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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 robust activity with high target specificity.The genome modification activities of fdCas9 were assayed at known Cas9 off-targets. (A) T7EI assays at HBD, a known off-target for the HBB genomic target, showed high modification frequencies using wtCas9 with various gRNAs targeting HBB. (B) T7EI assays at HBD showed no detectable modification activities using fdCas9. (C) T7EI assays at CCR2, a known off-target for the CCR5 genomic target showed high modification frequencies with wtCas9 using various gRNAs targeting CCR5. (D) T7EI assays at HBD showed no detectable modification using fdCas9 guided by different combinations of gRNAs.
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pone.0133373.g004: fdCas9 exhibited robust activity with high target specificity.The genome modification activities of fdCas9 were assayed at known Cas9 off-targets. (A) T7EI assays at HBD, a known off-target for the HBB genomic target, showed high modification frequencies using wtCas9 with various gRNAs targeting HBB. (B) T7EI assays at HBD showed no detectable modification activities using fdCas9. (C) T7EI assays at CCR2, a known off-target for the CCR5 genomic target showed high modification frequencies with wtCas9 using various gRNAs targeting CCR5. (D) T7EI assays at HBD showed no detectable modification using fdCas9 guided by different combinations of gRNAs.

Mentions: To investigate whether fdCas9 catalytic activity is reduced at potential genomic off-target sites, we selected several genomic targets with known off-targets. As we have previously reported, wtCas9 exhibits substantial genomic modification at off-target sequences [30]. Therefore, we designed gRNA pairs with optimal spacer lengths for efficient fdCas9 activity at HBB and CCR5 that were capable of binding to the HBD and CCR2 off-targets as single gRNAs. Furthermore, we selected gRNA pairs with spacer lengths ranging from 31–39 bp. We included paired nickases and wtCas9 to compare the specificities of these reagents at the on- and off-target sites to ensure that each gRNA could direct Cas9 cleavage at the intended sites. As expected, wtCas9 produced substantial off-target activities ranging from 25% to 30% for different targets, CCR5 and HBB sites, as previously reported (Fig 4A and 4C and A and Fig G in S1 File). In contrast, the fdCas9 variant did not exhibit any catalytic activity at the off-target sequence. Moreover, the Cas9 nickases did not exhibit detectable off-target activity, as previously reported [35]. It might be possible to detect off-target events using deep sequencing coverage of the target sites. Nevertheless, fdCas9 is highly specific when 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 robust activity with high target specificity.The genome modification activities of fdCas9 were assayed at known Cas9 off-targets. (A) T7EI assays at HBD, a known off-target for the HBB genomic target, showed high modification frequencies using wtCas9 with various gRNAs targeting HBB. (B) T7EI assays at HBD showed no detectable modification activities using fdCas9. (C) T7EI assays at CCR2, a known off-target for the CCR5 genomic target showed high modification frequencies with wtCas9 using various gRNAs targeting CCR5. (D) T7EI assays at HBD showed no detectable modification using fdCas9 guided by different combinations of gRNAs.
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pone.0133373.g004: fdCas9 exhibited robust activity with high target specificity.The genome modification activities of fdCas9 were assayed at known Cas9 off-targets. (A) T7EI assays at HBD, a known off-target for the HBB genomic target, showed high modification frequencies using wtCas9 with various gRNAs targeting HBB. (B) T7EI assays at HBD showed no detectable modification activities using fdCas9. (C) T7EI assays at CCR2, a known off-target for the CCR5 genomic target showed high modification frequencies with wtCas9 using various gRNAs targeting CCR5. (D) T7EI assays at HBD showed no detectable modification using fdCas9 guided by different combinations of gRNAs.
Mentions: To investigate whether fdCas9 catalytic activity is reduced at potential genomic off-target sites, we selected several genomic targets with known off-targets. As we have previously reported, wtCas9 exhibits substantial genomic modification at off-target sequences [30]. Therefore, we designed gRNA pairs with optimal spacer lengths for efficient fdCas9 activity at HBB and CCR5 that were capable of binding to the HBD and CCR2 off-targets as single gRNAs. Furthermore, we selected gRNA pairs with spacer lengths ranging from 31–39 bp. We included paired nickases and wtCas9 to compare the specificities of these reagents at the on- and off-target sites to ensure that each gRNA could direct Cas9 cleavage at the intended sites. As expected, wtCas9 produced substantial off-target activities ranging from 25% to 30% for different targets, CCR5 and HBB sites, as previously reported (Fig 4A and 4C and A and Fig G in S1 File). In contrast, the fdCas9 variant did not exhibit any catalytic activity at the off-target sequence. Moreover, the Cas9 nickases did not exhibit detectable off-target activity, as previously reported [35]. It might be possible to detect off-target events using deep sequencing coverage of the target sites. Nevertheless, fdCas9 is highly specific when 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.