020-06125-8 |
| format | Article |
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kcng4b
,
gdap1
, and
ghitm
genes in the zebrafish
Danio rerio
using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of
D. rerio
.</description><identifier>ISSN: 0301-4851</identifier><identifier>EISSN: 1573-4978</identifier><identifier>DOI: 10.1007/s11033-020-06125-8</identifier><identifier>PMID: 33481178</identifier><language>eng</language><publisher>Dordrecht: Springer Netherlands</publisher><subject>Animal Anatomy ; Animal Biochemistry ; Animals ; Biomedical and Life Sciences ; CRISPR ; CRISPR-Cas Systems ; Danio rerio ; Deletion mutant ; Gene Deletion ; Gene Editing - methods ; Genome editing ; Genomes ; gRNA ; Heterozygote ; Histology ; Life Sciences ; Morphology ; Mutagenesis ; Mutation ; Nerve Tissue Proteins - genetics ; Non-homologous end joining ; Nuclease ; Nucleic Acid Denaturation ; Nucleotide sequence ; Nucleotides ; RNA, Guide, CRISPR-Cas Systems - genetics ; Short Communication ; Voltage-Dependent Anion Channels - genetics ; Zebrafish - genetics ; Zebrafish Proteins - genetics</subject><ispartof>Molecular biology reports, 2021-02, Vol.48 (2), p.1951-1957</ispartof><rights>The Author(s) 2021</rights><rights>The Author(s) 2021. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c474t-6262f0857502cf8f45604b3650109b0cb4b40823348a1241bc8594c40a4eed73</citedby><cites>FETCH-LOGICAL-c474t-6262f0857502cf8f45604b3650109b0cb4b40823348a1241bc8594c40a4eed73</cites><orcidid>0000-0002-5020-9406</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s11033-020-06125-8$$EPDF$$P50$$Gspringer$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s11033-020-06125-8$$EHTML$$P50$$Gspringer$$Hfree_for_read</linktohtml><link.rule.ids>230,314,780,784,885,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33481178$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gasanov, Eugene V.</creatorcontrib><creatorcontrib>Jędrychowska, Justyna</creatorcontrib><creatorcontrib>Pastor, Michal</creatorcontrib><creatorcontrib>Wiweger, Malgorzata</creatorcontrib><creatorcontrib>Methner, Axel</creatorcontrib><creatorcontrib>Korzh, Vladimir P.</creatorcontrib><title>An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique</title><title>Molecular biology reports</title><addtitle>Mol Biol Rep</addtitle><addtitle>Mol Biol Rep</addtitle><description>Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the
kcng4b
,
gdap1
, and
ghitm
genes in the zebrafish
Danio rerio
using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of
D. rerio
.</description><subject>Animal Anatomy</subject><subject>Animal Biochemistry</subject><subject>Animals</subject><subject>Biomedical and Life Sciences</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems</subject><subject>Danio rerio</subject><subject>Deletion mutant</subject><subject>Gene Deletion</subject><subject>Gene Editing - methods</subject><subject>Genome editing</subject><subject>Genomes</subject><subject>gRNA</subject><subject>Heterozygote</subject><subject>Histology</subject><subject>Life Sciences</subject><subject>Morphology</subject><subject>Mutagenesis</subject><subject>Mutation</subject><subject>Nerve Tissue Proteins - genetics</subject><subject>Non-homologous end joining</subject><subject>Nuclease</subject><subject>Nucleic Acid Denaturation</subject><subject>Nucleotide sequence</subject><subject>Nucleotides</subject><subject>RNA, Guide, CRISPR-Cas Systems - genetics</subject><subject>Short Communication</subject><subject>Voltage-Dependent Anion Channels - genetics</subject><subject>Zebrafish - 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methods</topic><topic>Genome editing</topic><topic>Genomes</topic><topic>gRNA</topic><topic>Heterozygote</topic><topic>Histology</topic><topic>Life Sciences</topic><topic>Morphology</topic><topic>Mutagenesis</topic><topic>Mutation</topic><topic>Nerve Tissue Proteins - genetics</topic><topic>Non-homologous end joining</topic><topic>Nuclease</topic><topic>Nucleic Acid Denaturation</topic><topic>Nucleotide sequence</topic><topic>Nucleotides</topic><topic>RNA, Guide, CRISPR-Cas Systems - genetics</topic><topic>Short Communication</topic><topic>Voltage-Dependent Anion Channels - genetics</topic><topic>Zebrafish - genetics</topic><topic>Zebrafish Proteins - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gasanov, Eugene V.</creatorcontrib><creatorcontrib>Jędrychowska, Justyna</creatorcontrib><creatorcontrib>Pastor, Michal</creatorcontrib><creatorcontrib>Wiweger, Malgorzata</creatorcontrib><creatorcontrib>Methner, Axel</creatorcontrib><creatorcontrib>Korzh, Vladimir P.</creatorcontrib><collection>Springer Nature OA Free Journals</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Molecular biology reports</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gasanov, Eugene V.</au><au>Jędrychowska, Justyna</au><au>Pastor, Michal</au><au>Wiweger, Malgorzata</au><au>Methner, Axel</au><au>Korzh, Vladimir P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique</atitle><jtitle>Molecular biology reports</jtitle><stitle>Mol Biol Rep</stitle><addtitle>Mol Biol Rep</addtitle><date>2021-02-01</date><risdate>2021</risdate><volume>48</volume><issue>2</issue><spage>1951</spage><epage>1957</epage><pages>1951-1957</pages><issn>0301-4851</issn><eissn>1573-4978</eissn><abstract>Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the
kcng4b
,
gdap1
, and
ghitm
genes in the zebrafish
Danio rerio
using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of
D. rerio
.</abstract><cop>Dordrecht</cop><pub>Springer Netherlands</pub><pmid>33481178</pmid><doi>10.1007/s11033-020-06125-8</doi><tpages>7</tpages><orcidid>https://orcid.org/0000-0002-5020-9406</orcidid><oa>free_for_read</oa></addata></record> |
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| ispartof | Molecular biology reports, 2021-02, Vol.48 (2), p.1951-1957 |
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| language | eng |
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| subjects | Animal Anatomy Animal Biochemistry Animals Biomedical and Life Sciences CRISPR CRISPR-Cas Systems Danio rerio Deletion mutant Gene Deletion Gene Editing - methods Genome editing Genomes gRNA Heterozygote Histology Life Sciences Morphology Mutagenesis Mutation Nerve Tissue Proteins - genetics Non-homologous end joining Nuclease Nucleic Acid Denaturation Nucleotide sequence Nucleotides RNA, Guide, CRISPR-Cas Systems - genetics Short Communication Voltage-Dependent Anion Channels - genetics Zebrafish - genetics Zebrafish Proteins - genetics |
| title | An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique |
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