et al.

More documents

Recommendations

Info

CREDIT: K. SUTLIFF/SCIENCE A Restriction nucleases B Homing endonuclease C CRISPR-associated nuclease D Zinc finger nuclease E TAL effector nuclease F TFO nuclease Potential genome editing. Schematic comparison of nucleases with DNAbinding (blue) and DNA-cleaving domains (red). (Top) Natural nuclease classes: (A) Restriction enzyme (shown as a dimer), with separate DNA binding and DNA cleavage domains (left) and with a single dual-function domain (right); (B) homing endonuclease with an extended DNA binding domain; (C) type II the original sequence or result in different modifi cations. The latter changes range from exchange of designed DNA donor fragments by homologous recombination (thus, the editing of an existing gene or integration of a new gene) to small insertions and deletions by nonhomologous end joining of double-strand breaks (disruption of a gene). Classical restriction endonucleases are not suitable for genome engineering because the sites they recognize are very short [4 to 8 base pairs (bp); see the fi gure] and occur too frequently in the genome. Homing endonucleases recognize longer targets (20 to 30 bp), but adjusting their specifi city requires laborious protein design ( 3). More recently, artifi cial chimeric proteins have been created by fusing a nuclease domain to a DNA binding protein. One class, zinc fi nger nucleases, consists of a nuclease that is fused to a set of zinc fi nger domains that each interacts with three nucleotides; dimeric zinc fi nger nucleases can target up to 36-bp sequences. Adjusting the specifi city of zinc fi nger nucleases relies on the shuffl ing of domains with established triplet specifi city ( 4). Another class of DNA binding protein, transcription activator-like effector nucleases (TALENs), are composed of a nuclease that is fused to a protein consisting of an array of 12 to 26 domains, each of which specifi cally interacts with a single base; in dimer designs, this results in at least 24-bp binding sites ( 5). Thus, rational design toward any target DNA sequence NATURAL SYNTHETIC can be accomplished by relatively straightforward protein engineering. An attractive alternative strategy for binding DNA fragments with high specifi city may be the use of oligonucleotides as a recognition module. An initial attempt to synthesize such a tunable nuclease was the development of a short triple-helix–forming oligonucleotide conjugated to a restriction endonuclease ( 6). Application of this approach is mainly hampered by the restriction of triple-helix formation to DNA fragments with strands composed of either purines or pyrimidines. Recently, a natural RNA-guided DNA nuclease system has been discovered in bacteria and archaea. Analysis of clustered regularly interspaced short palindromic repeats (CRISPR) has elucidated a unique system for adaptive immunity by CRISPR-associated (Cas) proteins. The specifi city of the CRISPR-Cas system relies on tightly bound CRISPR RNA (crRNA), which effi ciently guides a nuclease to its target—a complementary DNA fragment ( 7, 8). A type II CRISPR-associated nuclease (Cas9) causes specifi c double-strand breaks in a DNA target. Processing of its crRNA guide involves a second, trans-acting crRNA (tracrRNA) and a ribonuclease (RNase III) ( 9). When loaded with both crRNA and tracrRNA (or an artifi cial chimera of the two RNAs), the Cas9 nuclease cleaves a DNA fragment that is complementary to the exposed part of the crRNA ( 10, 11). Dedicated cleavage of plasmids in vitro ( 9) demonstrates the promise PERSPECTIVES CRISPR-associated nuclease with a guiding crRNA (blue) and tracrRNA (red). (Bottom) Synthetic nuclease classes: (D) zinc fi nger nuclease dimer; (E) transcription activator-like effector nuclease (TALEN) dimer with nuclease domains; (F) triple-helix–forming oligonucleotides with a single-chain nuclease (shown as a dimer). of CRISPR-mediated gene targeting as a more generic molecular engineering tool. Cong et al. and Mali et al. report different applications of the bacterial Cas9 nuclease for RNA-guided engineering of mammalian genomes. Both groups describe the functional expression of Cas9 in the nucleus of cultured human cells. Cas9 that is loaded with guiding crRNA and assisting tracrRNA can carry out programmed DNA cleavage, which is eventually partly repaired by nonhomologous end joining, an event that frequently results in small insertions and deletions. Whereas the wild-type Cas9 generates double-strand breaks, Cong et al. and Mali et al. have used a Cas9 mutant in which one of the nuclease active sites is disrupted ( 10, 11). This variant generates breaks in only one of the DNA strands, which should diminish nonhomologous recombination. Indeed, both groups demonstrate a reduction in off-target mutations due to insertions and deletions. In this context, distinct donor DNA fragments were successfully integrated at the site of cleavage. Moreover, the simultaneous introduction of two adjacent double-strand breaks resulted in effi cient deletion of the intervening fragment. Notably, as Cong et al. and Mali et al. show, this also allows for scaling up by multiplex editing of distinct target loci. Similarly, Jinek et al. ( 12) have reported dedicated doublestrand DNA breaks based on Cas9-mediated genome editing in human cells. Cas9 cleaving effi ciencies depended mainly on the design of the chimeric RNA, suggesting www.sciencemag.org SCIENCE VOL 339 15 FEBRUARY 2013 769 Published by AAAS on February 14, 2013 www.sciencemag.org Downloaded from
PERSPECTIVES 770 a means for further optimizing the editing performance of Cas9. Effi cient strategies for directed editing of mammalian genomes will enable sophisticated genetic engineering for both fundamental and applied purposes. Especially in medical applications, high-fi delity target recognition is critical, as off-site nuclease activity will jeopardize the safety of the engineering operation; thus, long stretches of nucleotides should be specifi cally recognized. In addition, adjusting the system’s specifi city toward new target sequences should be easy and affordable; this is a major advantage of the Cas9 system, as it merely requires changing the sequence of the guide RNA. Further- PHYSICS Demonstrating Uncertainty Gerard J. Milburn Anyone using a modern camera is implementing an optical position measurement. In an active autofocus camera, a pulse of infrared light is emitted from the camera, and the time taken for it to be refl ected back to the camera is used to compute the distance between the object and the image plane. Imagine how diffi cult it would be to operate such a system if the object recoiled every time the infrared pulse was refl ected from it. Heisenberg suggested that this is precisely what would happen if light were used to determine the position of a quantum object as accurately as his famous uncertainty principle would allow. On page 801 of this issue, Purdy et al. ( 1) demonstrate this quantum back-action effect in an optical measurement of the position of a macroscopic mirror. The mechanical action of light has long been known ( 2). Kepler suggested that the reason comet tails point away from the Sun is due to the mechanical action of light. In the early 1970s, Arthur Ashkin of Bell Signal Laboratories showed that optical intensity gradients could exert a Probe force on micrometer-size parti- Centre for Engineered Quantum Systems, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia. E-mail: milburn@ physics.uq.edu.au more, the recombination should be fast, effi - cient, and scalable. Compared to the most promising currently available genome editing systems (zinc fi nger domains and TAL- ENs), the RNA-guided Cas9 nuclease probably is closest to meeting these requirements. However, effi ciency and specifi city still can be improved—for instance, by laboratory evolution. Applying these genome surgery techniques to correct human disease-associated genetic mutations, resulting in functional gene therapy and in curing genetic disorders, will therefore take time. The spectacular recent development of dedicated nucleases suggests, however, that we are entering the fi nal stage of this quest. cles. This observation eventually led to laser cooling and the fi eld of atom optics. The history of using optical transducers to monitor the quantized position of an object at the Heisenberg limit goes back to the early proposals for the optical detection of gravitational radiation ( 3). The relative length of the two orthogonal arms in a Michelson-Morley interferometer is Incident Reflected 15 FEBRUARY 2013 VOL 339 SCIENCE www.sciencemag.org Published by AAAS Transmitted Signal Probe References 1. L. Cong et al., Science 339, 819 (2013); 10.1126/science.1231143. 2. P. Mali et al., Science 339, 823 (2013); 10.1126/science.1232033. 3. B. L. Stoddard, Structure 19, 7 (2011). 4. F. D. Urnov, E. J. Rebar, M. C. Holmes, H. S. Zhang, P. D. Gregory, Nat. Rev. Genet. 11, 636 (2010). 5. A. J. Bogdanove, D. F. Voytas, Science 333, 1843 (2011). 6. K. Eisenschmidt et al., Nucleic Acids Res. 33, 7039 (2005). 7. B. Wiedenheft, S. H. Sternberg, J. A. Doudna, Nature 482, 331 (2012). 8. E. R. Westra et al., Annu. Rev. Genet. 46, 311 (2012). 9. E. Deltcheva et al., Nature 471, 602 (2011). 10. M. Jinek et al., Science 337, 816 (2012). 11. G. Gasiunas, R. Barrangou, P. Horvath, V. Siksnys, Proc. Natl. Acad. Sci. U.S.A. 109, E2579 (2012). 12. M. Jinek et al., elife 2013; 2:300471 (2013). 10.1126/science.1234726 Heisenberg’s uncertainty principle is demonstrated with a vibrating macroscopic mirror. changed by the tidal forces exerted by gravitational waves. This length difference leads to changes in the interference of light at the output mirror. Monitoring the output intensity can thus be used to measure the relative position of the end mirrors. The effect is small, however, and very small changes in the intensity need to be detected. This eventually runs into a problem caused by the essential granular nature of light (light pulses are made up of individual photons). Even the most carefully stabilized laser produces light with intensity fl uctuations due to the random arrival of individual photons, called shot noise. We rarely need to account for this as the relative size of the intensity fl uctuation falls off as the inverse square root of the intensity, so we can always increase the intensity to improve the signal-to-noise ratio. But there is another problem. If we take into account the mechanical action of light, we see that a price must be paid for An optical cavity with a vibrating mirror. As this mirror moves, the intensity of the transmitted light can be used to monitor its position. (Inset) When the quantum nature of light is included, the random reflection of individual photons shakes the mirror, adding radiation pressure noise to the position measurement. on February 14, 2013 www.sciencemag.org Downloaded from
Page 2 and 3: CONTENTS EDITORIAL 737 Designing Sc
Page 4 and 5: 736 EDITED BY STELLA HURTLEY The Bu
Page 6 and 7: CREDITS: (TOP LEFT) TOM KOCHEL; (RI
Page 8 and 9: CREDIT: WIKIMEDIA COMMONS and orbit
Page 10 and 11: CREDITS (TOP TO BOTTOM): EOS/UNIVER
Page 12 and 13: CREDIT: P. CARRIL/ESA European NEWS
Page 14 and 15: CREDIT: JOHN MACDOUGALL/AFP/GETTY I
Page 16 and 17: CREDIT: TITUS ET AL., SCIENCE TRANS
Page 18 and 19: CREDITS (TOP TO BOTTOM): ISTOCKPHOT
Page 20 and 21: CREDIT: NATIONAL SCIENCE FOUNDATION
Page 22 and 23: CREDITS (CLOCKWISE FROM TOP LEFT):
Page 24 and 25: Sensing cytosolic danger ture capit
Page 26 and 27: TECHNICAL COMMENT 757-c Fig. 2. Rel
Page 28 and 29: TECHNICAL COMMENT 757-d values ment
Page 30 and 31: policy ideas” could be rectifi ed
Page 32 and 33: ARMS CONTROL Beyond Arms-Control Mo
Page 34 and 35: CREDIT: K. SUTLIFF/SCIENCE IMMUNOLO
Page 36 and 37: A Field et al. B Partial modern tre
Page 38 and 39: depend on the binding strength of t
Page 42 and 43: increasing the accuracy of the meas
Page 44 and 45: Structural Biological Materials: Cr
Page 46 and 47: shown in Fig. 1C, illustrate an ene
Page 48 and 49: increasing toughness. These methods
Page 50 and 51: anches are connected by thin, folde
Page 52 and 53: experimental work has been conducte
Page 54 and 55: There are no other known achondrite
Page 56 and 57: δD (‰ V-SMOW) 400 300 200 100 0
Page 58 and 59: We estimated that we achieved a ran
Page 60 and 61: L929 cell lines stably expressing t
Page 62 and 63: Fig. 6. cGAS binds to DNA in the cy
Page 64 and 65: k1 and k2 (byconservationofenergyan
Page 66 and 67: How do we answer this trilemma? As
Page 68 and 69: (9). For n = 3, where we used three
Page 70 and 71: Fig. 2. Schematic and characterizat
Page 72 and 73: computation in the presence of erro
Page 74 and 75: REPORTS greatly eases the requireme
Page 76 and 77: dynamically, therefore, Sr3Ru2O7 ca
Page 78 and 79: 16. N. P. Ong, Phys. Rev. B 43, 193
Page 80 and 81: low-energy break in IC 443 and 21s
Page 82 and 83: 32. T. Kamae, N. Karlsson, T. Mizun
Page 84 and 85: ferent structures rather than the e
Page 86 and 87: Fig. 1. Fish behavioral response to
Page 88 and 89: charge distribution, and DNA bindin
Page 90 and 91:
ecause the efficiency of transcript
Page 92 and 93:
precision and efficiency. To invest
Page 94 and 95:
4. A. J. Wood et al., Science 333,
Page 96 and 97:
targeting ~40.5% exons of genes in
Page 98 and 99:
Fig. 1. DNA-dependent generation of
Page 100 and 101:
of the MS spectra revealed two ions
Page 102 and 103:
memories up to date with new learni
Page 104 and 105:
Fig. 3. PE is a necessary condition
Page 106 and 107:
Gordon Research Conferences: 2013
Page 108 and 109:
visit the frontiers of science. . .
Page 110 and 111:
Page 112 and 113:
Page 114 and 115:
Page 116 and 117:
Page 118 and 119:
Page 120 and 121:
Page 122 and 123:
Page 124 and 125:
Gordon Research Conferences: “Ses
Page 126 and 127:
Page 128 and 129:
Page 130 and 131:
MiSeq ® . Next-generation sequenci
Page 132 and 133:
“ % of baseline rate WE’RE BRIN
Page 134 and 135:
Support the sciences. Get rewarded.
Page 136 and 137:
CREDIT: (© ISTOCKPHOTO.COM/NANTELA
Page 138 and 139:
Produced by the Science New Product
Page 140 and 141:
Cancer Biology Thecomplexity ofcanc
Page 142 and 143:
RESEARCH WITH AN IMPACT The Helmhol
Page 144 and 145:
Scientists and Senior Scientists Su
Page 146 and 147:
Programme Leader Starting salary wi
Page 148 and 149:
AAAS is here - helping scientists a
show all

et al.

Create successful ePaper yourself

Delete template?

Save as template?