As a powerful genome-editing tool, the clustered regularly interspaced short 
palindromic repeats (CRISPR)-clustered regularly interspaced short palindromic 
repeats-associated protein 9 (Cas9) system has been quickly developed into a 
large-scale function-based screening strategy in mammalian cells. This new type 
of genetic library is constructed through the lentiviral delivery of 
single-guide RNA collections that direct Cas9 or inactive dead Cas9 fused with 
effectors to interrogate gene function or regulate gene transcription in 
targeted cells. Compared with RNA interference screening, the CRISPR-Cas9 system 
demonstrates much higher levels of effectiveness and reliability with respect to 
both loss-of-function and gain-of-function screening. Unlike the RNA 
interference strategy, a CRISPR-Cas9 library can target both protein-coding 
sequences and regulatory elements, including promoters, enhancers and elements 
transcribing microRNAs and long noncoding RNAs. This powerful genetic tool will 
undoubtedly accelerate the mechanistic discovery of various biological 
processes. In this mini review, we summarize the general procedure of 
CRISPR-Cas9 library mediated functional screening, system optimization 
strategies and applications of this new genetic toolkit.