often off-target effects, which pose a problem to obtaining consistent results [45].
CRISPR/Cas9 technology allows for permanent genetic modification of single or multiple genes simultaneously by transient or stable viral (retrovirus or lentivirus) delivery of the CRISPR/Cas9 components into cells [46–48]. Taking advantage of the indels generated in response to DSB and subsequent NHEJ, sgRNAs can be designed to target Cas9 to bind the initial protein-coding exons of a gene, which can generate LOF frameshift indel mutations upon DSB ( Fig. 1). This approach has been commonly used to generate LOF of genes of interest in the germline, somatically and in cell lines [49]. Recently, the Church group successfully edited 62 copies of the porcine endogenous retrovirus (PERV) in porcine cells, which opens up the road for safer porcine-to-human xenotransplantation by prevent-ing PERV transmission to human cells [50]. Alternatively, to introduce point mutations or complex knock-in alleles using precise genome editing can be achieved by designing sgRNAs against the locus of interest and co-delivering the sgRNAs with a template DNA used for HDR ( Fig. 1).