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CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.

Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G - Nucleic Acids Res. (2014)

Bottom Line: A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes.We further investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand.Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage.

View Article: PubMed Central - PubMed

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

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Activity for sgRNAs containing 5′-end truncations. (A) 1–6 bp truncations at the 5′ end of the guide sequence R-01 targeted to the HBB gene. (B) Activity for truncated sgRNAs. Truncated positions are highlighted in gray in the grid. Bar graph shows corresponding cleavage activity measured by T7E1 assay in HEK293T cells. Error bar, SEM (n = 2).
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Figure 3: Activity for sgRNAs containing 5′-end truncations. (A) 1–6 bp truncations at the 5′ end of the guide sequence R-01 targeted to the HBB gene. (B) Activity for truncated sgRNAs. Truncated positions are highlighted in gray in the grid. Bar graph shows corresponding cleavage activity measured by T7E1 assay in HEK293T cells. Error bar, SEM (n = 2).

Mentions: We further investigated if sgRNAs with small truncations at the 5′-end retain cleavage activity. One to six nucleotides were deleted from the 5′ end of R-01 except for the nucleotide at position 20, because the guanine here is required for the expression under the U6 promoter (Figure 3A). For these guide sequence truncations, we found that 1- to 2-bp 5′ truncations could still induce cleavage activities similar to the full-length sgRNA (Figure 3B).


CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.

Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G - Nucleic Acids Res. (2014)

Activity for sgRNAs containing 5′-end truncations. (A) 1–6 bp truncations at the 5′ end of the guide sequence R-01 targeted to the HBB gene. (B) Activity for truncated sgRNAs. Truncated positions are highlighted in gray in the grid. Bar graph shows corresponding cleavage activity measured by T7E1 assay in HEK293T cells. Error bar, SEM (n = 2).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066799&req=5

Figure 3: Activity for sgRNAs containing 5′-end truncations. (A) 1–6 bp truncations at the 5′ end of the guide sequence R-01 targeted to the HBB gene. (B) Activity for truncated sgRNAs. Truncated positions are highlighted in gray in the grid. Bar graph shows corresponding cleavage activity measured by T7E1 assay in HEK293T cells. Error bar, SEM (n = 2).
Mentions: We further investigated if sgRNAs with small truncations at the 5′-end retain cleavage activity. One to six nucleotides were deleted from the 5′ end of R-01 except for the nucleotide at position 20, because the guanine here is required for the expression under the U6 promoter (Figure 3A). For these guide sequence truncations, we found that 1- to 2-bp 5′ truncations could still induce cleavage activities similar to the full-length sgRNA (Figure 3B).

Bottom Line: A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes.We further investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand.Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage.

View Article: PubMed Central - PubMed

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

Show MeSH