Homo sapiens Raw sequence reads. Homo sapiens

The CRISPR/Cas9 systems have revolutionized the field of genome editing by providing unprecedented control over gene sequences and gene expression in many species, including humans. Lentiviral vectors (LVs) are one of the primary delivery platforms for the CRISPR/Cas9 system due to their ability to accommodate large DNA payloads and sustain robust expression in a wide range of dividing and non-dividing cells. However, long-term expression of LV-delivered Cas9/guide RNA may lead to undesirable off-target effects characterized by non-specific RNA-DNA interactions and off-target DNA cleavages.Integrase-deficient lentiviral vectors (IDLVs) present an attractive means for delivery of CRISPR/Cas9 components because: (i) they are capable of transducing a broad range of cells and tissues, (ii) the packaging capacity of IDLVs is superior to that of other vectors (e.g. adeno-associated vectors AAVs)), and (iii) they are expressed transiently and demonstrate very weak integration capability.In this manuscript, we aimed to establish IDLVs as a means for safe and efficient delivery of CRISPR/Cas9. To this end, we developed an all-in-one vector cassette with increased production efficacy and demonstrated that CRISPR/Cas9 delivered by the improved IDLV vectors can mediate rapid and robust gene editing in dividing fibroblasts and post-mitotic brain neurons in vivo. Importantly, CRISPR/Cas9 was expressed transiently and exhibited a higher level of specificity and accuracy of gene editing than the corresponding integrase-competent vectors.