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Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering.

Aouida M, Eid A, Ali Z, Cradick T, Lee C, Deshmukh H, Atef A, AbuSamra D, Gadhoum SZ, Merzaban J, Bao G, Mahfouz M - PLoS ONE (2015)

Bottom Line: Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9).Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites.Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Kingdom of Saudi Arabia.

ABSTRACT
The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15-39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

No MeSH data available.


Schematic representation of different dCas9 and FokI fusion variant architectures.(A) Schematic strategy used to test dCas9 and FokI fusion variants for homodimer formation and double-strand break (DSB) generation within the target sequence. A pair of gRNAs capable of guiding the dCas9 and FokI fusion variants and binding to the sense and antisense DNA strands to facilitate dimerization of the FokI catalytic domain is shown. (B) Schematic representation of dCas9 and FokI fusion variants. The FokI catalytic domain was fused to either the C- or N-terminus of dCas9 with different linker sequences to facilitate dimer formation. The fdCas9 variant was cloned under the CMV promoter with a linker of 16 amino acids and 4 NLSs, one in the N-terminal domain and three in the C-terminal domain. dCas9 was also cloned under the CMV promoter and used as a negative control. NLSs were included on either or both ends of the fusion protein to boost its nuclear localization.
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pone.0133373.g001: Schematic representation of different dCas9 and FokI fusion variant architectures.(A) Schematic strategy used to test dCas9 and FokI fusion variants for homodimer formation and double-strand break (DSB) generation within the target sequence. A pair of gRNAs capable of guiding the dCas9 and FokI fusion variants and binding to the sense and antisense DNA strands to facilitate dimerization of the FokI catalytic domain is shown. (B) Schematic representation of dCas9 and FokI fusion variants. The FokI catalytic domain was fused to either the C- or N-terminus of dCas9 with different linker sequences to facilitate dimer formation. The fdCas9 variant was cloned under the CMV promoter with a linker of 16 amino acids and 4 NLSs, one in the N-terminal domain and three in the C-terminal domain. dCas9 was also cloned under the CMV promoter and used as a negative control. NLSs were included on either or both ends of the fusion protein to boost its nuclear localization.

Mentions: A Cas9 nuclease dead (dCas9) version was generated by simultaneously disrupting the HNH and the RuvC catalytic domains [37]. The dCas9 protein variant was incapable of cleaving the DNA but retained the ability to be targeted by gRNAs. Since the Cas9 nuclease and the Cas9 nickases exhibit significant off-target activity, we attempted to design and constructed dCas9.FokI protein variants by using the dCas9 as a DNA targeting module and the non-specific catalytic domain of the FokI endonuclease as a cleaving module. Such biomodular protein variants, with architectures reminiscent of ZFN and TALEN architectures, can be used to generate homodimers on a DNA target sequence defined by the specificities of gRNA sequences on sense and antisense strands and the length of the intervening spacer sequence, (Fig 1A) [7]. We used the previously reported dCas9 backbone and generated different C- and N-terminus fusions of the FokI catalytic domain [37]. To generate the dCas9.FokI chimeric protein, we PCR-amplified and cloned the full-length dCas9 fragment into the pENTR/D TOPO vector (Life Technologies, Carlsbad, CA, USA) to facilitate subcloning and gateway recombination into a pDEST26 destination vector for mammalian cell expression (S1 File for sequencing data and map). To produce an in-frame dCas9 C-terminus fusion, we sub-cloned synthetic fragments composed of the catalytic domain of wild-type FokI preceded by three nuclear localization signals (3NLS) and a linker sequence (Fig 1B and S1 File). To produce an N-terminus fusion, we cloned a synthetic fragment composed of 3XFLAG, 1 NLS, 2GS, the wild-type FokI catalytic domain, and linker sequences into the N-terminus of dCas9 using the NcoI restriction enzyme (Fig 1B and S1 File). Subsequently, we cloned different C- and N-terminus fusions of dCas9 and FokI in pENTR/D into pDEST26 under the control of the CMV promoter for mammalian cell expression (Fig 1B). We tested dCas9 and FokI fusion variants for dimerization and catalytic activity on episomal and genomic DNA targets.


Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering.

Aouida M, Eid A, Ali Z, Cradick T, Lee C, Deshmukh H, Atef A, AbuSamra D, Gadhoum SZ, Merzaban J, Bao G, Mahfouz M - PLoS ONE (2015)

Schematic representation of different dCas9 and FokI fusion variant architectures.(A) Schematic strategy used to test dCas9 and FokI fusion variants for homodimer formation and double-strand break (DSB) generation within the target sequence. A pair of gRNAs capable of guiding the dCas9 and FokI fusion variants and binding to the sense and antisense DNA strands to facilitate dimerization of the FokI catalytic domain is shown. (B) Schematic representation of dCas9 and FokI fusion variants. The FokI catalytic domain was fused to either the C- or N-terminus of dCas9 with different linker sequences to facilitate dimer formation. The fdCas9 variant was cloned under the CMV promoter with a linker of 16 amino acids and 4 NLSs, one in the N-terminal domain and three in the C-terminal domain. dCas9 was also cloned under the CMV promoter and used as a negative control. NLSs were included on either or both ends of the fusion protein to boost its nuclear localization.
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Related In: Results  -  Collection

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pone.0133373.g001: Schematic representation of different dCas9 and FokI fusion variant architectures.(A) Schematic strategy used to test dCas9 and FokI fusion variants for homodimer formation and double-strand break (DSB) generation within the target sequence. A pair of gRNAs capable of guiding the dCas9 and FokI fusion variants and binding to the sense and antisense DNA strands to facilitate dimerization of the FokI catalytic domain is shown. (B) Schematic representation of dCas9 and FokI fusion variants. The FokI catalytic domain was fused to either the C- or N-terminus of dCas9 with different linker sequences to facilitate dimer formation. The fdCas9 variant was cloned under the CMV promoter with a linker of 16 amino acids and 4 NLSs, one in the N-terminal domain and three in the C-terminal domain. dCas9 was also cloned under the CMV promoter and used as a negative control. NLSs were included on either or both ends of the fusion protein to boost its nuclear localization.
Mentions: A Cas9 nuclease dead (dCas9) version was generated by simultaneously disrupting the HNH and the RuvC catalytic domains [37]. The dCas9 protein variant was incapable of cleaving the DNA but retained the ability to be targeted by gRNAs. Since the Cas9 nuclease and the Cas9 nickases exhibit significant off-target activity, we attempted to design and constructed dCas9.FokI protein variants by using the dCas9 as a DNA targeting module and the non-specific catalytic domain of the FokI endonuclease as a cleaving module. Such biomodular protein variants, with architectures reminiscent of ZFN and TALEN architectures, can be used to generate homodimers on a DNA target sequence defined by the specificities of gRNA sequences on sense and antisense strands and the length of the intervening spacer sequence, (Fig 1A) [7]. We used the previously reported dCas9 backbone and generated different C- and N-terminus fusions of the FokI catalytic domain [37]. To generate the dCas9.FokI chimeric protein, we PCR-amplified and cloned the full-length dCas9 fragment into the pENTR/D TOPO vector (Life Technologies, Carlsbad, CA, USA) to facilitate subcloning and gateway recombination into a pDEST26 destination vector for mammalian cell expression (S1 File for sequencing data and map). To produce an in-frame dCas9 C-terminus fusion, we sub-cloned synthetic fragments composed of the catalytic domain of wild-type FokI preceded by three nuclear localization signals (3NLS) and a linker sequence (Fig 1B and S1 File). To produce an N-terminus fusion, we cloned a synthetic fragment composed of 3XFLAG, 1 NLS, 2GS, the wild-type FokI catalytic domain, and linker sequences into the N-terminus of dCas9 using the NcoI restriction enzyme (Fig 1B and S1 File). Subsequently, we cloned different C- and N-terminus fusions of dCas9 and FokI in pENTR/D into pDEST26 under the control of the CMV promoter for mammalian cell expression (Fig 1B). We tested dCas9 and FokI fusion variants for dimerization and catalytic activity on episomal and genomic DNA targets.

Bottom Line: Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9).Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites.Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Kingdom of Saudi Arabia.

ABSTRACT
The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15-39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

No MeSH data available.