Journal: Nature Communications
Article Title: CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion
doi: 10.1038/s41467-021-22927-1
Figure Lengend Snippet: a Scheme depicting a generic CopyCatcher element. Light blue boxes: exons of a targeted gene on the donor chromosome; dark blue boxes: exons on the receiver chromosome; black lines: genomic DNA; red box: splice acceptor site (SA); yellow box: T2A self-cleavage peptide; light red arrow: DsRed reporter; dark purple arrow: gRNA; light blue arrow: selection marker mCerulean ; red hourglass marks: point mutations at or near initiator ATG codons of endogenous genes; short black line: Cas9/gRNA cleavage site; light blue circle and dark lines: Cas9/gRNA complex. Scissors in ( a ) denote insertion of cargo cassettes into DSBs on homologous chromosomes including SA, T2A, gRNA, and selection marker mCerulean . b – d Distinct outcomes of three DSB repair mechanisms followed by Cas9/gRNA cleavage of the homologous “receiver” chromosome. Slashes on the second exons in ( b )–( d ) indicate loss of function on the marked chromosomes. Labels indicate resulting phenotypes. e, f Mosaic clones of somatic gene conversion (SGC) in two CopyCatcher lines. Photographs show the phenotypes and fluorescence patterns of two CopyCatcher elements inserted into the intron of white and ple loci in flies without (( e ) w [3XP3-CC] , ( f ) ple [CC] ) or with (( e ) w [ATG-,3XP3-CC] , ( f ) ple [ATG-,CC] ) associated ATG – point mutations, or F1 mosaics resulting from Cas9-mediated copying (( e ) w [ATG-,3XP3-CC] / w +; Cas9/+, ( f ) ple [ATG-,CC] /Cas9). SGC clones generated by ple [ATG-,CC] are outlined with white dotted lines. Rightmost panels in ( e ) and ( f ) are higher magnification views of areas delineated by white boxes in the lower magnification views immediately to their left. At least five independent flies were imaged and observed in ( e ) and ( f ) with similar results. Scale bars stand for 150 pixels in ( e ) and ( f ).
Article Snippet: The SA-T2A sequence was amplified from pBS-KS-attB2-SA-T2A-3XGal80-Hsp70 series plasmids (Addgene 62951 and 62952) , gRNAs-expressing cassette targeting to the first intron of yellow , white , and ple were assembled following online published protocols (CRISPR fly Design - http://www.crisprflydesign.org/ ) and mCerulean selection marker was amplified from Addgene 27795 plasmid.
Techniques: Selection, Marker, Clone Assay, Fluorescence, Generated
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![a Scheme depicting a generic CopyCatcher element. Light blue boxes: exons of a targeted gene on the donor chromosome; dark blue boxes: exons on the receiver chromosome; black lines: genomic DNA; red box: splice acceptor site (SA); yellow box: <t>T2A</t> self-cleavage peptide; light red arrow: DsRed reporter; dark purple arrow: gRNA; light blue arrow: selection marker mCerulean ; red hourglass marks: point mutations at or near initiator ATG codons of endogenous genes; short black line: Cas9/gRNA cleavage site; light blue circle and dark lines: Cas9/gRNA complex. Scissors in ( a ) denote insertion of cargo cassettes into DSBs on homologous chromosomes including SA, T2A, gRNA, and selection marker mCerulean . b – d Distinct outcomes of three DSB repair mechanisms followed by Cas9/gRNA cleavage of the homologous “receiver” chromosome. Slashes on the second exons in ( b )–( d ) indicate loss of function on the marked chromosomes. Labels indicate resulting phenotypes. e, f Mosaic clones of somatic gene conversion (SGC) in two CopyCatcher lines. Photographs show the phenotypes and fluorescence patterns of two CopyCatcher elements inserted into the intron of white and ple loci in flies without (( e ) w [3XP3-CC] , ( f ) ple [CC] ) or with (( e ) w [ATG-,3XP3-CC] , ( f ) ple [ATG-,CC] ) associated ATG – point mutations, or F1 mosaics resulting from Cas9-mediated copying (( e ) w [ATG-,3XP3-CC] / w +; Cas9/+, ( f ) ple [ATG-,CC] /Cas9). SGC clones generated by ple [ATG-,CC] are outlined with white dotted lines. Rightmost panels in ( e ) and ( f ) are higher magnification views of areas delineated by white boxes in the lower magnification views immediately to their left. At least five independent flies were imaged and observed in ( e ) and ( f ) with similar results. Scale bars stand for 150 pixels in ( e ) and ( f ).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3449/pmc08113449/pmc08113449__41467_2021_22927_Fig1_HTML.jpg)