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CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity.

Cradick TJ, Fine EJ, Antico CJ, Bao G - Nucleic Acids Res. (2013)

Bottom Line: The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering.We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations.Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites, including those with mismatches to the 12-bases proximal to the guide strand protospacer-adjacent motif.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.

ABSTRACT
The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering. However, the target specificity of CRISPR systems is largely unknown. Here we demonstrate that CRISPR/Cas9 systems targeting the human hemoglobin β and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin δ and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions. The guide strands of the CRISPR/Cas9 systems were designed to have a range of mismatches with the sequences of potential off-target sites. Off-target analysis was performed using the T7 endonuclease I mutation detection assay and Sanger sequencing. We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations. Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites, including those with mismatches to the 12-bases proximal to the guide strand protospacer-adjacent motif.

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Chromosomal deletions in HBB and HBD induced by CRISPR/Cas9 systems. HEK-293T cells were transfected with each CRISPR construct, and their genomic DNA harvested after 3 days in culture. The (a) on- and (b) off-target loci for guide strands R-03 were amplified with flanking PCR primers, cloned and Sanger sequenced. Sequencing reads are given for each guide strand and aligned to the wild-type sequence. The number of times each read occurred is indicated to the left of the alignment. Unmodified reads are indicated by ‘WT’. In (b) the guide strand mismatch is boxed. In (a) and (b), the A, C, T and G nucleotides are shown in green, blue, red and black, respectively, for clarity. (c) Genomic DNA from cells treated with R-03 was amplified using an HBD forward primer and reverse primer downstream of the HBB site. The PCR products were sequenced and aligned to ‘HBB-HBD’ with the bases unique to HBB or HBD indicated in blue or green, respectively, surrounding an identical area found in both genes. Sequencing detected that each product contained indels and mutations consistent with NHEJ, near the target sites for R-03. Insertions and point mutations are marked in yellow and deletions (:) are highlighted in gray.
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gkt714-F3: Chromosomal deletions in HBB and HBD induced by CRISPR/Cas9 systems. HEK-293T cells were transfected with each CRISPR construct, and their genomic DNA harvested after 3 days in culture. The (a) on- and (b) off-target loci for guide strands R-03 were amplified with flanking PCR primers, cloned and Sanger sequenced. Sequencing reads are given for each guide strand and aligned to the wild-type sequence. The number of times each read occurred is indicated to the left of the alignment. Unmodified reads are indicated by ‘WT’. In (b) the guide strand mismatch is boxed. In (a) and (b), the A, C, T and G nucleotides are shown in green, blue, red and black, respectively, for clarity. (c) Genomic DNA from cells treated with R-03 was amplified using an HBD forward primer and reverse primer downstream of the HBB site. The PCR products were sequenced and aligned to ‘HBB-HBD’ with the bases unique to HBB or HBD indicated in blue or green, respectively, surrounding an identical area found in both genes. Sequencing detected that each product contained indels and mutations consistent with NHEJ, near the target sites for R-03. Insertions and point mutations are marked in yellow and deletions (:) are highlighted in gray.

Mentions: Guide strands directed toward HBB resulted in high rates of on-target activity, with an average mutation frequency of 54% measured by the T7E1 assay (Figure 1c, Supplementary Figure S2). Because the T7E1 assay may not cleave the PCR product completely and assumptions must be made about the indel diversity to calculate the mutation percentages (21), we verified the mutation frequencies using Sanger sequencing. We found that for some guide strands and loci, Sanger sequencing gave much higher mutation frequencies than the T7E1 measurements. For example, Sanger sequencing of the HBB loci indicated that R-02 and R-03 resulted, respectively, in 60 of 80 (75%) and 31 of 44 (70%) sequences with insertions or deletions (indels) indicative of the error-prone nonhomologous end-joining (NHEJ) DNA repair pathway (Supplementary Figure S1a, Figure 3a). Similarly, HEK-293T cells transfected with CRISPR constructs containing guide strands targeting CCR5 resulted in high rates of on-target activity, with an average of 57% mutation frequency measured by the T7E1 assay (Figure 2c, Supplementary Figure S2).Figure 3.


CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity.

Cradick TJ, Fine EJ, Antico CJ, Bao G - Nucleic Acids Res. (2013)

Chromosomal deletions in HBB and HBD induced by CRISPR/Cas9 systems. HEK-293T cells were transfected with each CRISPR construct, and their genomic DNA harvested after 3 days in culture. The (a) on- and (b) off-target loci for guide strands R-03 were amplified with flanking PCR primers, cloned and Sanger sequenced. Sequencing reads are given for each guide strand and aligned to the wild-type sequence. The number of times each read occurred is indicated to the left of the alignment. Unmodified reads are indicated by ‘WT’. In (b) the guide strand mismatch is boxed. In (a) and (b), the A, C, T and G nucleotides are shown in green, blue, red and black, respectively, for clarity. (c) Genomic DNA from cells treated with R-03 was amplified using an HBD forward primer and reverse primer downstream of the HBB site. The PCR products were sequenced and aligned to ‘HBB-HBD’ with the bases unique to HBB or HBD indicated in blue or green, respectively, surrounding an identical area found in both genes. Sequencing detected that each product contained indels and mutations consistent with NHEJ, near the target sites for R-03. Insertions and point mutations are marked in yellow and deletions (:) are highlighted in gray.
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gkt714-F3: Chromosomal deletions in HBB and HBD induced by CRISPR/Cas9 systems. HEK-293T cells were transfected with each CRISPR construct, and their genomic DNA harvested after 3 days in culture. The (a) on- and (b) off-target loci for guide strands R-03 were amplified with flanking PCR primers, cloned and Sanger sequenced. Sequencing reads are given for each guide strand and aligned to the wild-type sequence. The number of times each read occurred is indicated to the left of the alignment. Unmodified reads are indicated by ‘WT’. In (b) the guide strand mismatch is boxed. In (a) and (b), the A, C, T and G nucleotides are shown in green, blue, red and black, respectively, for clarity. (c) Genomic DNA from cells treated with R-03 was amplified using an HBD forward primer and reverse primer downstream of the HBB site. The PCR products were sequenced and aligned to ‘HBB-HBD’ with the bases unique to HBB or HBD indicated in blue or green, respectively, surrounding an identical area found in both genes. Sequencing detected that each product contained indels and mutations consistent with NHEJ, near the target sites for R-03. Insertions and point mutations are marked in yellow and deletions (:) are highlighted in gray.
Mentions: Guide strands directed toward HBB resulted in high rates of on-target activity, with an average mutation frequency of 54% measured by the T7E1 assay (Figure 1c, Supplementary Figure S2). Because the T7E1 assay may not cleave the PCR product completely and assumptions must be made about the indel diversity to calculate the mutation percentages (21), we verified the mutation frequencies using Sanger sequencing. We found that for some guide strands and loci, Sanger sequencing gave much higher mutation frequencies than the T7E1 measurements. For example, Sanger sequencing of the HBB loci indicated that R-02 and R-03 resulted, respectively, in 60 of 80 (75%) and 31 of 44 (70%) sequences with insertions or deletions (indels) indicative of the error-prone nonhomologous end-joining (NHEJ) DNA repair pathway (Supplementary Figure S1a, Figure 3a). Similarly, HEK-293T cells transfected with CRISPR constructs containing guide strands targeting CCR5 resulted in high rates of on-target activity, with an average of 57% mutation frequency measured by the T7E1 assay (Figure 2c, Supplementary Figure S2).Figure 3.

Bottom Line: The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering.We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations.Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites, including those with mismatches to the 12-bases proximal to the guide strand protospacer-adjacent motif.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.

ABSTRACT
The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering. However, the target specificity of CRISPR systems is largely unknown. Here we demonstrate that CRISPR/Cas9 systems targeting the human hemoglobin β and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin δ and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions. The guide strands of the CRISPR/Cas9 systems were designed to have a range of mismatches with the sequences of potential off-target sites. Off-target analysis was performed using the T7 endonuclease I mutation detection assay and Sanger sequencing. We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations. Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites, including those with mismatches to the 12-bases proximal to the guide strand protospacer-adjacent motif.

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