sequence on the DNA of interest. Editing specificity can therefore be changed by modifying this spacer sequence. Upon system delivery to a cell, Cas9 and the gRNA bind, forming a ribonucleoprotein complex. This causes a conformational change in Cas9, allowing it to cleave DNA if the gRNA spacer sequence binds with sufficient homology to a particular sequence in the host genome. When the gRNA binds to the target sequence, Cas will cleave the locus, causing a double-strand break (DSB). The resulting DSB can be repaired by one of two mechanisms – Non-Homologous End Joining (NHEJ) - an efficient but error-prone mechanism, which often introduces insertions and deletions (indels) at the DSB site. This means it is often used in knockout experiments to disrupt genes and introduce loss of function mutations. Homology Directed Repair (HDR) - a less efficient but high-fidelity process which is used to introduce precise modifications into the target sequence. The process requires adding a DNA repair template including a desired sequence, which the cell's machinery uses to repair the DSB, incorporating the sequence of interest into the genome. Since NHEJ is more efficient than HDR, most DSBs will be repaired via NHEJ, introducing gene knockouts. To increase frequency of HDR, inhibiting genes associated with NHEJ and performing the process in particular cell cycle phases (primarily S and G2) appear effective. CRISPR/Cas9 is an effective way of manipulating the genome in vivo in animals as well as in human cells in vitro, but some issues with the efficiency of delivery and editing mean that it is not considered safe for use in viable human embryos or the body's germ cells. As well as the higher efficiency of NHEJ making inadvertent knockouts likely, CRISPR can introduce DSBs to unintended parts of the genome, called off-target effects. These arise due to the spacer sequence of the gRNA conferring sufficient sequence homology to random loci in the genome, which can introduce random mutations throughout. If performed in germline cells, mutations could be introduced to all the cells of a developing embryo. There are developments to prevent unintended consequences otherwise known as off-target effects due to gene editing. There is a race to develop new gene editing technologies that prevent off-target effects from occurring with some of the technologies being known as biased off-target detection, and Anti-CRISPR Proteins. For biased off-target effects detection, there are several tools to predict the locations where