title: Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells.
abstract: Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from Escherichia coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells.
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: Trine Skov
authors.initials: TS
authors.lastname: Petersen
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark; University of Cambridge, UK.
authors.forename: Kristopher Torp
authors.initials: KT
authors.lastname: Jensen
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Bolund
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany; Berlin Institute of Health, 10117 Berlin, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Kühn
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark. Electronic address: alun@biomed.au.dk.
authors.forename: Yonglun
authors.initials: Y
authors.lastname: Luo
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques / Q000379:methods*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D054643:INDEL Mutation; D008322:Mammals / Q000235:genetics*; D011938:Rec A Recombinases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D011938:Rec A Recombinases; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR; DSB; Rec A; SSA; SpCas9; SpCas9(1.1)