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The utility of CRISPR-Cas9 and TALENs for genome editing may be compromised by their off-target activity. We show that integrase-defective lentiviral vectors (IDLVs) can detect such off-target cleavage with a frequency as low as 1%. In the case of Cas9, we find frequent off-target sites with a one-base bulge or up to 13 mismatches between the single guide RNA (sgRNA) and its genomic target, which refines sgRNA design.
Several groups have reported that the linear double-stranded IDLV genome could be incorporated preferentially into DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ). In this study, the scientists report the use of IDLVs to measure the off-target activities of CRISPR-Cas9 and TALENs. These results suggest that TALEN and CRISPR–D10A Cas9 (a mutant nuclease with nickase activity), have improved target specificity over CRISPR-Cas9.
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