Genom Düzenlemede CRISPR/Cas9 Çağı ve Lösemideki Uygulamaları

Yıl 2018, Cilt: 8 Sayı: 3, 232 - 248, 01.12.2018

Nurcan Gümüş , Burçin Tezcanlı Kaymaz

Öz

Prokaryotik
canlıların immün sistemden adapte edilmiş olan CRISPR/Cas9  (Düzenli aralıklarla bölünmüş kısa palindromik
tekrar kümeleri/CRISPR ilişkili nukleaz 9) sistemi genom mühendisliği araçlarının arasına en son eklenen
RNA/protein kompleksidir. CRISPR/Cas9 teknolojisi, endonukleaz Cas9 ve kılavuz
RNA (sgRNA) aracılığı ile genomun istenen bir bölgesinde çift zincir
kırıklarının oluşumunu katalizler. Bu kırıkların (HR: Homolog Rekombinasyon [Homologous Recombination ya da HDR: Homology-Directed Repair]) ve (NHEJ:
Non-Homolog Uç Birleşmesi [Non-Homologous
End-Joining]) DNA tamir mekanizmaları kullanılarak, genom düzenlemesi
gerçekleştirilmektedir. CRISPR/Cas9 teknolojisi; bakteri suşlarının
tanımlanması, gen ve miRNA fonksiyonlarının belirlenmesi, genoma DNA fragmenti eklenmesi/çıkartılması,
gen susturma, transkripsiyonel ve epigenetik hedefleme ya da hastalık
modellerinin oluşturulması gibi çok geniş bir platformda kullanılmaktadır.

Kan/Kemik
iliği hastalıklarında lökositoz ile karakterize olan malignite gösteren lösemiler,
kromozomal yeniden düzenlemeler ya da mutasyonlar sonucunda gelişmektedir.
Günümüzde löseminin moleküler biyolojisi ile patogenezinin anlaşılması,
gelecekte çok daha etkin ve kişiye özgül tedavi imkanları sağlayacağından;
genom mühendisliği bu noktada önem kazanmaktadır. Çok geniş kullanım alanına
sahip olan CRISPR/Cas9 genom tasarımı teknolojisi, hastalık modellerinin
oluşturulması, gen ekleme ve susturulması, epigenetik regülasyon gibi alanlarda
kullanılarak, lösemi tedavisinde yeni bir fonksiyonel hedef ve tedavi çağı
başlangıcı olarak karşımıza çıkmaktadır. Bu derlemede, CRISPR/Cas9
teknolojisinin; tarihçesi, lokus birleşenleri, alt tipleri, adaptif bağışıklık
yanıtının oluşum aşamaları, Cas9 özgüllüğü ile bu teknolojinin çeşitli lösemi
tiplerinde kullanımıyla elde edilen tedavi kazanımlar ele alınacaktır. Ayrıca
bu derlemede CRISPR/Cas9 teknolojisinin, etik açısından değerlendirmelerine de
yer verilecektir.

Anahtar Kelimeler

CRISPR/Cas9, genom düzenleme teknolojisi, lösemi, etik

CRISPR/Cas9 Age in Genome Editing and Leukemia Applications

Öz

The
CRISPR/Cas9 ( clustered regularly interspaced short palindromic repeat/CRISPR-associated
nuclease-9) system which adapted from the prokaryotic immune system, is the
latest RNA/protein complex to be included among genomic engineering tools. CRISPR/Cas9
technology catalyzes the formation of double-strand breaks in DNA, according to
the Watson-Crick base pairing in a interested region of the genome, via
endonuclease Cas9 and guide RNA (sgRNA). Genomic regulation is performed by
repairing these fractures using (Homologous
Recombination or HDR:
Homology-Directed Repair)  and (NHEJ:
Non-Homologous End-Joining) DNA
repair mechanisms. CRISPR/Cas9 technology is used on a wide range of platforms,
 starting from identification of
bacterial strains, identification of gene and miRNA functions, genomic DNA
fragment insertion/deletion, gene silencing, transcriptional and epigenetic
targeting to creation of disease models. Leukemia, a malignancy characterized
by leukocytosis in the blood/bone marrow, is caused by chromosomal rearrangements
or mutations. Nowadays, genomic engineering is gaining an accelerating importance
in order to elucidate the pathogenesis and molecular biology of leukemia; thus
to provide more effective and personalised treatment opportunities in the
future. The widely used CRISPR/Cas9 genome design technologyrepresent a new
functional object for treatment of leukemia an the begining of a therapeutic
new era by being applied in areas such as creation of disease models, gene
insertion and silencing, epigenetic regulation. In this review, CRISPR/Cas9
technology; locus components, subtypes, stages of development of the adaptive
immune response, Cas9 specificity and therapeutic gains obtained via using this
technology in various types of leukemia will be discused. Also In this review,
the evaluation of CRISPR/Cas9 technology in terms of ethics will be included.

Anahtar Kelimeler

CRISPR/Cas9, genome editing technology, leukemia, ethics

Kaynakça

• 1. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science (80- ) 2007;315(5819):1709–12. 2. Sorek R, Kunin V, Hugenholtz P. CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol. 2008;6(3):181–6. 3. Breitbart M, Rohwer F. Here a virus, there a virus, everywhere the same virus? Trends Microbiol. 2005;13(6):278–84. 4. Karginov F V., Hannon GJ. The CRISPR System: Small RNA-Guided Defense in Bacteria and Archaea. Mol Cell.; 2010;37(1):7–19. 5. Hyman P, Abedon ST. Bacteriophage host range and bacterial resistance. Advances in applied microbiology. Elsevier Inc.; 2010;70: 217-48 . 6. Barrangou R, Marraffini LA. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Molecular Cell. 2014;54(2): 234–44. 7. Singh V, Braddick D, Dhar PK. Exploring the potential of genome editing CRISPR-Cas9 technology. Gene. 2017;599:1–18. 8. Terns MP, Terns RM. CRISPR-based adaptive immune systems. Curr Opin Microbiol. 2011;14(3):321–7. 9. Rath D, Amlinger L, Rath A, Lundgren M. The CRISPR-Cas immune system: Biology, mechanisms and applications. Biochimie. 2015;117: 119–28. 10. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 1987;169(12):5429–33. 11. Mojica FJM, Díez-Villaseñor C, Soria E, Juez G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol. 2000;36(1):244–6. 12. Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43(6):1565–75. 13. Al-Attar S, Westra ER, Van Der Oost J, Brouns SJJ. Clustered regularly interspaced short palindromic repeats (CRISPRs): The hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol Chem. 2011;392(4):277–89. 14. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005;151(8):2551–61. 15. Mojica FJM, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005;60(2):174–82. 16. Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. 2005;151(3):653–63. 17. Bozok Çetintaş V, Kotmakçı M, Tezcanlı Kaymaz B. From the Immune Response to the Genome Design; CRISPR-Cas9 System: Review. Turkiye Klin J Med Sci. 2017;37(1):27–42. 18. Horvath P, Barrangou R. CRISPR/Cas, the Immune System of Bacteria and Archaea. Science. 2010;327(5962):167–70. 19. Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, et al. RNA-Guided RNA Cleavage by a Cris RNA-Cas Protein Complex. Cell. 2009;139(5):945–56. 20. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH, Snijders AP, et al. Small CRİSPR RNAs Guide Antiviral Defense in Prokaryotes. Science 2008;321(5891):960–4. 21. Garneau JE, Dupuis M-È, Villion M, Romero DA, Barrangou R, Boyaval P, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010;468(7320):67–71. 22. Deltcheva E, Chylinski K, Sharma CM, Gonzales K. CRISPR RNA maturation by trans -encoded small RNA and host factor RNase III. Nature. 2011;471(7340):602–7. 23. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 2011;39(21):9275–82. 24. Zhang J, Rouillon C, Kerou M, Reeks J, Brugger K, Reimann J, et al. Structure and mechanism of the CMR complex for CRISPR- mediated antiviral immunity. Mol Cell. 2012;45(3):303–13. 25. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012;337(6096):816–21. 26. Ju X Da, Xu J, Sun ZS. CRISPR Editing in Biological and Biomedical Investigation. J Cell Biochem. 2018;119(1):52-61 27. Cong L, Ran FA, Cox D, Lin S, Barretto R, Hsu PD, et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 2013;339(6121):819–23. 28. Mali PL, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, et al. RNA-Guided Human Genome Engineering via Cas9. 2013;339(6121):823–6. 29. Osborn MJ, Belanto JJ, Tolar J, Voytas DF. Gene editing and its application for hematological diseases. Int J Hematol. 2016;104(1):18–28. 30. Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics. 2007;8(1):172. 31. Kunin V, Sorek R, Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol. 2007;8(4):R61. 32. Barrangou R. CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA. 2013;4(3):267–78. 33. Lillestøl R, Redder P, Garrett RA, Brügger K. A putative viral defence mechanism in archaeal cells. Archaea. 2006;2(1):59–72. 34. Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, et al. Essential Features and Rational Design of CRISPR RNAs that Function with the Cas RAMP Module Complex to Cleave RNAs. Mol Cell. 2012;45(3):292–302. 35. Pul Ü, Wurm R, Arslan Z, Geissen R, Hofmann N, Wagner R. Identification and characterization of E. coli CRISPR-Cas promoters and their silencing by H-NS. Mol Microbiol. 2010;75(6):1495–512. 36. Spilman M, Cocozaki A, Hale C, Shao Y, Ramia N, Terns R, et al. Structure of an RNA Silencing Complex of the CRISPR-Cas Immune System. Mol Cell. 2013;52(1):146–52. 37. Haft DH, Selengut J, Mongodin EF, Nelson KE. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol. 2005;1(6):474–83. 38. Makarova KS, Grishin N V, Shabalina SA, Wolf YI, Koonin E V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006;16(1):7. 39. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E , Horvath P, et al.. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 2011;9(6):467–77. l 40. Jiang F, Doudna JA. The structural biology of CRISPR-Cas systems. Curr Opin Struct Biol. 2015;30:100–11. 41. Makarova KS, Wolf YI, Koonin E V. The basic building blocks and evolution of CRISPR–Cas systems. Biochem Soc Trans. 2013;41(6):1392–400. 42. Hatoum-Aslan A, Maniv I, Marraffini LA. Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc Natl Acad Sci U S A. 2011;108(52):21218–22. 43. Staals RHJ, Agari Y, Maki-yonekura S, Zhu Y, David W Taylor, Duijn E Van, et al. Structure and activity of the RNA-targeting Type III-B CRISPR- Cas complex of Thermus thermophilus. Molecular Cell 2013;52(1):135–45. 44. Hatoum-Aslan A, Samai P, Maniv I, Jiang W, Marraffini LA. A ruler protein in a complex for antiviral defense determines the length of small interfering CRISPR RNAs. J Biol Chem. 2013;288(39):27888–97. 45. Karvelis T, Gasiunas G, Miksys A, Barrangou R, Horvath P, Siksnys V. crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. RNA Biol. 2013;10(5):841–51. 46. Carte J, Christopher RT, Smith JT, Olson S, Barrangou R, Moineau S, et al. The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus. 2014;93(1):98–112. 47. Hsu PD, Lander ES, Zhang F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell. 2014;157(6):1262–78. 48. Hille F, Charpentier E. CRISPR-Cas : biology , mechanisms and relevance. Philos Trans R Soc Lond B Biol Sci. 2016 5;371(1707). 49. Oost J, Jore MM, Westra ER, Lundgren M, Brouns SJ. CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci. 2009;34(8):401–7. 50. Fineran PC, Charpentier E. Memory of viral infections by CRISPR-Cas adaptive immune systems: Acquisition of new information. Virology.2012;434(2):202–9. 51. Yosef I, Goren MG, Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 2012;40(12):5569–76. 52. Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012;482(7385):331–8. 53. Barrangou R, Horvath P. CRISPR: New Horizons in Phage Resistance and Strain Identification. Annu Rev Food Sci Technol. 2012;3(1):143–62. 54. Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096 55. Westra ER, Swarts DC, Staals RHJ, Jore MM, Brouns SJJ, van der Oost J. The CRISPRs, They Are A-Changin’: How Prokaryotes Generate Adaptive Immunity. Annu Rev Genet. 2012;46(1):311–39. 56. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31(9):827–32. 57. Koo T, Lee J, Kim JS. Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9. Mol Cells. 2015;38(6):475–81. 58. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;156(5):935–49. 59. Shen S, Loh TJ, Shen H, Zheng X, Shen H. CRISPR as a strong gene editing tool. BMB Rep. 2017;50(1):20–4. 60. Tsai SQ, Joung JK. Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases. Nat Rev Genet. 2016;17(5):300–12. 61. Riballo E, Woodbine L, Stiff T, Walker SA, Goodarzi AA, Jeggo PA. XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation. Nucleic Acids Res. 2009;37(2):482–92. 62. Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015;33(5):543–8. 63. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. 64. Gori JL, Hsu PD, Maeder ML, Shen S, Welstead GG, Bumcrot D. Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy. Hum Gene Ther. 2015;26(7):443–51. 65. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013;31(9):822–6. 66. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380–9. 67. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014;32(3):279–84. 68. Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, et al. Analysis of off-target effects of CRISPR Cas-derived RNA-guided endonucleases and nickases. Genome Res. 2014;24(1):132-41 69. Kim S, Kim D, Cho SW, Kim J, Kim J-S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014; 24(6): 1012–19 70. Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 2014;24(6):1020–7. 71. Pliatsika V, Rigoutsos I. “Off-Spotter”: very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/Cas guide RNAs. Biol Direct. 2015;10(1):4. 72. Bae S, Park J, Kim JS. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014;30(10):1473–5. 73. Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E. CHOPCHOP: A CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 2014;42(W1):401–7. 74. Cradick TJ, Qiu P, Lee CM, Fine EJ, Bao G. COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites. Mol Ther - Nucleic Acids. 2014;3:e214. 75. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013;152(5):1173–83. 76. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 2013;154(2):442–51. 77. Khatodia S, Bhatotia K, Passricha N, Khurana SMP, Tuteja N. The CRISPR/Cas Genome-Editing Tool: Application in Improvement of Crops. Front Plant Sci. 2016;7:1–13. 78. Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41(15):7429–37. 79. Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, et al. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Res. 2016;44(12):5615–28. 80. Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol. 2014; 32(6): 577–582. 81. Wyvekens N, Topkar V V., Khayter C, Joung JK, Tsai SQ. Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing. Hum Gene Ther. 2015;26(7):425–31. 82. Rodriguez E. Ethical Issues in Genome Editing using CRISPR/Cas9 System. J Clin Res Bioeth . 2016;7(2):7–10. 83. Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, et al. CRISPR/Cas9 -mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6(5):363–72. 84. Otieno MO. CRISPR-Cas9 Human Genome Editing: Challenges, Ethical Concerns and Implications. J Clin Res Bioeth. 2015;06(06):5–7. 85. Tang LE. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. Molecular Genetics and Genomics. 2017; 292, 525–533. 86. Ma, H. Gutierrez NM, Park ZW. Correction of a pathogenic gene mutation in human embryos. Nature. 2017;548, 413–419. 87. T.C. Resmi Gazete http://www.resmigazete.gov.tr/eskiler/2010/08/20100813-4.htm/;2018 [accessed 04.11.18] 88. Juliusson G, Hough R. Leukemia. Prog Tumor Res. 2016;43:87–100. 89. Wagner G, Fenchel K, Back W, Schulz A, Sachse MM. Leukemia cutis - epidemiology, clinical presentation, and differential diagnoses. J Dtsch Dermatol Ges. 2012;10(1):27–36. 90. Fröhling S, Döhner H. Chromosomal Abnormalities in Cancer. N Engl J Med. 2008;359(7):722–34. 91. Mitelman F, Johansson B, Mertens F. The impact of translocations and gene fusions on cancer causation. Nat Rev Cancer. 2007;7(4):233–45. 92. Torres R, Martin MC, Garcia A, Cigudosa JC, Ramirez JC, Rodriguez-Perales S. Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system. Nat Commun. 2014;5:3964 93. Heckl D, Kowalczyk S M, Yudovich D, Belizaire RP, Rishi VP, McConkey EM, et al. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. 2014;32(9):941–6. 94. Brabetz O, Alla V, Angenendt L, Schliemann C, Berdel WE, Arteaga MF, et al. RNA-Guided CRISPR-Cas9 System-Mediated Engineering of Acute Myeloid Leukemia Mutations. Mol Ther - Nucleic Acids. 2017;6:243–8. 95. Valletta S, Dolatshad H, Bartenstein M, Yip BH, Bello E, Gordon S, et al. ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts. Oncotarget. 2015;6(42):44061–71. 96. García-Tuñón I, Hernández-Sánchez M, Ordoñez JL, Alonso-Pérez V, Álamo-Quijada M, Benito R, et al. The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia. Oncotarget. 2017;8(16):26027–40. 97. Randhawa S, Cho BS, Ghosh D, Sivina M, Koehrer S, Müschen M, et al. Effects of pharmacological and genetic disruption of CXCR4 chemokine receptor function in B-cell acute lymphoblastic leukaemia. Br J Haematol. 2016;174(3):425–36. 98. Xie H, Peng C, Huang J, Li BE, Kim W, Smith EC, et al. Chronic myelogenous leukemia-initiating cells require polycomb group protein EZH2. Cancer Discov. 2016;6(11):1237–47. 99. Tzelepis K, Koike-Yusa H, De Braekeleer E, Li Y, Metzakopian E, Dovey OM, et al. A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia. Cell Rep. 2016;17(4):1193–205. 100.Chen C, Liu Y, Rappaport AR, Kitzing T, Schultz N, Shroff AS, et al. MLL3 Is a Haploinsufficient 7q Tumor Suppressor in Acute Myeloid Leukemia. Cancer Cell. 2014;25(5):652–65. 101.Wan L, Wen H, Li Y, Lyu J, Xi Y, Hoshii T, et al. ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia. Nature. 2017;543:265-9 102.Arya D, Sachithanandan SP, Ross C, Palakodeti D, Li S, Krishna S. MiRNA182 regulates percentage of myeloid and erythroid cells in chronic myeloid leukemia. Cell Death Dis. 2017;8(1):e2547. 103.Wallace J, Hu R, Mosbruger TL, Dahlem TJ, Stephens WZ, Rao DS, et al. Genome wide CRISPR-Cas9 screen identifies microRNAs that regulate myeloid leukemia cell growth. PLoS One. 2016;11(4):1–11. 104.Vukovic M, Guitart A V., Sepulveda C, Villacreces A, O’Duibhir E, Panagopoulou TI, et al. Hif-1α and Hif-2α synergize to suppress AML development but are dispensable for disease maintenance. J Exp Med. 2015;212(13):2223–34. 105.Rathe SK, Moriarity BS, Stoltenberg CB, Kurata M, Aumann NK, Rahrmann EP, et al. Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia. Sci Rep. 2014;4:1–9. 106.Cooper S, Guo H, Friedman AD. The +37 kb Cebpa enhancer is critical for Cebpa myeloid gene expression and contains functional sites that bind SCL, GATA2, C/EBPα, PU.1, and additional Ets factors. PLoS One. 2015;10(5):1–18. 107.Navarro JM, Touzart A, Pradel LC, Loosveld M, Koubi M, Fenouil R, et al. Site- and allele-specific polycomb dysregulation in T-cell leukaemia. Nat Commun. 2015;6. 108.Miyajima C, Itoh Y, Inoue Y, Hayashi H. Positive Regulation of Interleukin-2 Expression by a Pseudokinase, Tribbles 1, in Activated T Cells. Biol Pharm Bull. 2015;38(8):1126–33. 109.Kampa-Schittenhelm KM, Salitzky O, Akmut F, Illing B, Kanz L, Salih HR, et al. Dronabinol has preferential antileukemic activity in acute lymphoblastic and myeloid leukemia with lymphoid differentiation patterns. BMC Cancer.2016;16(1):1–12. 110.Wang T, Wei J, Sabatini D, Lander E. Genetic screens in human cells using the CRISPR/Cas9 system. Science. 2014;343(6166):80–4. 111.Shi J, Wang E, Milazzo JP, Wang Z, Kinney JB, Vakoc CR. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. 2015;33(6):661–7 112.Zhang H, McCarty Nami. CRISPR-Cas9 technology and its application in haematological disorders. Br J Haematol. 2016 Oct;175(2):208-25