GENOME EDITING The new frontier of genome …

1 RESEA RCH. in the DNA. The dual tracrRNA:crRNA was REVIEW SUMMARY engineered as a single guide RNA (sgRNA). that retains two critical features: a sequence GENOME EDITING at the 5 side that determines the DNA tar- get site by Watson-Crick base-pairing and The new frontier of GENOME a duplex RNA structure at the 3 side that binds to Cas9. This finding created a simple two-component system in which changes in engineering with CRISPR-Cas9 the guide sequence of the sgRNA program Cas9 to target any DNA sequence of interest. Jennifer A. Doudna* and Emmanuelle Charpentier* The simplicity of CRISPR-Cas9 program- ming, together with a unique DNA cleaving BACKGROUND: Technologies for mak- widespread adoption of these engineered mechanism, the capacity for multiplexed tar- ing and manipulating DNA have enabled nucleases for routine use.

2 Get recognition, and the existence of many advances in biology ever since the discov- natural type II CRISPR-Cas system variants, ery of the DNA double helix. But intro- ADVANCES: The field of biology is now ex- has enabled remarkable developments using ducing site-specific modifications in the periencing a transformative phase with the this cost-effective and easy-to-use technol- genomes of cells and organisms remained advent of facile GENOME engineering in ani- ogy to precisely and efficiently target, edit, elusive. Early approaches relied on the mals and plants using RNA-programmable modify, regulate, and mark genomic loci of a principle of site-specific recognition of CRISPR-Cas9. The CRISPR-Cas9 technology wide array of cells and organisms. DNA sequences by oligonucleotides, small originates from type II CRISPR-Cas systems, molecules, or self-splicing introns.

3 More which provide bacteria with adaptive immu- OUTLOOK: CRISPR-Cas9 has triggered a Downloaded from on February 13, 2016. recently, the site-directed zinc finger nu- nity to viruses and plasmids. The CRISPR- revolution in which laboratories around cleases (ZFNs) and TAL effector nucleases associated protein Cas9 is an endonuclease the world are using the technology for in- (TALENs) using the principles of DNA- that uses a guide sequence within an RNA novative applications in biology. This Re- protein recognition were developed. How- duplex, tracrRNA:crRNA, to form base pairs view illustrates the power of the technology ever, difficulties of protein design, synthe- with DNA target sequences, enabling Cas9 to to systematically analyze gene functions sis, and validation remained a barrier to introduce a site-specific double-strand break in mammalian cells, study genomic rear- rangements and the progression of cancers sgRNA.

4 Or other diseases, and potentially correct binding to genetic mutations responsible for inherited Cas9. disorders. CRISPR-Cas9 is having a major Matching DNA PAM impact on functional target sequence sequence ON OUR WEB SITE genomics conducted Read the full article in experimental sys- at tems. Its application in . GENOME -wide studies will enable large-scale screening for drug tar- gets and other phenotypes and will facili- tate 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 CRISPR-Cas9 development CRISPR-Cas9 applications and course of agricultural research. Future DNA deletion Biological research research directions to improve the technol- DNA insertion Research and development ogy will include engineering or identifying DNA replacement Human medicine smaller Cas9 variants with distinct specific- DNA modifcation Biotechnology ity that may be more amenable to delivery DNA labeling Agriculture in human cells.

5 Understanding the homol- ogy-directed repair mechanisms that follow Transcription modulation .. Cas9-mediated DNA cleavage will enhance RNA targeting insertion of new or corrected sequences into .. genomes . The development of specific meth- The Cas9 enzyme (blue) generates breaks in double-stranded DNA by using its two ods for efficient and safe delivery of Cas9. ILLUSTRATION: K. SUTLIFF/SCIENCE. catalytic centers (blades) to cleave each strand of a DNA target site (gold) next to a and its guide RNAs to cells and tissues will PAM sequence (red) and matching the 20-nucleotide sequence (orange) of the single also be critical for applications of the tech- guide RNA (sgRNA). The sgRNA includes a dual-RNA sequence derived from CRISPR RNA nology in human gene therapy.

6 (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 The list of author affiliations is available in the full article online. that triggers repair enzymes to disrupt or replace DNA sequences at or near the cleavage *Corresponding author. E-mail: ( ); ( ). site. Catalytically inactive forms of Cas9 can also be used for programmable regulation of Cite this article as J. A. Doudna, E. Charpentier, Science 346, transcription and visualization of genomic loci. 1258096 (2014). DOI: SCIENCE Corrected 25 November, 2014; see full text. 28 NOVEMBER 2014 VOL 346 ISSUE 6213 1077. Published by AAAS. R ES E A RC H. The use of self-splicing introns for GENOME REVIEW EDITING also suggested the possibility of using intron-encoded nucleases homing endonucleases.

7 That are capable of site-specific DNA cleavage and GENOME EDITING integration of the intron sequence. By inserting desired sequences into the intron first, researchers The new frontier of GENOME could incorporate selected genetic information into a GENOME at sites recognized by the homing en- engineering with CRISPR-Cas9. donuclease (24, 25). At around the same time, the initial reports of zinc finger mediated DNA bind- ing (26, 27) led to the creation of modular DNA. recognition proteins that, when coupled to the Jennifer A. Doudna1,2,3* and Emmanuelle Charpentier4,5,6* sequence-independent nuclease domain of the re- striction enzyme FokI, could function as site- The advent of facile GENOME engineering using the bacterial RNA-guided CRISPR-Cas9 specific nucleases (28).

8 When designed to recog- system in animals and plants is transforming biology. We review the history of CRISPR nize a chromosomal sequence, such zinc finger (clustered regularly interspaced palindromic repeat) biology from its initial discovery nucleases (ZFNs) were found to be effective at in- through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage ducing genomic sequence changes in Drosophila for remarkable developments using this technology to modify, regulate, or mark genomic and mammalian cells (29, 30). Although ZFNs loci in a wide variety of cells and organisms from all three domains of life. These results are effective GENOME EDITING reagents for some highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, they were not widely adopted be- experiments, paving the way toward fundamental discoveries in biology, with applications cause of the difficulty inherent in designing and in all branches of biotechnology, as well as strategies for human therapeutics.

9 Validating such proteins for a specific DNA locus of interest. Thus, the field was primed for the T. first reports of transcription activator like (TAL). echnologies for making and manipulating GENOME engineering effectors, which occur naturally in bacteria that DNA have enabled many of the advances A decades-long goal infect plants, enabling rapid creation of FokI- in biology over the past 60 years. This era Ever since the discovery of the DNA double coupled versions that could be used similarly to began with the discovery of the DNA double helix, researchers and clinicians have been con- ZFNs for site-directed GENOME EDITING (31 33). helix and continued with the development templating the possibility of making site-specific Such TAL effector nucleases (TALENs) were easier of chemical methods for solid-phase DNA syn- changes to the genomes of cells and organisms.

10 Than ZFNs to produce and validate, generating thesis, enabling detection and exploration of Many of the earliest approaches to what has widespread excitement about the possibility of GENOME organization. Enzymes (including poly- been referred to as GENOME EDITING relied on the facile GENOME EDITING that would be fast and merases, ligases, and restriction endonucleases) principle of site-specific recognition of DNA se- inexpensive. But difficulties of protein design, and the polymerase chain reaction (PCR) pro- quences (Fig. 1). The study of natural DNA re- synthesis, and validation remained a barrier to vided ways to isolate genes and gene fragments, pair pathways in bacteria and yeast, as well as widespread adoption of these engineered nu- as well as to introduce mutations into genes in the mechanisms of DNA recombination (1 5), cleases for routine use.