The CRISPR/Cas9 system

originally derived from the prokaryotic adaptive immune system

has been developed as efficient genome editing tools. It enables precise gene manipulation on chromosomal DNA through the specific binding of programmable sgRNA to target DNA

and the Cas9 protein

which has endonuclease activity

will cut a double strand break at specific locus. However

Cas9 is a foreign protein in mammalian cells

and the potential risks associated with its introduction into mammalian cells are not fully understood. In this study

we performed pull-down and mass spectrometry (MS) analysis of

Streptococcus pyogenes

Cas9 (SpyCas9) interacting proteins in HEK293T cells and showed that the majority of Cas9-associated proteins identified by MS were localized in the nucleolus. Interestingly

we further discovered that the Cas9 protein contains a sequence encoding a nucleolus detention signal (NoDS). Compared with wild-type (WT) Cas9

NoDS-mutated variants of Cas9 (mCas9) are less stable

although their gene editing activity is minimally affected. Overexpression of WT Cas9

but not mCas9

causes general effects on transcription and protein translation in the host cell. Overall

identification of NoDS in Cas9 will improve the understanding of Cas9's biological function

in vivo

and the removal of NoDS in Cas9 may enhance its safety for future clinical use.