The new frontier of genome engineering with CRISPR-Cas9

Background Technologies for making and manipulating DNA have enabled advances in biology ever since the discovery of the DNA double helix. But introducing site-specific modifications in the genomes of cells and organisms remained elusive. Early approaches relied on the principle of site-specific recognition of DNA sequences by oligonucleotides, small molecules, or self-splicing introns. More recently, the site-directed zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) using the principles of DNA-protein recognition were developed. However, difficulties of protein design, synthesis, and validation remained a barrier to widespread adoption of these engineered nucleases for routine use. The Cas9 enzyme (blue) generates breaks in double-stranded DNA by using its two catalytic centers (blades) to cleave each strand of a DNA target site (gold) next to a PAM sequence (red) and matching the 20-nucleotide sequence (orange) of the single guide RNA (sgRNA). The sgRNA includes a dual-RNA sequence derived from CRISPR RNA (light green) and a separate transcript (tracrRNA, dark green) that binds and stabilizes the Cas9 protein. Cas9-sgRNA–mediated DNA cleavage produces a blunt double-stranded break that triggers repair enzymes to disrupt or replace DNA sequences at or near the cleavage site. Catalytically inactive forms of Cas9 can also be used for programmable regulation of transcription and visualization of genomic loci. Advances The field of biology is now experiencing a transformative phase with the advent of facile genome engineering in animals and plants using RNA-programmable CRISPR-Cas9. The CRISPR-Cas9 technology originates from type II CRISPR-Cas systems, which provide bacteria with adaptive immunity to viruses and plasmids. The CRISPR-associated protein Cas9 is an endonuclease that uses a guide sequence within an RNA duplex, tracrRNA:crRNA, to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific double-strand break in the DNA. The dual tracrRNA:crRNA was engineered as a single guide RNA (sgRNA) that retains two critical features: a sequence at the 5′ side that determines the DNA target site by Watson-Crick base-pairing and a duplex RNA structure at the 3′ side that binds to Cas9. This finding created a simple two-component system in which changes in the guide sequence of the sgRNA program Cas9 to target any DNA sequence of interest. The simplicity of CRISPR-Cas9 programming, together with a unique DNA cleaving mechanism, the capacity for multiplexed target recognition, and the existence of many natural type II CRISPR-Cas system variants, has enabled remarkable developments using this cost-effective and easy-to-use technology to precisely and efficiently target, edit, modify, regulate, and mark genomic loci of a wide array of cells and organisms. Outlook CRISPR-Cas9 has triggered a revolution in which laboratories around the world are using the technology for innovative applications in biology. This Review illustrates the power of the technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders. CRISPR-Cas9 is having a major impact on functional genomics conducted in experimental systems. Its application in genome-wide studies will enable large-scale screening for drug targets and other phenotypes and will facilitate the generation of engineered animal models that will benefit pharmacological studies and the understanding of human diseases. CRISPR-Cas9 applications in plants and fungi also promise to change the pace and course of agricultural research. Future research directions to improve the technology will include engineering or identifying smaller Cas9 variants with distinct specificity that may be more amenable to delivery in human cells. Understanding the homology-directed repair mechanisms that follow Cas9-mediated DNA cleavage will enhance insertion of new or corrected sequences into genomes. The development of specific methods for efficient and safe delivery of Cas9 and its guide RNAs to cells and tissues will also be critical for applications of the technology in human gene therapy. The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics. CRISPR-cas: A revolution in genome engineering The ability to engineer genomic DNA in cells and organisms easily and precisely will have major implications for basic biology research, medicine, and biotechnology. Doudna and Charpentier review the history of genome editing technologies, including oligonucleotide coupled to genome cleaving agents that rely on endogenous repair and recombination systems to complete the targeted changes, self-splicing introns, and zinc-finger nucleases and TAL effector nucleases. They then describe how clustered regularly interspaced palindromic repeats (CRISPRs), and their associated (Cas) nucleases, were discovered to constitute an adaptive immune system in bacteria. They document development of the CRISPR-Cas system into a facile genome engineering tool that is revolutionizing all areas of molecular biology. Science, this issue 10.1126/science.1258096