基于TIDE和TIDER的绵羊囊胚基因组编辑效率分析方法的研究

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中图分类号：S813.3 文献标志码：A

Abstract：ObjectiveThereliabilityofTIDE（Trackof IndelsbyDecomposition）andTIDER（Trackingof Insertion，DEletionsand Recombinationevents）inanalyzingthegenomeeditingresultsofshpblastocystshasbeenverified.Theaimistoprovideanaurate andtimelyanalyticalmethodfordetectingthemutationeficiencyofgenome-editedshpembryosMethodsTDEisasimpleandaccurateassay topreciselydeterminethespectrumandfrequencyof targeted mutations generated inapoolofcelsby CRISPR/Cas9genomeeditingtols．TheTDEplatformfacilitatesthecharacterizationandquantificationofinducedmutationsinsheepblastocysts. Results The genomicsamplesofMSTNmutantsheepwereusedas materialsforPCRamplification.Flowing amplification，the DNA fragmentsareinsertedintoT-vectors，andsinglecloneswererandomlyselectedforsequening.Thesequencingresultsverfdtheaccuracyof thegenomeeditingresultsofTDEanalysis.TheeditingresultsgeneratedfromTIDERanalysisofsingle-strandedoligoucleotidetemplateintegrationwerecomparedwiththeDNArestritionenzymemap，soastoverifytheaccuracyofTIDERanalysisofforeignDNAshortfragmentintroductionConclusionTIDEandTIDERtoolscanefectivelyquantifytemutationrateofshepblastocysts andidentifythemaintypesofinsertionsanddeletions.Theyarehighlyeficientandcostefectivemetodsforanalyinggenoeediing mutations.

KeyWords：Cas9 RNPs；TIDE;TIDER;blastocysts; sheep

CRISPR/Cas9技术是1种强大的基因编辑工 （doublestrandbreaks，DSBs）。（剩余11363字）