SUMMARY CRISPR-Cas systems provide bacteria and archaea with sequence-specific protection against invading mobile genetic elements. In the presence of divalent metal ions, Cas9 and Cas12a (formerly Cpf1) proteins target and cleave DNA that is complementary to a cognate guide RNA. The recognition of a protospacer adjacent motif (PAM) sequence in the target DNA by Cas9 and Cas12a is essential for cleavage. This RNA-guided DNA targeting is widely used for gene-editing methods. Here, we show that Francisella tularensis novicida (Fno) Cas12a, FnoCas9, and Streptococcus pyogenes Cas9 (SpyCas9) cleave DNA without a guide RNA in the presence of Mn2+ ions. Substrate requirements for the RNA-independent activity vary. FnoCas9 preferentially nicks double-stranded plasmid, SpyCas9 degrades single-stranded plasmid, and FnoCas12a cleaves both substrates. These observations suggest that the identities and levels of intracellular metals, along with the Cas9/Cas12a ortholog employed, could have significant impacts in genome editing applications, In Brief CRISPR-mediated gene editing involves DNA targeting using complementary guide RNAs (gRNAs). Sundaresan et al. find that Cas9/Cas12a orthologs cause RNA-independent, non-sequence-specific DNA cleavage in the presence of Mn2+ ions. These observations suggest that the type of Cas9/Cas12a and levels of intracellular metal ions may affect CRISPR-based genome editing.