GUIDE-Seq has been shown to miss some off-targets, when compared to the genome-wide sequencing DIGENOME-Seq method, due to the nature of its targeting. Another caveat is that GUIDE-Seq has been observed to generate slightly different off-target sites depending on the cell line. This could be due to cell lines having different parental genetic origins, cell line specific mutations, or, in the case of some immortal cell lines such as K562s, having aneuploidy. This suggests that it would be pertinent for researchers to test multiple cell lines to validate efficacy and accuracy. GUIDE-Seq cannot be used to identify off-targets in vivo.