Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced 
short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool 
for genome editing, transcriptional regulation, and cellular imaging 
applications. Structures of Streptococcus pyogenes Cas9 alone or bound to 
single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture 
that undergoes major conformational changes upon guide RNA and DNA binding. To 
investigate the molecular determinants and relevance of the interlobe 
rearrangement for target recognition and cleavage, we designed a split-Cas9 
enzyme in which the nuclease lobe and α-helical lobe are expressed as separate 
polypeptides. Although the lobes do not interact on their own, the sgRNA 
recruits them into a ternary complex that recapitulates the activity of 
full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified 
sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the 
development of an inducible dimerization system. We propose that split-Cas9 can 
act as a highly regulatable platform for genome-engineering applications.