The CRISPR-Cas9 system is perhaps the most remarkable recent breakthrough in genome editing technology. CRISPR is a ubiquitous family of clustered repetitive DNA elements present in 90% of Archaea and 40% of sequenced Bacteria. CRISPR arrays consist of interspersed identical REPEAT sequences (21-48bp) and several unique invader-targeting SPACER sequences (26-72bp). The CRISPR-Cas9 genome editing system consists of two components: a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9). The Cas9 protein is an endonuclease that uses gRNA to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific double-stranded break in the DNA. Through RNA-directed Cas9 nucleases, the CRISPR-Cas9 system can modify DNA with greater precision than existing technologies like TALEN and ZFN.
An advantage the CRISPR-Cas9 system offers over other mutagenic techniques like ZFN and TALEN is the relative simplicity of its plasmid design and construction. For each target site, the specificity of CRISPR-Cas9 relies on the formation of a ribonucleotide complex of sgRNA and the target DNA as opposed to protein/DNA recognition. CRISPR-Cas9 is easily programmable by changing the guide sequence (20 nucleotides in the native RNA) of the sgRNA to any DNA sequence of interest. Additionally, CRISPR is capable of modifying chromosomal targets with high fidelity whereas ZFN/TALEN are prone to CpG methylation. Last but not least, multiplexed genome editing with CRISPR-Cas9 library can be easily achieved with the monomeric Cas9 protein and any number of different sequence-specific gRNAs. The simplicity of CRISPR-Cas9 programming and its capacity for multiplexed target recognition have fueled the popularity of this cost-effective and easy-to-use technology.