In all cases, it is critical to ensure that upon correct HDR, the sgRNA is unable to guide further DSB events on the newly modified allele, since these could result in further undesirable mutagenesis through NHEJ-mediated indels. This ideally requires that the sgRNA is chosen to span the SNP of interest. Since the guide RNA can tolerate up to three mismatches whilst still retaining the ability to direct the Cas9 endonuclease to this site, it is often necessary or advisable to introduce additional base changes outside the SNP of interest to ensure this is the case. For SNPs within protein coding genes, this can be achieved by introducing silent mutations into the DNA whilst maintaining its protein coding capacity. Even in this case, there may still be a role of such synonymous mutations in protein translation efficiency (Quax et al. 2015), exonic transcription factor binding (Stergachis et al. 2013) or other as yet unknown functions. However, for those changes outside of protein coding sequence, it is usually impossible to predict the effects of such secondary mutations. This makes it necessary
