CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

Abstract

With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.

Keywords

CAS9 / DNA-PK / DNA double-stranded breaks / genetic instability / DNA repair

References

[1] Barrangou R, Doudna JA (2016) Applications of CRISPR technologies in research and beyond. Nat Biotechnol 34:933–941

[2] Chen J, Li WJ, Cui K, Ji KY, Xu SX, Xu Y(2018) Artemisitene suppresses tumorigenesis by inducing DNA damage through deregulating c-Myc-topoisomerase pathway. Oncogene 37:5079–5087

[3] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W,Marraffini LA (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823

[4] Davis AJ, Chen BPC, Chen DJ (2014) DNA-PK: a dynamic enzyme in a versatile DSB repair pathway. DNA Repair 17:21–29

[5] Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, Pavel-Dinu M, Saxena N, Wilkens AB, Mantri S (2016) CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature 539:384–389

[6] Gilbert Luke A, Larson Matthew H, Morsut L, Liu Z, Brar Gloria A, Torres Sandra E, Stern-Ginossar N, Brandman O, Whitehead Evan H,Doudna Jennifer A (2013) CRISPR-mediated modular RNAguided regulation of transcription in eukaryotes. Cell 154:442–451

[7] Gomez-Cabello D, Jimeno S, Fernández-Ávila MJ, Huertas P (2013) New tools to study DNA double-strand break repair pathway choice. PLoS ONE 8:e77206

[8] Guo XG, Chavez A, Tung A, Chan Y, Kaas C, Yin Y, Cecchi R, Garnier SL, Kelsic ED, Schubert M (2018) High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast. Nat Biotechnol 36:540–546

[9] Haapaniemi E, Botla S, Persson J,Schmierer B, Taipale J (2018) CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med 24:927–930

[10] Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP, Cofsky JC, Kyrpides NC, Banfield JF, Doudna JA (2018) Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science 362:839–842

[11] Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z (2018) Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556:57–63

[12] Ihry RJ, Worringer KA, Salick MR, Frias E, Ho D, Theriault K, Kommineni S, Chen J, Sondey M, Ye CY (2018) p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med 24:939–946

[13] Jackson SP, Bartek J (2009) The DNA-damage response in human biology and disease. Nature 461:1071–1078

[14] Jinek M, Chylinski K, Fonfara I, Hauer M,Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

[15] Kang J,Bronson RT, Xu Y (2002) Targeted disruption of NBS1 reveals its roles in mouse development and DNA repair. EMBO J 21:1447–1455

[16] Kim J,Yu LL, Chen WC, Xu YX, Wu M, Todorova D, Tang QS, Feng BB, Jiang L, He JJ (2019) Wild-type p53 promotes cancer metabolic switch by inducing PUMA-dependent suppression of oxidative phosphorylation. Cancer Cell 35:191–203

[17] Komor AC, Badran AH, Liu DR (2017) CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168:20–36

[18] Kosicki M, Tomberg K, Bradley A (2018) Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36:765–771

[19] Lei L,Chen H, Xue W, Yang B,Hu B, Wei J, Wang L, Cui Y, Li W, Wang J (2018) APOBEC3 induces mutations during repair of CRISPR–Cas9-generated DNA breaks. Nat Struct Mol Biol 25:45–52

[20] Lin T, Chao C, Saito S, Mazur SJ, Murphy ME, Appella E, Xu Y (2005) p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression. Nat Cell Biol 7:165–171 Epub 2004 Dec 2026

[21] Maeder ML, Linder SJ, Cascio VM, Fu YF, Ho QH, Joung JK (2013) CRISPR RNA-guided activation of endogenous human genes. Nat Methods 10:977–979

[22] Mali P, Esvelt KM, Church GM (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10:957–963

[23] Mladenov E, Iliakis G (2011) Induction and repair of DNA double strand breaks: the increasing spectrum of non-homologous end joining pathways. Mut Res 711:61–72

[24] Murovec J, Pirc Z, Yang B (2017) New variants of CRISPR RNAguided genome editing enzymes. Plant Biotechnol J 15:917–926

[25] Song H, Chung SK, Xu Y (2010) Modeling disease in human ESCs using an efficient BAC-based homologous recombination system. Cell Stem Cell 6:80–89

[26] Tan EP, Li YL, Velasco-Herrera MD, Yusa K, Bradley A (2015) Offtarget assessment of CRISPR-Cas9 guiding RNAs in human iPS and mouse ES cells. Genesis 53:225–236

[27] Uematsu N, Weterings E,Yano K,Morotomi-Yano K, Jakob B, Taucher-Scholz G, Mari PO, van Gent DC, Chen BPC, Chen DJ (2007) Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks. J Cell Biol 177:219–229

[28] Urnov FD (2018) Ctrl-Alt-inDel: genome editing to reprogram a cell in the clinic. Curr Opin Genet Dev 52:48–56

[29] WareJoncas Z, Campbell JM, Martínez-Gálvez G, Gendron WAC, Barry MA, Harris PC, Sussman CR, Ekker SC (2018) Precision gene editing technology and applications in nephrology. Nat Rev Nephrol 14:663–677

[30] Xiong J, Todorova D, Su NY, Kim J, Lee PJ, Shen Z, Briggs SP, Xu Y (2015) Stemness factor Sall4 is required for DNA damage response in embryonic stem cells. J Cell Biol 208:513–520

[31] Zetsche B, Gootenberg Jonathan S, Abudayyeh Omar O, Slaymaker Ian M, Makarova Kira S, Essletzbichler P,Volz Sara E, Joung J, van der Oost J, Regev A (2015) Cpf1 Is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771

[32] Zhu J, Ming C,Fu X, Duan YO, Hoang DA, Rutgard J, Zhang RZ, Wang WQ, Hou R, Zhang D (2019) Gene and mutation independent therapy via CRISPR-Cas9 mediated cellular reprogramming in rod photoreceptors (vol 27, pg 830, 2017). Cell Res 29:337–337

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