ACKNOWLEDGMENTSWe thank Malcolm Brenner for encouraging this piece; Christen Rachul, Zubin Master, and Lisa Belanger for their research assistance; and the Stem Cell Network (Networks of Centres of Excellence Canada) and the Cancer Stem Cell Consortium for funding support.
REFERENCES1. Caulfi eld, T and Condit, C (2012). Science and
the sources of hype. Public Health Genomics 15: 209–217.
2. Kovaleski, SF (2011). Baseball gets records on pitcher’s procedure. New York Times, 13 July.
3. Armstrong, K (2011). Peyton Manning, Colts QB, underwent stem cell therapy in Europe for nag-ging neck injury: report. New York Daily News, 18 September.
4. Weissman, I (2012). Stem cell therapies could change medicine…if they get the chance. Cell Stem Cell 10: 663–665.
5. Lau, U, Ogbogu, B, Taylor, T, Stafi nski, D, Menon, D and Caulfi eld, T (2008). Stem cell clinics online: the direct-to-consumer portrayal of stem cell medi-cine. Cell Stem Cell 3: 591–594.
6. Regenberg, A, Hutchinson, L, Schanker, B and Mathews, D (2009). Medicine on the fringe: stem cell–based interventions in advance of evidence. Stem Cells 27: 2312–2319.
7. Baker, M (2008). Stem cell society condemns unprov-en treatments. Nat Rep Stem Cells 26 June <http://www.nature.com/stemcells/2008/0806/080626/full/stemcells.2008.98.html>.
8. Zarzeczny, A, Rachul, C, Nisbet, M and Caulfi eld, T (2010). Stem cell clinics in the news. Nat Biotechnol 28: 1243–1246.
9. Bubela, T, Nisbet, MC, Borchelt, R, Brunger, F, Critchley, C, Einsiedel, E et al. (2009). Science communication reconsidered. Nat Biotechnol 27: 514–518.
10. Nolen, S (2010). The stem-cell black market: Delhi doctor claims wonder cure. Globe and Mail, 19 September.
11. Kiatpongsan, S and Sipp, D (2008). Offshore stem cell treatments. Nat Rep Stem Cells, 3 December <http://www.nature.com/stem-cells/2008/0812/081203/full/stemcells.2008.151.html>.
12. US Food and Drug Administration (2012). FDA warns about stem schemes <http://www.fda.gov/forconsumers/consumerupdates/ucm286155.htm>.
13. Brown, WJ, Basil, MD and Bocarnea, MC (2003). The infl uence of famous athletes on health beliefs and practices: Mark McGwire, child abuse preven-tion, and androstenedione. J Health Commun 8: 41–57.
14. Kuehn, BM (2009). Teen steroid, supplement use targeted. JAMA 302: 2301–2303.
15. Perko, M, Bartee, R, Dunn, M, Wang, M and Eddy, JM (2000). Giving new meaning to the term “taking one for the team”: infl uences on the use/non-use of dietary supplements among adolescent athletes. Am J Health Studies 16: 99–106.
16. Rosenfi eld, C (2011). The use of ergogenic agents in high school athletes. Am J Lifestyle Med 5: 320–327.
17. Perez, AJ (2011). Athletes go to great lengths for recovery. Fox Sports, 7 October <http://msn.foxsports.com/nfl /story/American-athletes-go-to-great-lengths-to-recover-from-injury-100611>.
18. Barling, M (2011). Stem cell and platelet-rich plasma (PRP) treatments. Bleacher Report <http://bleacherreport.com/articles/857012-stem-cell-and-platelet-rich-plasma-prp-treatments>.
19. Affordable Stem Cell Therapy (2011). Retired NFL great Bart Oates has joined the lineup of professional athletes seeking stem cell treatment abroad <http://www.affordablestemcelltherapy.com/cell-medicine/retired-nfl -great-bart-oates-has-joined-the-lineup-of-professional-athletes-seeking-stem-cell-treatment-abroad.php>.
20. Regenexx (2010). Jarvis Green gets contract with Houston Texans, attributes it to Regenexx stem cell injections <http://www.regenexx.com/2010/12/jarvis-green-gets-contract-with-houston-texans-attributes-it-to-regenexx-stem-cell-injections>.
A CRISPR Approach to Gene TargetingDana Carroll1
doi:10.1038/mt.2012.171
1Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah, USACorrespondence: Dana Carroll, Department of Biochemistry, University of Utah School of Medi-cine, 15 N. Medical Dr. East, Room 4100, Salt Lake City, Utah 84112, USA. E-mail: [email protected]
It is getting easier and easier to determine complete genome sequences—of model
organisms, animals and plants of commer-cial importance, and humans: Craig Venter, Jim Watson, the 1000 Genome Project, soon you and me. Now that researchers have all this information at hand, the focus has shift ed in many cases to manipulating par-ticular sequences to determine their func-tion or to alter their impact. A new study by Jinek et al.1 proposes a new approach—based on the oldest of DNA recognition principles—to the design of reagents that can target specifi c genomic sequences.
Precision genome engineering has been enhanced substantially in recent years by the development of targetable DNA cleav-age reagents.2 A double-strand break (DSB) made at a specifi c genomic location by, for example, zinc-fi nger nucleases (ZFNs) will oft en be repaired inaccurately by non-homologous end joining (NHEJ), creating a targeted mutation (Figure 1). When a mod-ifi ed donor DNA is also provided, repair by homologous recombination will lead to in-troduction of donor sequences at the target. Th ese break-induced modifi cations can be very effi cient, in the range of 10% or more of all targets in a single treatment. Gene-ed-iting nucleases such as ZFNs, not only have been used for engineering precise genomic changes in experimental organisms but are being tested in current clinical trials.3
ZFNs are hybrid proteins that have sever-al favorable properties as targeting reagents.2 Th e zinc-fi nger modules that comprise their DNA-binding domain can be assembled in many combinations to recognize a wide range of genomic sequences (Figure 2). Th e FokI-derived cleavage domain is not active as a monomer, so the nuclease is assembled only when two ZFNs bind at the designed target. Th e binding and cleavage domains can be manipulated separately to alter recognition and cleavage properties independently.
A problem with ZFNs has been the unpredictability of their recognition capabilities. Some fi ngers apparently bind their corresponding DNA triplet (or quartet) reliably in diff erent contexts, but others do not. Even procedures that select fi nger combinations explicitly for new targets are not always suc-cessful, and they can be dauntingly laborious.4
Th is design challenge has recently been addressed with the adoption of an alterna-tive set of DNA-binding modules derived from Xanthomonas, a genus of proteo-bacteria.5,6 Each transcription activator–like eff ector (TALE) module recognizes a single base pair, and standard modules for each of the four possibilities seem to be-have well in essentially any sequence con-text. TALENs (TALE nucleases) (Figure 2) consist of multiple TALE domains fused to the FokI cleavage domain, and they have out performed ZFNs in many early trials.
Although TALE modules make design for new targets much easier and apparently more reliable, some questions about speci-fi city remain. Ask any biochemist or mole-cular biologist what the gold standard is for DNA sequence recognition and the answer will be: Watson–Crick base pairing. Th is is the key to the proposal by Jinek et al.1
21. Martin, A and Coxe, F (2012). Stem cell therapy plays a crucial role for athletes in the 2012 Olympic games: Kobe Bryant, Dara Torres, and David Payne. MetroMD Inst Regen Med (http://metromd.net/stem-cell-therapy-plays-a-crucial-role-for-athletes-in-the-2012-olympic-games).
22. Ryan, KA, Sanders, AM, Wang, DD and Levine, AD (2010). Tracking the rise of stem cell tourism. Regen Med 5: 27–33.
23. Lysaght, T and Campbell, A (2011). Regulating autologous adult stem cells: the FDA steps up. Cell Stem Cell 9: 393–396.
Molecular Th erapy vol. 20 no. 9 september 2012 1659
Th e reagents described in the new article derive from a novel system of adap-tive immunity, found in many species of bacteria and archaea, called CRISPR (for the clustered regularly interspersed short palindromic repeats that characterize the genomic loci involved). Th is system is rather complex, and several variations exist, but the common features can be out-lined as follows.7
When a viral genome or plasmid en-ters one of these microbial hosts, a few fragments of the invading DNA are cap-tured as “spacers” between identical “re-peats” that are specifi c to the particular CRISPR system (Figure 3). Both the re-peats and spacers are typically a few tens of base pairs in length. Transcription of the locus produces a precursor RNA that is processed into smaller fragments, each carrying one spacer linked to a portion of a repeat. When the same viral or plasmid sequence invades the host again, the corre-sponding spacer RNA guides destruction of the invading RNA or DNA, depending on the particular system. Cas (CRISPR-associated) proteins mediate both produc-tion of the spacer RNAs and cleavage of the invading target.
In the type II CRISPR systems the Cas9 protein forms a complex with the spacer-containing RNA and a second RNA, trans-activating CRISPR RNA (tracrRNA), that is partially complementary to the repeat se-quence, and this complex catalyzes destruc-tion of the invading DNA (Figure 3). Jinek
et al. have shown that this DNA cleavage reaction can be recapitulated in vitro with purifi ed Cas9 and a single RNA molecule that has the minimal required features of both spacer and tracr (Figure 4). Only a target DNA that matches the spacer RNA sequence is cleaved. Diff erent spacer RNAs direct cleavage to diff erent DNA sequences, and both strands of the target are cut.
Jinek et al. used spacers of 20–30 nucleo-tides to demonstrate the effi ciency and specifi city of cleavage by Cas9–RNA com-plexes. Both supercoiled plasmid DNA and short, double-stranded oligonucleotides are good substrates. Each DNA strand is cut by one of the two separate nuclease domains of
Cas9; mutation of either active site leads to single-strand cleavage. Th e critical region of RNA–DNA duplex is at the downstream end of the spacer DNA, which corresponds to the 3ʹ side of the RNA in the match. A minimum of 16 base pairs is required. In addition, Cas9 recognizes 2 or 3 base pairs in the DNA just to the right of the hybrid region, called PAM (protospacer adjacent motif), which is probably also recognized during the establishment phase of immuni-ty. Finally, a region of RNA duplex between the repeat segment and its complement in tracrRNA is necessary for cleavage. Using information about all these requirements, Jinek et al. produced a single RNA mol-ecule (Figure 4) that guides cleavage in conjunction with Cas9.
All the experiments described above were performed in vitro with purifi ed components, but several aspects have been confi rmed in bacteria. Th e authors make the bold prediction that this system can potentially be used in place of ZFNs or TALENs for targeted genomic cleavage in higher organisms. Let’s think about how this might work.
Cas9 protein and the targeting RNA would need to be expressed in the cells or organism of interest. Presumably both could be produced from DNA vectors with ap-propriate promoters; Cas9 messenger RNA and the targeting RNA could be produced in vitro and introduced into cells; or purifi ed protein and synthetic targeting RNA could be introduced. Th e optimal choice would depend on the experimental situation.
ZFNsTALENs HEs
CRISPR?
+ Donor DNA
NHEJ
HR
Figure 1 Consequences of targeted genomic cleavage. A double-strand break made by any type of cleavage reagent can be repaired by error-prone nonhomologous end joining (NHEJ), leaving small insertions and/or deletions at the site. An alternative mode of repair is homologous recombination (HR), which can use a manipulated donor DNA as a template, resulting in replace-ment of genomic sequences. The break can be made by any targetable nuclease: zinc-fi nger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), homing endonucleases (HEs), or, perhaps, the new CRISPR reagents.
ZFNs
TALENs
Figure 2 ZFNs and TALENs. (Top) Each zinc fi nger (small ovals) in a zinc-fi nger nuclease (ZFN) binds primarily to three consecutive base pairs; a minimum of three fi ngers is required to provide suffi cient affi nity. Different colors indicate fi ngers recognizing different DNA triplets. Each set of fi ngers is joined to a FokI-derived cleavage domain (large ovals) by a short linker. (Bottom) In transcription activator–like effector nucleases (TALENs), each module (small ovals) binds a single base pair; the four colors indicate modules for each of the four base pairs. The minimum effective number of modules is 10–12, but more are typically used. The linker to the FokI domain (large ovals) is longer than for ZFNs and contains additional TALE-derived sequences.
1660 www.moleculartherapy.org vol. 20 no. 9 september 2012
Recognition specifi city is provided by the match between the targeting RNA and the DNA target. Watson–Crick pairing can be very specifi c, and a match of 16–20 base pairs is suffi cient to ensure recognition of a unique sequence in a complex genome. Discrimination could therefore be more precise than with either zinc fi ngers or TALE modules.
A key issue for all gene-targeting re-agents is how delivery to the target cells or organisms will be accomplished. In many animals, direct injection of nuclease-en-coding messenger RNAs into early embry-os has proved quite eff ective in generating germline modifi cations. For example, this approach has added a very welcome tool to
the arsenal of rat geneticists.8 For human somatic therapy, targeting is most eas-ily applied to situations that allow ex vivo treatment of cells before reinfusion. Cells of the hematopoietic lineages are obvious targets, and as more pluripotent cell types are identifi ed or generated, the applica-tions will expand.
What about activity of the system in eukaryotic cells? Both zinc fi ngers and TALE modules come from natural transcription factors that bind their targets in a chromatin context. Th is is not true of the CRISPR components. Th ere is no guarantee that Cas9 will work eff ectively on a chromatin target or that the required DNA–RNA hybrid can be stabilized in that
context. Th is structure may be a substrate for RNA hydrolysis by ribonuclease H and/or FEN1, both of which function in the re-moval of RNA primers during DNA repli-cation. Only attempts to apply the system in eukaryotes will address these concerns.
Intriguingly, some eukaryotic cells ap-pear to have an inherent system to make double-strand breaks in the region of DNA–RNA hybrids. Th is was revealed by disabling ribonuclease H in yeast.9 Such a maneuver might enhance the activity of CRISPR cleavage as well, but with the potential side eff ect of inducing breaks at multiple regions of transcription.
Gene editing through base pairing has been attempted many times and is still being pursued. Th e effi ciency of modifi -cation by introduction of simple oligonu-cleotides, chemically modifi ed oligos, or oligo mimics such as peptide nucleic acids remains discouragingly low in most cas-es.10,11 Triplex-forming oligonucleotides12 have shown activity, but with a limited range of targets and less effi ciency than ZFN or TALEN cleavage. Whether the CRISPR system will provide the next-next generation of targetable cleavage reagents remains to be seen, but it is clearly well worth a try. Stay tuned.
REFERENCES 1. Jinek, M et al. (2012). A programmable dual-RNA-
guided DNA endonuclease in adaptive bacterial immunity. Science, e-pub ahead of print 28 June 2012.
2. Carroll, D (2011). Genome engineering with zinc-fi nger nucleases. Genetics 188: 773–782.
3. Urnov, FD, Rebar, EJ, Holmes, MC, Zhang, HS and Gregory, PD (2010). Genome editing with engineered zinc fi nger nucleases. Nat Rev Genet 11: 636–646.
4. Maeder, ML et al. (2008). Rapid “Open-Source” engineering of customized zinc-fi nger nucleases for highly effi cient gene modifi cation. Mol Cell 31: 294–301.
5. Bogdanove, AJ and Voytas, DF (2011). TAL effectors: customizable proteins for DNA targeting. Science 333: 1843–1846.
6. Scholze, H and Boch, J (2011). TAL effectors are remote controls for gene activation. Curr Opin Micro-biol 14: 47–53.
7. Wiedenheft, B, Sternberg, SH and Doudna, JA (2012). RNA-guided genetic silencing systems in bacteria and archaea. Nature 482: 331–338.
8. Geurts, AM et al. (2009). Knockout rats via embryo microinjection of zinc-fi nger nucleases. Science 325: 433.
9. Wahba, L, Amon, JD, Koshland, D and Vuica-Ross, M (2011). RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability. Mol Cell 44: 978–988.
10. Aarts, M and te Riele, H (2011). Progress and pros-pects: oligonucleotide-directed gene modifi cation in mouse embryonic stem cells: a route to therapeutic application. Gene Ther 18: 213–219.
11. Nielsen, PE (2010). Sequence-selective targeting of duplex DNA by peptide nucleic acids. Curr Opin Mol Ther 12: 184–191.
12. Mukherjee, A and Vasquez, KM (2011). Triplex tech-nology in studies of DNA damage, DNA repair and mutagenesis. Biochimie 93: 1197–1208.
Spacer Spacer Spacer Spacer
Repeat Repeat Repeat Repeat Repeat
Spacer
tracr
Invading DNA
Spacer
tracr
Spacer
tracr
Spacer
tracr
Transcription
Processing
+ Cas9
Figure 3 The type II CRISPR system. In a bacterial genome, identical repeats fl ank virus- or plasmid-derived spacer sequences in tandem arrays (blue). Long transcripts (green line) are pro-cessed into short RNAs containing a single spacer and a partial repeat. These short RNAs form partial duplexes with tracrRNAs and are bound by the Cas9 protein (orange oval). The complex then cleaves invading viral or plasmid DNA directed by the spacer RNAs. tracrRNA, trans-activated CRISPR RNA.
Figure 4 The CRISPR minimal-cleavage elements described by Jinek et al.1 A single RNA (green lines) with the critical elements of spacer and tracrRNA binds Cas9 protein (orange oval) and directs cleavage (arrowheads) to a sequence in DNA (blue) that has homology to the spacer. The region of RNA–DNA base pairing provides cleavage specifi city. The target must also have a particular two– to three–base pair sequence adjacent to the region of homology, called PAM, which is recognized by the complex. PAM, protospacer adjacent motif; tracrRNA, trans-activated CRISPR RNA.
