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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 CCR5 and CCR2 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-25 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-25 was amplified using a CCR2 forward primer and reverse primer downstream of the CCR5 site. The PCR products were sequenced and aligned to ‘CCR2-CCR5’ with the bases unique to CCR2 or CCR5 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-25. Insertions and point mutations are marked in yellow and deletions (:) are highlighted in gray.
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gkt714-F4: Chromosomal deletions in CCR5 and CCR2 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-25 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-25 was amplified using a CCR2 forward primer and reverse primer downstream of the CCR5 site. The PCR products were sequenced and aligned to ‘CCR2-CCR5’ with the bases unique to CCR2 or CCR5 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-25. Insertions and point mutations are marked in yellow and deletions (:) are highlighted in gray.

Mentions: Similarly, CCR5 is located ∼8 kb upstream of CCR2 on chromosome 3; thus, chromosomal rearrangements may occur with cleavages at both CCR5 and CCR2. These gross chromosomal deletions were detected with the R-25 CRISPR/Cas9 system, which cleaved both genes at high rates (Figure 4a and b). Here again, PCR amplification and sequence analysis revealed two cleavage events in (or near) a conserved region of the CCR5 and CCR2 genes, as indicated by indels consistent with NHEJ (Figure 4c). Cells transfected with the R-30 CRISPR/Cas9 system also had chromosomal deletions between CCR5 and CCR2 (Supplementary Figure S1h).Figure 4.


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 CCR5 and CCR2 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-25 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-25 was amplified using a CCR2 forward primer and reverse primer downstream of the CCR5 site. The PCR products were sequenced and aligned to ‘CCR2-CCR5’ with the bases unique to CCR2 or CCR5 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-25. Insertions and point mutations are marked in yellow and deletions (:) are highlighted in gray.
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gkt714-F4: Chromosomal deletions in CCR5 and CCR2 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-25 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-25 was amplified using a CCR2 forward primer and reverse primer downstream of the CCR5 site. The PCR products were sequenced and aligned to ‘CCR2-CCR5’ with the bases unique to CCR2 or CCR5 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-25. Insertions and point mutations are marked in yellow and deletions (:) are highlighted in gray.
Mentions: Similarly, CCR5 is located ∼8 kb upstream of CCR2 on chromosome 3; thus, chromosomal rearrangements may occur with cleavages at both CCR5 and CCR2. These gross chromosomal deletions were detected with the R-25 CRISPR/Cas9 system, which cleaved both genes at high rates (Figure 4a and b). Here again, PCR amplification and sequence analysis revealed two cleavage events in (or near) a conserved region of the CCR5 and CCR2 genes, as indicated by indels consistent with NHEJ (Figure 4c). Cells transfected with the R-30 CRISPR/Cas9 system also had chromosomal deletions between CCR5 and CCR2 (Supplementary Figure S1h).Figure 4.

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.

Show MeSH