Limits...
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.


Robust catalytic activity of the fdCas9 variant on endogenous genomic targets.(A, C, E, and G) T7EI assays for the EMX1, AAVS1, CCR5, and HBB genomic targets, respectively with fdCas9 using several combinations of gRNAs in PAM-in and PAM-out orientations. Arrows in (C) indicate the expected size of the DNA bands of AAVS1 amplicons cleaved by T7EI. (B, D, F, and H) Alignment of Sanger sequencing reads of PCR amplicons encompassing the EMX1, AAVS1, CCR5, and HBB target sequences showing indels within the 37-, 20-, 17-, and 28-bp spacer sequences, respectively. gRNA targets are highlighted in green, the PAM sequence is shown in bold and underlined, dashes indicate nucleotide deletions, nucleotides highlighted in red indicate insertions, and nucleotides highlighted in blue indicate substitutions. Mutation frequencies were estimated as the number of mutant clones divided by the total number of sequenced clones. (I) Catalytic activities of fdCas9 on different genomic targets using gRNA pairs with different spacer sizes (represented in percentage).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520497&req=5

pone.0133373.g003: Robust catalytic activity of the fdCas9 variant on endogenous genomic targets.(A, C, E, and G) T7EI assays for the EMX1, AAVS1, CCR5, and HBB genomic targets, respectively with fdCas9 using several combinations of gRNAs in PAM-in and PAM-out orientations. Arrows in (C) indicate the expected size of the DNA bands of AAVS1 amplicons cleaved by T7EI. (B, D, F, and H) Alignment of Sanger sequencing reads of PCR amplicons encompassing the EMX1, AAVS1, CCR5, and HBB target sequences showing indels within the 37-, 20-, 17-, and 28-bp spacer sequences, respectively. gRNA targets are highlighted in green, the PAM sequence is shown in bold and underlined, dashes indicate nucleotide deletions, nucleotides highlighted in red indicate insertions, and nucleotides highlighted in blue indicate substitutions. Mutation frequencies were estimated as the number of mutant clones divided by the total number of sequenced clones. (I) Catalytic activities of fdCas9 on different genomic targets using gRNA pairs with different spacer sizes (represented in percentage).

Mentions: We included all FokI.dCas9 fusion variants to test and confirm whether the catalytic activity and target specificity were similar to those observed in the surrogate reporter system assays. To this end, we selected four genes, CCR5, HBB, AAVS1, and EMX1, and employed the T7 endonuclease I (T7EI) surveyor assay to determine the catalytic activities of the dCas9 and FokI fusion variants, as previously described [30]. Our T7EI assays demonstrated activity of wtCas9 and some activity of the paired nickases, the positive controls, and no detectable activity of dCas9, the negative control, confirming correct gRNA designs and as well as the validity of the T7EI assay (Figs A and D in S1 File). Furthermore, our data on the modification of the four genomic targets corroborate our previous data obtained using the surrogate reporter system (Fig 2B). None of the dCas9f variants exhibited any catalytic activity on any genomic target for various spacer lengths in PAM-in and PAM-out orientations (Fig D in S1 File). In contrast, the fdCas9 variant produced robust catalytic activity on the genomic targets only in the PAM-out orientation of the dual gRNAs, consistent with our data using the surrogate reporter system (Fig 3A, 3C, 3E and 3G for EMX1, AAVS1, CCR5, and HBB, respectively). It is worth noting that the fdCas9 variant exhibited robust catalytic activity for different spacer lengths (17–37 bp) on genomic targets (Fig 3). No detectable activity was observed for the fdCas9 variant with a single gRNA, verifying that fdCas9 monomers were catalytically inactive (Fig C in S1 File). To precisely determine the cleavage site and the nature of indels, we PCR-amplified fragments encompassing the target site only with the combination of gRNAs that exhibited the highest fdCas9 activity for EMX1, AAVS1, CCR5, and HBB and cloned the amplicons into plasmid vectors using the TOPO TA Cloning Kit for Sequencing (Life Technologies). Based on Sanger sequencing data for these clones, fdCas9-induced DSBs were within the spacer sequence and resulted in indels of various lengths (Fig 3B, 3D, 3F and 3H for EMX1, AAVS1, CCR5, and HBB, respectively).


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)

Robust catalytic activity of the fdCas9 variant on endogenous genomic targets.(A, C, E, and G) T7EI assays for the EMX1, AAVS1, CCR5, and HBB genomic targets, respectively with fdCas9 using several combinations of gRNAs in PAM-in and PAM-out orientations. Arrows in (C) indicate the expected size of the DNA bands of AAVS1 amplicons cleaved by T7EI. (B, D, F, and H) Alignment of Sanger sequencing reads of PCR amplicons encompassing the EMX1, AAVS1, CCR5, and HBB target sequences showing indels within the 37-, 20-, 17-, and 28-bp spacer sequences, respectively. gRNA targets are highlighted in green, the PAM sequence is shown in bold and underlined, dashes indicate nucleotide deletions, nucleotides highlighted in red indicate insertions, and nucleotides highlighted in blue indicate substitutions. Mutation frequencies were estimated as the number of mutant clones divided by the total number of sequenced clones. (I) Catalytic activities of fdCas9 on different genomic targets using gRNA pairs with different spacer sizes (represented in percentage).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520497&req=5

pone.0133373.g003: Robust catalytic activity of the fdCas9 variant on endogenous genomic targets.(A, C, E, and G) T7EI assays for the EMX1, AAVS1, CCR5, and HBB genomic targets, respectively with fdCas9 using several combinations of gRNAs in PAM-in and PAM-out orientations. Arrows in (C) indicate the expected size of the DNA bands of AAVS1 amplicons cleaved by T7EI. (B, D, F, and H) Alignment of Sanger sequencing reads of PCR amplicons encompassing the EMX1, AAVS1, CCR5, and HBB target sequences showing indels within the 37-, 20-, 17-, and 28-bp spacer sequences, respectively. gRNA targets are highlighted in green, the PAM sequence is shown in bold and underlined, dashes indicate nucleotide deletions, nucleotides highlighted in red indicate insertions, and nucleotides highlighted in blue indicate substitutions. Mutation frequencies were estimated as the number of mutant clones divided by the total number of sequenced clones. (I) Catalytic activities of fdCas9 on different genomic targets using gRNA pairs with different spacer sizes (represented in percentage).
Mentions: We included all FokI.dCas9 fusion variants to test and confirm whether the catalytic activity and target specificity were similar to those observed in the surrogate reporter system assays. To this end, we selected four genes, CCR5, HBB, AAVS1, and EMX1, and employed the T7 endonuclease I (T7EI) surveyor assay to determine the catalytic activities of the dCas9 and FokI fusion variants, as previously described [30]. Our T7EI assays demonstrated activity of wtCas9 and some activity of the paired nickases, the positive controls, and no detectable activity of dCas9, the negative control, confirming correct gRNA designs and as well as the validity of the T7EI assay (Figs A and D in S1 File). Furthermore, our data on the modification of the four genomic targets corroborate our previous data obtained using the surrogate reporter system (Fig 2B). None of the dCas9f variants exhibited any catalytic activity on any genomic target for various spacer lengths in PAM-in and PAM-out orientations (Fig D in S1 File). In contrast, the fdCas9 variant produced robust catalytic activity on the genomic targets only in the PAM-out orientation of the dual gRNAs, consistent with our data using the surrogate reporter system (Fig 3A, 3C, 3E and 3G for EMX1, AAVS1, CCR5, and HBB, respectively). It is worth noting that the fdCas9 variant exhibited robust catalytic activity for different spacer lengths (17–37 bp) on genomic targets (Fig 3). No detectable activity was observed for the fdCas9 variant with a single gRNA, verifying that fdCas9 monomers were catalytically inactive (Fig C in S1 File). To precisely determine the cleavage site and the nature of indels, we PCR-amplified fragments encompassing the target site only with the combination of gRNAs that exhibited the highest fdCas9 activity for EMX1, AAVS1, CCR5, and HBB and cloned the amplicons into plasmid vectors using the TOPO TA Cloning Kit for Sequencing (Life Technologies). Based on Sanger sequencing data for these clones, fdCas9-induced DSBs were within the spacer sequence and resulted in indels of various lengths (Fig 3B, 3D, 3F and 3H for EMX1, AAVS1, CCR5, and HBB, respectively).

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.