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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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Related in: MedlinePlus

On- and off-target cleavage by CRISPR/Cas9 systems targeting the HBB gene. (a) Guide strands are aligned to their target sites and corresponding region in HBB and HBD. Forward direction guide strands (marked ‘greater than’) are shown adjacent to NGG, representing the PAM sequence. Guide strands complementary to the reverse strand (marked ‘less than’) are listed to the right of CCN. Asterisks between HBB and HBD indicate nucleotides that differentiate the two genes. The first base shown in HBB is the sickle cell anemia mutation site. For clarity, the A, C, T and G nucleotides are shown in green, blue, red and black, respectively. (b) R-03-induced cleavage at HBB (on-target) and HBD (off-target) measured by the T7E1 assay. Cells were transfected with 100, 200, 400 or 800 ng of the R-03 CRISPR plasmid. (c) Guide strands ranked in order of the off-target mutation rates at HBD. Differences between the guide sequence and HBD are in red. A lowercase g indicates that the first base in HBB does not match the guide strands’ initial G (for all but R-01). The 12 bases closest to the PAM are boxed in blue and numbered on top.
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gkt714-F1: On- and off-target cleavage by CRISPR/Cas9 systems targeting the HBB gene. (a) Guide strands are aligned to their target sites and corresponding region in HBB and HBD. Forward direction guide strands (marked ‘greater than’) are shown adjacent to NGG, representing the PAM sequence. Guide strands complementary to the reverse strand (marked ‘less than’) are listed to the right of CCN. Asterisks between HBB and HBD indicate nucleotides that differentiate the two genes. The first base shown in HBB is the sickle cell anemia mutation site. For clarity, the A, C, T and G nucleotides are shown in green, blue, red and black, respectively. (b) R-03-induced cleavage at HBB (on-target) and HBD (off-target) measured by the T7E1 assay. Cells were transfected with 100, 200, 400 or 800 ng of the R-03 CRISPR plasmid. (c) Guide strands ranked in order of the off-target mutation rates at HBD. Differences between the guide sequence and HBD are in red. A lowercase g indicates that the first base in HBB does not match the guide strands’ initial G (for all but R-01). The 12 bases closest to the PAM are boxed in blue and numbered on top.

Mentions: There were no CRISPR target sites in the human HBB gene sequence with their proximal 12 bases unique in the human genome (8); therefore, we chose CRISPR/Cas9 guide strands targeting HBB by comparing the similar regions in the human hemoglobin δ (HBD) gene. We designed eight 20-base guide strands to target sites near the sickle mutation in the HBB gene (Figure 1a), each adjacent to a PAM sequence that contains the canonical trinucleotide NGG. We also designed five guide strands to target two segments in the human CCR5 gene (Figure 2a), and tested the corresponding CRISPR/Cas9 systems to determine their on-target cleavage and potential off-target activity at the human C-C chemokine receptor type 2 (CCR2) gene. Herein we use the name of the guide strand (such as R-03) to represent the CRISPR/Cas9 system with the specified guide strand.Figure 1.


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)

On- and off-target cleavage by CRISPR/Cas9 systems targeting the HBB gene. (a) Guide strands are aligned to their target sites and corresponding region in HBB and HBD. Forward direction guide strands (marked ‘greater than’) are shown adjacent to NGG, representing the PAM sequence. Guide strands complementary to the reverse strand (marked ‘less than’) are listed to the right of CCN. Asterisks between HBB and HBD indicate nucleotides that differentiate the two genes. The first base shown in HBB is the sickle cell anemia mutation site. For clarity, the A, C, T and G nucleotides are shown in green, blue, red and black, respectively. (b) R-03-induced cleavage at HBB (on-target) and HBD (off-target) measured by the T7E1 assay. Cells were transfected with 100, 200, 400 or 800 ng of the R-03 CRISPR plasmid. (c) Guide strands ranked in order of the off-target mutation rates at HBD. Differences between the guide sequence and HBD are in red. A lowercase g indicates that the first base in HBB does not match the guide strands’ initial G (for all but R-01). The 12 bases closest to the PAM are boxed in blue and numbered on top.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814385&req=5

gkt714-F1: On- and off-target cleavage by CRISPR/Cas9 systems targeting the HBB gene. (a) Guide strands are aligned to their target sites and corresponding region in HBB and HBD. Forward direction guide strands (marked ‘greater than’) are shown adjacent to NGG, representing the PAM sequence. Guide strands complementary to the reverse strand (marked ‘less than’) are listed to the right of CCN. Asterisks between HBB and HBD indicate nucleotides that differentiate the two genes. The first base shown in HBB is the sickle cell anemia mutation site. For clarity, the A, C, T and G nucleotides are shown in green, blue, red and black, respectively. (b) R-03-induced cleavage at HBB (on-target) and HBD (off-target) measured by the T7E1 assay. Cells were transfected with 100, 200, 400 or 800 ng of the R-03 CRISPR plasmid. (c) Guide strands ranked in order of the off-target mutation rates at HBD. Differences between the guide sequence and HBD are in red. A lowercase g indicates that the first base in HBB does not match the guide strands’ initial G (for all but R-01). The 12 bases closest to the PAM are boxed in blue and numbered on top.
Mentions: There were no CRISPR target sites in the human HBB gene sequence with their proximal 12 bases unique in the human genome (8); therefore, we chose CRISPR/Cas9 guide strands targeting HBB by comparing the similar regions in the human hemoglobin δ (HBD) gene. We designed eight 20-base guide strands to target sites near the sickle mutation in the HBB gene (Figure 1a), each adjacent to a PAM sequence that contains the canonical trinucleotide NGG. We also designed five guide strands to target two segments in the human CCR5 gene (Figure 2a), and tested the corresponding CRISPR/Cas9 systems to determine their on-target cleavage and potential off-target activity at the human C-C chemokine receptor type 2 (CCR2) gene. Herein we use the name of the guide strand (such as R-03) to represent the CRISPR/Cas9 system with the specified guide strand.Figure 1.

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
Related in: MedlinePlus