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A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC-Cas9 or cleavage by Cas9 in living cells.

St Martin A, Salamango D, Serebrenik A, Shaban N, Brown WL, Donati F, Munagala U, Conticello SG, Harris RS
Nucleic Acids Res. 2018 May 9. pii: 4994269. doi: 10.1093/nar/gky332. (Link opens in a new window) PubMed (Link opens in a new window) Article

Plasmids from Article

ID Plasmid Purpose
109425A3Ai-Cas9n-UGI-NLSExpresses human APOBEC3A containing an L1 intron fused to Cas9n, Uracil DNA Glycosylase Inhibitor, with a nuclear localization signal.
109426A3Bi-ctd-Cas9n-UGI-NLSExpresses the C-terminal catalytic domain of human APOBEC3B containing an L1 intron fused to Cas9n, Uracil DNA Glycosylase Inhibitor, with a nuclear localization signal.
109427plenti-CMV-mCherry-T2A-GFPA lentiviral backbone expressing mCherry-T2A-eGFP off of a CMV promoter.
109428ACE Reporter (pLenti-CMV-mCherry (+43) -T2A-eGFP)A lentiviral backbone expressing mCherry with a 43 basepair insert and eGFP off of a CMV promoter. A reporter for both Cas9 nuclease and APOBEC-mediated base-editing activity.
109429A3Bi-ctd E255A -Cas9n-UGI-NLSExpresses the C-terminal catalytically inactive mutant of human APOBEC3B containing an L1 intron fused to Cas9n, Uracil DNA Glycosylase Inhibitor, with a nuclear localization signal.
109430A3Ai E72A-Cas9n-UGI-NLSExpresses catalytically inactive human APOBEC3A with an L1 intron fused to Cas9n, Uracil DNA Glycosylase Inhibitor, with a nuclear localization signal.
109431mCherry Codon 59 sgRNAMLM3636 backbone containing a gRNA that guides Cas9 to codon 59 of mCherry in the ACE reporter
109432Non-specific sgRNAMLM3636 backbone containing a gRNA that does not bind to any sequence in the human genome.

Antibodies from Article