In order to introduce such delicate changes to the genome, it is necessary to employ the HDR pathway of DNA repair, making this process less efficient in general than simple gene knockouts (Fig. 4d). Templates for DNA repair can be supplied as either double-stranded DNA plasmids with approximately 500–1000 nt of homology either side of the introduced mutation, or more typically as chemically synthesised short single-stranded DNA oligonucleotides (ssODN) of 100–200 nt in length. The latter are simple to design and synthesise and have a comparable efficiency of HDR to longer dsDNA fragments due to the higher recombinogenic activity of ssDNA (Chen et al. 2015). Although there is substantial variability in the absolute efficiencies reported in the literature, we find similarly to others that a combination of chemically synthesised crRNA and tracrRNA, recombinant Cas9 protein and approximately 100 nt ssODN highly effective for introduction of SNPs (Kim et al. 2014; Liang et al. 2015; Lin et al. 2014; Niu et al. 2016; Richardson et al. 2016; Song et al. 2015). This obviates the need for any cloning or DNA manipulation,
