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(a) Double nicking by RNA-Guided CRISPR Cas9 for enhanced genome-editing speci?city. Cas9 protein is shown in orange. Scissors indicate cleavage sites. (b) selleck products Fusion ... Table 1 Common CRISPR/Cas9 design tools Cas9 Protein and Other Factors Direct delivery of purified Cas9 protein and sgRNA into cells has been reported to result in reduced off-target effects compared to the delivery of plasmid sequences encoding Cas9 and sgRNA, because Cas9-sgRNA ribonucleoprotein complexes cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells.33,34 Off-target effects might be cell-type-specific and highly depending on the integrity of double-stranded breaks (DSBs) repair pathways of particular cell type.22 For example, nuclease off-target effects can occur in transformed human cell lines with dysregulated DSBs repair pathways, while whole-genome sequencing of healthy human pluripotent stem cell clones with relatively intact DSBs repairing capability have revealed very few off-target mutations attributable to the nucleases.35,36 Furthermore, it has been reported that methylation of DNA at CpG sites may impede the binding efficiency of Cas9 in cells, and small molecules that enhance CRISPR genome editing by promoting LDK378 precise genome editing via homology-directed repair (e.g., L755507, a 73-adrenergic receptor agonist, and Brefeldin A, an inhibitor of intracellular protein transport from the ER to the Golgi apparatus) or sequence-specific Quetiapine gene knockout via nonhomologous end-joining (NHEJ) (e.g., azidothymidineorTrifluridine) in pluripotent stem cells are being studied.21,22,30,37 Moreover, toward the goal of achieving more efficient genome editing with CRISPR/Cas9, more explorative studies by employing epigenetic, DSBs repairing pathways modulators, such as small molecules or RNAi strategies that can further enhance or inhibit specific genome-editing pathways of Cas9 via homology-directed repair or NHEJ, need to be more extensively examined. In summary, the seed sequence and the PAM, which are indispensable components of CRISPR/Cas9, need to be carefully designed. Moreover, the application of purified Cas9 protein as well as modulatory small molecules of DSBs pathways, which influence both on-target efficiency and off-target specificity, should be considered in individual application. Methods of Off-Target Detection Detecting off-target sites in a highly sensitive and comprehensive manner remains a key challenge in the field of gene editing.38 The T7 endonuclease I assay was initially used to detect off-target mutations, but this assay suffers poor sensitivity (it cannot detect off-target mutations that occur at frequencies