The RNA-guided nuclease function of CRISPR-Cas is reconstituted in mammalian cells through the heterologous expression of human codon–optimized Cas9 and the requisite RNA components22–25. Furthermore, the crRNA and tracrRNA can be fused together to create a chimeric, single-guide RNA (sgRNA)27 (Fig. 1). Cas9 can thus be re-directed toward almost any target of interest in immediate vicinity of the PAM sequence by altering the 20-nt guide sequence within the sgRNA.
