While off-target activity at least may be reduced by high-fidelity variants or, for DSB-based approaches, by paired CRISPR/Cas nickases or CRISPR/FokI dimeric nucleases, approval for clinical trials will be difficult to obtain without comprehensive experimental analysis of potential off-target effects, so as to minimize the residual risk to patients [ 149 ]. Another safety concern is that of pre-existing immunity against Cas molecules in humans [ 39, 150, 151 ]. While this issue may be of minor importance for most ex vivo approaches and may be overcome for in vivo approaches by alternative enzymes or transient immunosuppression [ 39 ], it nevertheless casts a shadow of doubt over in vivo applications of CRISPR/Cas, which are already at risk of adverse reactions or lowered efficiency by pre-existing immunity to AAV and other delivery vectors [ 152, 153, 154 ].