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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 exhibited significantly improved specificity.T7EI assays to determine the catalytic activity of fdCas9 on potential off-targets, identified by PROGNOS web-tool, for EMX1, AAVS1, CCR5 and HBB genomic targets. T7EI mutation detection assays for potential off-targets of EMX1.4 and EMX1.14 gRNA pair (Fig 5A). T7EI mutation detection assays for potential off-targets of AAVS1.2 and AAVS1.10 gRNA pair (Fig 5B). T7EI mutation detection assays for potential off-targets of CCR5.13 and CCR5.15 gRNA pair (Fig 5C). T7EI mutation detection assays for potential off-targets of HBB9 and HBB10 gRNA pair (Fig 5D). Note: * indicates the expected size of the DNA bands of corresponding amplicons cleaved by T7EI.
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pone.0133373.g005: fdCas9 exhibited significantly improved specificity.T7EI assays to determine the catalytic activity of fdCas9 on potential off-targets, identified by PROGNOS web-tool, for EMX1, AAVS1, CCR5 and HBB genomic targets. T7EI mutation detection assays for potential off-targets of EMX1.4 and EMX1.14 gRNA pair (Fig 5A). T7EI mutation detection assays for potential off-targets of AAVS1.2 and AAVS1.10 gRNA pair (Fig 5B). T7EI mutation detection assays for potential off-targets of CCR5.13 and CCR5.15 gRNA pair (Fig 5C). T7EI mutation detection assays for potential off-targets of HBB9 and HBB10 gRNA pair (Fig 5D). Note: * indicates the expected size of the DNA bands of corresponding amplicons cleaved by T7EI.

Mentions: Moreover, we employed our recently developed PROGNOS web-tool to interrogate the genome for potential off-targets by allowing different mismatches (up to 6 nts) and spacer lengths (0–50 nts). We used the gRNA pair that produced the highest catalytic activity on each genomic target (Fig 3I). We identified 10 potential off-targets for EMX1, 19 off-targets for AAVS1, 9 off-targets for CCR5, and 24 off-targets for HBB (Tables I-L in S2 File). We used the T7EI assay to test the catalytic activity at each potential off-target. Our data reveal that the fdCas9 exhibited significant improvement of specificity. For example, fdCas9 did not exhibit any catalytic activity on CCR5 and HBB off-targets (Fig 5C and 5D). However, very weak activity has been observed with 1 off-target of EMX1 and AVVS1 (Fig 5A and 5B). Therefore, fdCas9 exhibited a significant specificity compared to wtCas9.


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 exhibited significantly improved specificity.T7EI assays to determine the catalytic activity of fdCas9 on potential off-targets, identified by PROGNOS web-tool, for EMX1, AAVS1, CCR5 and HBB genomic targets. T7EI mutation detection assays for potential off-targets of EMX1.4 and EMX1.14 gRNA pair (Fig 5A). T7EI mutation detection assays for potential off-targets of AAVS1.2 and AAVS1.10 gRNA pair (Fig 5B). T7EI mutation detection assays for potential off-targets of CCR5.13 and CCR5.15 gRNA pair (Fig 5C). T7EI mutation detection assays for potential off-targets of HBB9 and HBB10 gRNA pair (Fig 5D). Note: * indicates the expected size of the DNA bands of corresponding amplicons cleaved by T7EI.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520497&req=5

pone.0133373.g005: fdCas9 exhibited significantly improved specificity.T7EI assays to determine the catalytic activity of fdCas9 on potential off-targets, identified by PROGNOS web-tool, for EMX1, AAVS1, CCR5 and HBB genomic targets. T7EI mutation detection assays for potential off-targets of EMX1.4 and EMX1.14 gRNA pair (Fig 5A). T7EI mutation detection assays for potential off-targets of AAVS1.2 and AAVS1.10 gRNA pair (Fig 5B). T7EI mutation detection assays for potential off-targets of CCR5.13 and CCR5.15 gRNA pair (Fig 5C). T7EI mutation detection assays for potential off-targets of HBB9 and HBB10 gRNA pair (Fig 5D). Note: * indicates the expected size of the DNA bands of corresponding amplicons cleaved by T7EI.
Mentions: Moreover, we employed our recently developed PROGNOS web-tool to interrogate the genome for potential off-targets by allowing different mismatches (up to 6 nts) and spacer lengths (0–50 nts). We used the gRNA pair that produced the highest catalytic activity on each genomic target (Fig 3I). We identified 10 potential off-targets for EMX1, 19 off-targets for AAVS1, 9 off-targets for CCR5, and 24 off-targets for HBB (Tables I-L in S2 File). We used the T7EI assay to test the catalytic activity at each potential off-target. Our data reveal that the fdCas9 exhibited significant improvement of specificity. For example, fdCas9 did not exhibit any catalytic activity on CCR5 and HBB off-targets (Fig 5C and 5D). However, very weak activity has been observed with 1 off-target of EMX1 and AVVS1 (Fig 5A and 5B). Therefore, fdCas9 exhibited a significant specificity compared to wtCas9.

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.