LAUSR

To date, it has been broadly demonstrated and well proven that the CRISPR-derived genome and epigenome edit ing technologies have completely revolutionised and significantly accelerated discoveries and breakthroughs in gene therapy, animal modelling, drug screening, functional genomics etc. Similar to other gene editing tools such as ZFN and TALEN, the CRISPR gene editing tool has two important characteristics: (i) programmable binding to a specific locus in the genome of a living cell/organism; (ii) enzymatic introduction of a double-strand break (DSB) within or adjacent to the binding site. The beauty of CRISPR gene editing system is that it contains a programmable small RNA, known as the guide RNA (gRNA), which directs a sole CRISPR-associated protein (Cas) to the target site where it introduces a DSB. In mammalian cells, unrepaired DSBs are detrimental, subsequently leading to genomic instability and chromosomal aberrations. Cells have thus developed self-protecting and systematic DNA damage repair mechanisms to repair the DSBs. In mammalian cells, DSBs are mainly repaired by the non-homologous end joining (NHEJ) pathway leading to introduction of small deletion or insertions (indels) at the break site after repairing. The introduction of DSBs also stimulates the homology-directed repair (HDR) machinery in mammalian cells. However, compared with the NHEJ pathway, the efficiency of DSB repair by HDR is approximately 40 folds lower (Weinstock and Jasin 2006). The principle of all current gene editing technologies is based on hijacking programmable DNA endonucleases to introduce specific DSBs and activate the cellular DSB repair machinery. While CRISPR technology has been so broadly used, its specificity versus risk of off-target effects is consistently ranked as the top concern especially in clinically orientated applications. In this editorial, I would like to focus the CRISPR/ Cas9 off-targets into two main categories: (1) Off-target cleavage and (2) Off-target modification. The first problem, off-target cleavage, refers to the introduction of either single-strand nickings by Cas9 nickase or DSBs by wild-type Cas9 at unintended genomic loci. This is the typical category of off-targets that most applications are addressing. The Cas9 protein contains two catalytic domains, RuvC and HNH, which are responsible for cleaving the non-complementary and complementary strand of the target DNA, respectively (Nishimasu et al. 2014). Introducing catalytically inactivating mutations to one or both domains has generated Cas9 nickase (nCas9) or dead Cas9 (dCas9). The Cell Biol Toxicol https://doi.org/10.1007/s10565-019-09482-8