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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.


fdCas9 fusion variant exhibited robust catalytic activity in surrogate reporter assays.(A) Schematic representation of the pMRS plasmid used in surrogate reporter assays. Different versions were used including those with different spacer lengths in the intervening sequence between the monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP) reporters. gRNA pairs in PAM-in and PAM-out orientations were tested. (B) Flow cytometry of HEK293 cells at 3 d post-co-transfection with fdCas9 and the PCR amplicon of gRNA under the U6 promoter with a 36-bp spacer target. Paired Cas9 nickases and wtCas9 were used as positive controls and dCas9 as a negative control. The percentage of cells expressing both mRFP and eGFP is shown in the Q2 area of each panel.
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pone.0133373.g002: fdCas9 fusion variant exhibited robust catalytic activity in surrogate reporter assays.(A) Schematic representation of the pMRS plasmid used in surrogate reporter assays. Different versions were used including those with different spacer lengths in the intervening sequence between the monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP) reporters. gRNA pairs in PAM-in and PAM-out orientations were tested. (B) Flow cytometry of HEK293 cells at 3 d post-co-transfection with fdCas9 and the PCR amplicon of gRNA under the U6 promoter with a 36-bp spacer target. Paired Cas9 nickases and wtCas9 were used as positive controls and dCas9 as a negative control. The percentage of cells expressing both mRFP and eGFP is shown in the Q2 area of each panel.

Mentions: The catalytic activities of heterodimeric ZFNs and TALENs are sensitive to spacer length [5, 7]; therefore, we sought to determine the genome editing efficiency of different N- and C-terminus variants. We used a surrogate reporter system with various spacers (i.e., sizes) and orientations (i.e., protospacer adjacent motif (PAM)-in and PAM-out) (Fig 2A and S1 File) [38–40]. Briefly, this system was composed of two red and green fluorescent proteins (RFPs and GFPs) with their coding sequences separated by an intervening target sequence that contains a stop codon and renders GFP out of frame (Fig 2A). Targeted DSBs in the intervening sequence due to nuclease activity followed by non-homologous end joining makes 1/3 of the repair events in frame with the second GFP reporter, resulting in functional copies and green fluorescence. We transfected HEK293 cells with the DNA of dCas9 and FokI variants, pairs of gRNAs targeting the fusion proteins to the intervening sequence, and the pMRS reporter plasmid. All gRNA pairs were designed to bind in either PAM-in or PAM-out orientations, bringing the two dCas9.FokI monomers in close proximity to allow FokI dimer formation and subsequent catalytic activity (S1 File). Since FokI activity is dependent on dimerization of the FokI domain, we designed various combinations of forward and reverse gRNAs to test various spacer lengths with PAM-in and PAM-out orientations (S1 File). It has been reported previously that the optimal spacer length for ZFNs is 6–8 bp and for TALENs is 16–24 bp [7]. We therefore designed spacer lengths that ranged from 2–39 bp (S1 File). Binding of the dCas9 and FokI fusion monomers that permit dimer formation of the FokI domain results in DSBs, frameshift mutations, and the rescue of GFP expression.


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)

fdCas9 fusion variant exhibited robust catalytic activity in surrogate reporter assays.(A) Schematic representation of the pMRS plasmid used in surrogate reporter assays. Different versions were used including those with different spacer lengths in the intervening sequence between the monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP) reporters. gRNA pairs in PAM-in and PAM-out orientations were tested. (B) Flow cytometry of HEK293 cells at 3 d post-co-transfection with fdCas9 and the PCR amplicon of gRNA under the U6 promoter with a 36-bp spacer target. Paired Cas9 nickases and wtCas9 were used as positive controls and dCas9 as a negative control. The percentage of cells expressing both mRFP and eGFP is shown in the Q2 area of each panel.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520497&req=5

pone.0133373.g002: fdCas9 fusion variant exhibited robust catalytic activity in surrogate reporter assays.(A) Schematic representation of the pMRS plasmid used in surrogate reporter assays. Different versions were used including those with different spacer lengths in the intervening sequence between the monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP) reporters. gRNA pairs in PAM-in and PAM-out orientations were tested. (B) Flow cytometry of HEK293 cells at 3 d post-co-transfection with fdCas9 and the PCR amplicon of gRNA under the U6 promoter with a 36-bp spacer target. Paired Cas9 nickases and wtCas9 were used as positive controls and dCas9 as a negative control. The percentage of cells expressing both mRFP and eGFP is shown in the Q2 area of each panel.
Mentions: The catalytic activities of heterodimeric ZFNs and TALENs are sensitive to spacer length [5, 7]; therefore, we sought to determine the genome editing efficiency of different N- and C-terminus variants. We used a surrogate reporter system with various spacers (i.e., sizes) and orientations (i.e., protospacer adjacent motif (PAM)-in and PAM-out) (Fig 2A and S1 File) [38–40]. Briefly, this system was composed of two red and green fluorescent proteins (RFPs and GFPs) with their coding sequences separated by an intervening target sequence that contains a stop codon and renders GFP out of frame (Fig 2A). Targeted DSBs in the intervening sequence due to nuclease activity followed by non-homologous end joining makes 1/3 of the repair events in frame with the second GFP reporter, resulting in functional copies and green fluorescence. We transfected HEK293 cells with the DNA of dCas9 and FokI variants, pairs of gRNAs targeting the fusion proteins to the intervening sequence, and the pMRS reporter plasmid. All gRNA pairs were designed to bind in either PAM-in or PAM-out orientations, bringing the two dCas9.FokI monomers in close proximity to allow FokI dimer formation and subsequent catalytic activity (S1 File). Since FokI activity is dependent on dimerization of the FokI domain, we designed various combinations of forward and reverse gRNAs to test various spacer lengths with PAM-in and PAM-out orientations (S1 File). It has been reported previously that the optimal spacer length for ZFNs is 6–8 bp and for TALENs is 16–24 bp [7]. We therefore designed spacer lengths that ranged from 2–39 bp (S1 File). Binding of the dCas9 and FokI fusion monomers that permit dimer formation of the FokI domain results in DSBs, frameshift mutations, and the rescue of GFP expression.

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.