1. Department of Cell Biology, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China 2. Yunnan Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China 3. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 4. State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China 5. Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
OO Abudayyeh, JS Gootenberg, P Essletzbichler, S Han, J Joung, JJ Belanto, V Verdine, DBT Cox, MJ Kellner, A Regevet al. (2017) RNA targeting with CRISPR-Cas13. Nature 550:280–284 https://doi.org/10.1038/nature24049
2
OO Abudayyeh, JS Gootenberg, B Franklin, J Koob, MJ Kellner, A Ladha, J Joung, P Kirchgatterer, DBT Cox, F Zhang (2019) A cytosine deaminase for programmable single-base RNA editing. Science 365:382–386 https://doi.org/10.1126/science.aax7063
3
Allergan (2019) Single ascending dose study in participants with LCA10. ClinicalTrial.gov Identifier: NCT03872479. (clinicaltrials.-gov/ct2/show/NCT03872479)
4
L Amoasii, JCW Hildyard, H Li, E Sanchez-Ortiz, A Mireault, D Caballero, R Harron, TR Stathopoulou, C Massey, JM Sheltonet al. (2018) Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science 362:86–91 https://doi.org/10.1126/science.aau1549
5
AV Anzalone, PB Randolph, JR Davis, AA Sousa, LW Koblan, JM Levy, PJ Chen, C Wilson, GA Newby, A Raguramet al. (2019) Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576:149–157 https://doi.org/10.1038/s41586-019-1711-4
6
P Cameron, MM Coons, SE Klompe, AM Lied, SC Smith, B Vidal, PD Donohoue, T Rotstein, BW Kohrs, DB Nyeret al. (2019) Harnessing type I CRISPR-Cas systems for genome engineering in human cells. Nat Biotechnol 37(12):1471–1477 https://doi.org/10.1038/s41587-019-0310-0
7
B Chen, LA Gilbert, BA Cimini, J Schnitzbauer, W Zhang, GW Li, J Park, EH Blackburn, JS Weissman, LS Qiet al. (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479–1491 https://doi.org/10.1016/j.cell.2013.12.001
B Chen, J Hu, R Almeida, H Liu, S Balakrishnan, C Covill-Cooke, WA Lim, B Huang (2016b) Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci. Nucleic Acids Res 44:e75 https://doi.org/10.1093/nar/gkv1533
10
B Chen, W Zou, H Xu, Y Liang, B Huang (2018) Efficient labeling and imaging of protein-coding genes in living cells using CRISPRTag. Nat Commun 9:5065 https://doi.org/10.1038/s41467-018-07498-y
11
AW Cheng, N Jillette, P Lee, D Plaskon, Y Fujiwara, W Wang, A Taghbalout, H Wang (2016) Casilio: a versatile CRISPR-Cas9Pumilio hybrid for gene regulation and genomic labeling. Cell Res 26:254–257 https://doi.org/10.1038/cr.2016.3
12
Chinese PLA General Hospital (2018) Study of PD-1 gene-knocked out mesothelin-directed CAR-T cells with the conditioning of PC in mesothelin positive multiple solid tumors. Identifier: NCT03747965.
13
AE Dolan, Z Hou, Y Xiao, MJ Gramelspacher, J Heo, SE Howden, PL Freddolino, A Ke, Y Zhang (2019) Introducing a spectrum of long-range genomic deletions in human embryonic stem cells using type I CRISPR-Cas. Mol Cell 74(5):936–950 https://doi.org/10.1016/j.molcel.2019.03.014
14
S Dreissig, S Schiml, P Schindele, O Weiss, T Rutten, V Schubert, E Gladilin, MF Mette, H Puchta, A Houben (2017) Live-cell CRISPR imaging in plants reveals dynamic telomere movements. Plant J 91:565–573 https://doi.org/10.1111/tpj.13601
15
J Duan, G Lu, Y Hong, Q Hu, X Mai, J Guo, X Si, F Wang, Y Zhang (2018) Live imaging and tracking of genome regions in CRISPR/ dCas9 knock-in mice. Genome Biol 19:192 https://doi.org/10.1186/s13059-018-1530-1
16
A Edraki, A Mir, R Ibraheim, I Gainetdinov, Y Yoon, CQ Song, Y Cao, J Gallant, W Xue, JA Rivera-Perezet al. (2018) A compact, highaccuracy Cas9 with a dinucleotide PAM for in vivo genome editing. Mol Cell 73(4):714–726 https://doi.org/10.1016/j.molcel.2018.12.003
17
Y Fu, PP Rocha, VM Luo, R Raviram, Y Deng, EO Mazzoni, JA Skok (2016) CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci. Nat Commun 7:11707 https://doi.org/10.1038/ncomms11707
18
X Gao, Y Tao, V Lamas, M Huang, WH Yeh, B Pan, YJ Hu, JH Hu, DB Thompson, Y Shuet al. (2018) Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature 553:217–221 https://doi.org/10.1038/nature25164
19
NM Gaudelli, AC Komor, HA Rees, MS Packer, AH Badran, DI Bryson, DR Liu (2017) Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551:464–471 https://doi.org/10.1038/nature24644
20
J Grünewald, R Zhou, S Iyer, CA Lareau, SP Garcia, MJ Aryee, J Keith Joung (2019) CRISPR adenine and cytosine base editors with reduced RNA off-target activities. bioRxiv. https://doi.org/10.1101/631721
21
B Gu, T Swigut, A Spencley, MR Bauer, M Chung, T Meyer, J Wysocka (2018) Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements. Science 359:1050–1055 https://doi.org/10.1126/science.aao3136
22
D Han, Y Hong, X Mai, Q Hu, G Lu, J Duan, J Xu, X Si, Y Zhang (2019) Systematical study of the mechanistic factors regulating genome dynamics in vivo by CRISPRsie. J Mol Cell Biol 11:1018–1020 https://doi.org/10.1093/jmcb/mjz074
23
S Jin, Y Zong, Q Gao, Z Zhu, Y Wang, P Qin, C Liang, D Wang, JL Qiu, F Zhanget al. (2019) Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364:292–295 https://doi.org/10.1126/science.aaw7166
24
T Karvelis, G Bigelyte, JK Young, Z Hou, R Zedaveinyte, K Pociute, A Silanskas, Č Venclovas, V Siksnys (2019) PAM recognition by miniature CRISPR-Cas14 triggers programmable doublestranded DNA cleavage. bioRxiv. https://doi.org/10.1101/654897
25
T Kelliher, D Starr, X Su, G Tang, Z Chen, J Carter, PE Wittich, S Dong, J Green, E Burchet al. (2019) One-step genome editing of elite crop germplasm during haploid induction. Nat Biotechnol 37:287–292 https://doi.org/10.1038/s41587-019-0038-x
26
I Khanday, D Skinner, B Yang, R Mercier, V Sundaresan (2019) A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature 565:91–95 https://doi.org/10.1038/s41586-018-0785-8
27
D Kim, K Lim, ST Kim, SH Yoon, K Kim, SM Ryu, JS Kim (2017) Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat Biotechnol 35:475–480 https://doi.org/10.1038/nbt.3852
28
SE Klompe, PLH Vo, TS Halpin-Healy, SH Sternberg (2019) Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature 571:219–225 https://doi.org/10.1038/s41586-019-1323-z
29
SC Knight, L Xie, W Deng, B Guglielmi, LB Witkowsky, L Bosanac, ET Zhang, M El Beheiry, JB Masson, M Dahanet al. (2015) Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science 350:823–826 https://doi.org/10.1126/science.aac6572
30
SC Knight, R Tjian, JA Doudna (2018) Genomes in focus: development and applications of CRISPR-Cas9 imaging technologies. Angew Chem Int Ed Engl 57:4329–4337 https://doi.org/10.1002/anie.201709201
31
AC Komor, YB Kim, MS Packer, JA Zuris, DR Liu (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420–424 https://doi.org/10.1038/nature17946
32
EV Koonin, KS Makarova, F Zhang (2017) Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol 37:67–78 https://doi.org/10.1016/j.mib.2017.05.008
33
C-T Kwon, J Heo, ZH Lemmon, Y Capua, SF Hutton, J Van Eck, SJ Park, ZB Lippman (2019) Rapid customization of Solanaceae fruit crops for urban agriculture. Nat Biotechnol 38:182–188 https://doi.org/10.1038/s41587-019-0361-2
34
Z Liu, Y Cai, Y Wang, Y Nie, C Zhang, Y Xu, X Zhang, Y Lu, Z Wang, M Pooet al. (2018) Cloning of macaque monkeys by somatic cell nuclear transfer. Cell 172(881–887):e887 https://doi.org/10.1016/j.cell.2018.01.020
35
C Liu, Y Zhong, X Qi, M Chen, Z Liu, C Chen, X Tian, J Li, Y Jiao, D Wanget al. (2019a) Extension of the in vivo haploid induction system from diploid maize to hexaploid wheat. Plant Biotechnol J 18:316–318 https://doi.org/10.1111/pbi.13218
36
J-J Liu, N Orlova, BL Oakes, E Ma, HB Spinner, KLM Baney, J Chuck, D Tan, GJ Knott, LB Harringtonet al. (2019b) CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566:218–223 https://doi.org/10.1038/s41586-019-0908-x
37
H Ma, LC Tu, A Naseri, M Huisman, S Zhang, D Grunwald, T Pederson (2016a) CRISPR-Cas9 nuclear dynamics and target recognition in living cells. J Cell Biol 214:529–537 https://doi.org/10.1083/jcb.201604115
38
H Ma, LC Tu, A Naseri, M Huisman, S Zhang, D Grunwald, T Pederson (2016b) Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol 34:528–530 https://doi.org/10.1038/nbt.3526
39
H Ma, LC Tu, A Naseri, YC Chung, D Grunwald, S Zhang, T Pederson (2018) CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods 15:928–931 https://doi.org/10.1038/s41592-018-0174-0
40
KS Makarova, YI Wolf, J Iranzo, SA Shmakov, OS Alkhnbashi, SJJ Brouns, E Charpentier, D Cheng, DH Haft, P Horvathet al. (2019) Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 18:67–83 https://doi.org/10.1038/s41579-019-0299-x
41
S Mao, Y Ying, X Wu, CJ Krueger, AK Chen (2019) CRISPR/dualFRET molecular beacon for sensitive live-cell imaging of nonrepetitive genomic loci. Nucleic Acids Res 47:e131 https://doi.org/10.1093/nar/gkz752
42
T Merkle, S Merz, P Reautschnig, A Blaha, Q Li, P Vogel, J Wettengel, JB Li, T Stafforst (2019) Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat Biotechnol 37:133–138 https://doi.org/10.1038/s41587-019-0013-6
43
H Morisaka, K Yoshimi, Y Okuzaki, P Gee, Y Kunihiro, E Sonpho, H Xu, N Sasakawa, Y Naito, S Nakadaet al. (2019) CRISPR-Cas3 induces broad and unidirectional genome editing in human cells. Nat Commun 10:5302 https://doi.org/10.1038/s41467-019-13226-x
44
DA Nelles, MY Fang, MR O’Connell, JL Xu, SJ Markmiller, JA Doudna, GW Yeo (2016) Programmable RNA Tracking in live cells with CRISPR/Cas9. Cell 165:488–496 https://doi.org/10.1016/j.cell.2016.02.054
45
CE Nelson, Y Wu, MP Gemberling, ML Oliver, MA Waller, JD Bohning, JN Robinson-Hamm, K Bulaklak, RM Castellanos Rivera, JH Collieret al. (2019) Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat Med 25:427–432 https://doi.org/10.1038/s41591-019-0344-3
46
P Qin, M Parlak, C Kuscu, J Bandaria, M Mir, K Szlachta, R Singh, X Darzacq, A Yildiz, M Adli (2017) Live cell imaging of lowand non-repetitive chromosome loci using CRISPR-Cas9. Nat Commun 8:14725 https://doi.org/10.1038/ncomms14725
47
PY Qiu, J Jiang, Z Liu, YJ Cai, T Huang, Y Wang, QM Liu, YH Nie, F Liu, JM Chenget al. (2019) BMAL1 knockout macaque monkeys display reduced sleep and psychiatric disorders. Natl Sci Rev 6:87–100 https://doi.org/10.1093/nsr/nwz002
48
L Qu, Z Yi, S Zhu, C Wang, Z Cao, Z Zhou, P Yuan, Y Yu, F Tian, Z Liuet al. (2019) Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs. Nat Biotechnol 37:1059–1069 https://doi.org/10.1038/s41587-019-0178-z
49
HA Rees, DR Liu (2018) Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet 19 (12):770–780 https://doi.org/10.1038/s41576-018-0059-1
50
S Shao, W Zhang, H Hu, B Xue, J Qin, C Sun, Y Sun, W Wei, Y Sun (2016) Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system. Nucleic Acids Res 44:e86 https://doi.org/10.1093/nar/gkw066
51
J Strecker, S Jones, B Koopal, J Schmid-Burgk, B Zetsche, L Gao, KS Makarova, EV Koonin, F Zhang (2019a) Engineering of CRISPR-Cas12b for human genome editing. Nat Commun 10:1–8 https://doi.org/10.1038/s41467-018-08224-4
52
J Strecker, A Ladha, Z Gardner, JL Schmid-Burgk, KS Makarova, EV Koonin, F Zhang (2019b) RNA-guided DNA insertion with CRISPR-associated transposases. Science 365:48–53 https://doi.org/10.1126/science.aax9181
53
ME Tanenbaum, LA Gilbert, LS Qi, JS Weissman, RD Vale (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:635–646 https://doi.org/10.1016/j.cell.2014.09.039
54
F Teng, J Li, T Cui, K Xu, L Guo, Q Gao, G Feng, C Chen, D Han, Q Zhouet al. (2019) Enhanced mammalian genome editing by new Cas12a orthologs with optimized crRNA scaffolds. Genome Biol 20:1–6 https://doi.org/10.1186/s13059-019-1620-8
55
Vertex (2018a) A safety and efficacy study evaluating CTX001 in subjects with severe sickle cell disease. ClinicalTrial.gov Identi fier: NCT03745287. (clinicaltrials.gov/ct2/show/NCT03745287)
56
Vertex (2018b) A safety and efficacy study evaluating CTX001 in subjects with transfusion-dependent β-thalassemia. ClinicalTrial. gov Identifier: NCT03655678. (clinicaltrials.gov/ct2/show/ NCT03655678)
57
S Wang, JH Su, F Zhang, X Zhuang (2016) An RNA-aptamer-based two-color CRISPR labeling system. Sci Rep 6:26857 https://doi.org/10.1038/srep26857
58
B Wang, L Zhu, B Zhao, Y Zhao, Y Xie, Z Zheng, Y Li, J Sun, H Wang (2019a) Development of a haploid-inducer mediated genome editing system for accelerating maize breeding. Mol Plant 12:597–602 https://doi.org/10.1016/j.molp.2019.03.006
59
C Wang, Q Liu, Y Shen, Y Hua, J Wang, J Lin, M Wu, T Sun, Z Cheng, R Mercieret al. (2019b) Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes. Nat Biotechnol 37:283–286 https://doi.org/10.1038/s41587-018-0003-0
60
H Wang, M Nakamura, TR Abbott, D Zhao, K Luo, C Yu, CM Nguyen, A Lo, TP Daley, M La Russaet al. (2019c) CRISPR-mediated live imaging of genome editing and transcription. Science 365:1301–1305 https://doi.org/10.1126/science.aax7852
61
X Wu, S Mao, Y Ying, CJ Krueger, AK Chen (2019) Progress and Challenges for Live-cell Imaging of Genomic Loci Using CRISPR based Platforms. Genom Proteom Bioinform 17:119–128 https://doi.org/10.1016/j.gpb.2018.10.001
S Yan, Z Tu, Z Liu, N Fan, H Yang, S Yang, W Yang, Y Zhao, Z Ouyang, C Laiet al. (2018) A Huntingtin Knockin PIG model recapitulates features of selective neurodegeneration in Huntington’s disease. Cell 173(989–1002):e1013 https://doi.org/10.1016/j.cell.2018.03.005
64
WX Yan, P Hunnewell, LE Alfonse, JM Carte, E Keston-Smith, S Sothiselvam, AJ Garrity, S Chong, KS Makarova, EV Kooninet al. (2019) Functionally diverse type V CRISPR-Cas systems. Science 363:88–91 https://doi.org/10.1126/science.aav7271
65
LZ Yang, Y Wang, SQ Li, RW Yao, PF Luan, H Wu, GG Carmichael, LL Chen (2019) Dynamic imaging of RNA in living cells by CRISPR-Cas13 systems. Mol Cell 76(981–997):e987 https://doi.org/10.1016/j.molcel.2019.10.024
66
H Ye, Z Rong, Y Lin (2017) Live cell imaging of genomic loci using dCas9-SunTag system and a bright fluorescent protein. Protein Cell 8:853–855 https://doi.org/10.1007/s13238-017-0460-0
W Zhang, H Wan, G Feng, J Qu, J Wang, Y Jing, R Ren, Z Liu, L Zhang, Z Chenet al. (2018) SIRT6 deficiency results in developmental retardation in cynomolgus monkeys. Nature 560:661–665 https://doi.org/10.1038/s41586-018-0437-z
69
R Zhang, J Liu, Z Chai, S Chen, Y Bai, Y Zong, K Chen, J Li, L Jiang, C Gao (2019) Generation of herbicide tolerance traits and a new selectable marker in wheat using base editing. Nat Plants 5:480–485 https://doi.org/10.1038/s41477-019-0405-0
70
Y Zhong, C Liu, X Qi, Y Jiao, D Wang, Y Wang, Z Liu, C Chen, B Chen, X Tianet al. (2019) Mutation of ZmDMP enhances haploid induction in maize. Nat Plants 5:575–580 https://doi.org/10.1038/s41477-019-0443-7
71
Y Zhou, P Wang, F Tian, G Gao, L Huang, W Wei, XS Xie (2017) Painting a specific chromosome with CRISPR/Cas9 for live-cell imaging. Cell Res 27:298–301 https://doi.org/10.1038/cr.2017.9
72
C Zhou, Y Sun, R Yan, Y Liu, E Zuo, C Gu, L Han, Y Wei, X Hu, R Zenget al. (2019a) Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571:275–278 https://doi.org/10.1038/s41586-019-1314-0
73
Y Zhou, J Sharma, Q Ke, R Landman, JL Yuan, H Chen, DS Hayden, JW Fisher, MQ Jiang, W Menegaset al. (2019b) Atypical behaviour and connectivity in SHANK3-mutant macaques. Nature 570:326–331 https://doi.org/10.1038/s41586-019-1278-0
74
E Zuo, Y Sun, W Wei, T Yuan, W Ying, H Sun, L Yuan, LM Steinmetz, Y Li, H Yang (2019) Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364:289 https://doi.org/10.1126/science.aav9973