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Shortening the Half-Life of the CRISPR/dCas9 System


Shortening the Half-Life of the CRISPR/dCas9 System

Jaunky, Brandon Boodhai (2023) Shortening the Half-Life of the CRISPR/dCas9 System. Masters thesis, Concordia University.

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Shortening the half-life of genetic engineering tools such as dCas9-VP64 can become a pivotal component to regulate many cellular networks, allowing periodic gene expression and protein levels, thereby governing vital cellular processes. However, currently this effector protein has a prolonged half-life in cells and can cause adverse effects by binding to other regions or interacting with other transcriptional or translational machinery. This thesis aims to explore the N-End Rule as a novel approach to achieve shorter half-life dCas9-VP64, offering tighter control for future applications in synthetic circuits.
The need for shorter half-life gene effectors arises from the cytotoxic effects observed with persistence exposure that can lead to off-target effects and unintended modifications, hampering their accuracy and reliability in genetic manipulation. The introduction of short-lived effector proteins also holds significant promise for enhancing temporal control and predictable timing of protein degradation.
The central hypothesis driving this research is that the N-End Rule can be harnessed to shorten the half-life of the artificial transcriptional activator dCas9-VP64 (Varshavsky, 1998). Through site-directed mutagenesis, four amino acids were introduced at the N-terminus of dCas9-VP64, including alanine, valine, serine, and cysteine. These amino acid residues were identified as destabilizing when positioned at N-termini based on the N-acetylation N-End Rule (Nguyen, K.T. et al., 2018).
Experimental design involved genetic construct development, fusion with a fluorescent protein tag for visualization, and validation of N-terminus mutations. Using cycloheximide, a protein synthesis and translation inhibitor, the steady-state level of the dCas9-VP64-eGFP protein was monitored through eGFP fluorescence from a microplate reader assay and through western blot analysis. We observed different phenotypes during induction and tracking of fusion proteins' signals. Protein half-life was measured using the first order rate kinetic equation for protein degradation.
The results revealed the differential half-life of dCas9-VP64 variants, showcasing the potential of the N-End Rule in achieving shorter half-life. The cysteine variant demonstrated the shortest half-life of 37 minutes, indicating its potential suitability for synthetic circuits. dCas9-VP64-eGFP with the wild-type N-terminus exhibited a half-life of 57 minutes, while serine and alanine variants displayed approximately 54 and 61 minutes, respectively.
In conclusion, this thesis contributes to the advancement of strategies for shortening the half-life of proteins. The implementation of the N-End Rule to achieve this goal exemplifies its potential in optimizing genetic behavior control in future applications with synthetic circuits. By harnessing the N-End Rule, researchers can pave the way for future innovations and advancements in the realm of genetic engineering and molecular biology.

Divisions:Concordia University > Faculty of Arts and Science > Biology
Item Type:Thesis (Masters)
Authors:Jaunky, Brandon Boodhai
Institution:Concordia University
Degree Name:M. Sc.
Date:17 August 2023
Thesis Supervisor(s):Zerges, William and Kharma, Nawwaf
Keywords:CRISPR/dCas9, VP64, N-End Rule, Half-Life
ID Code:992960
Deposited By: Brandon Jaunky
Deposited On:14 Nov 2023 19:20
Last Modified:14 Nov 2023 19:20


“Fluorescence Microplate-Based Cycloheximide Chase Assay: A Technique to Monitor
the Degradation Kinetics of Fluorescent Nuclear Misfolded Proteins.” Protocol,
to. Accessed 13 Sept. 2023.
● Aksnes, H., Van Damme, P., Goris, M., Starheim, K. K., Marie, M., Støve, S. I., ... &
Arnesen, T. (2015). An organellar Nα-acetyltransferase, naa60, acetylates cytosolic N
termini of transmembrane proteins and maintains Golgi integrity. Cell Reports, 10(8),
● Arnesen, T. Protein N-terminal acetylation: NAT 2007–2008 Symposia. BMC Proc 3
(Suppl 6), S1 (2009). https://doi.org/10.1186/1753-6561-3-S6-S1
● Arnesen, T., Van Damme, P., Polevoda, B., Helsens, K., Evjenth, R., Colaert, N., Varhaug,
J. E., Vandekerckhove, J., Lillehaug, J. R., Sherman, F., Gevaert, K. (2009). Proteomics
analyses reveal the evolutionary conservation and divergence of N-terminal
acetyltransferases from yeast and humans. Proceedings of the National Academy of
Sciences of the United States of America, 106(20), 8157–8162.
● Balboa D, Weltner J, Eurola S, Trokovic R, Wartiovaara K, Otonkoski T. Conditionally
Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell
Reprogramming and Differentiation. Stem Cell Reports. 2015 Sep 8;5(3):448-59. doi:
10.1016/j.stemcr.2015.08.001. PMID: 26352799; PMCID: PMC4618656.
● Bard JAM, Goodall EA, Greene ER, Jonsson E, Dong KC, Martin A. Structure and
Function of the 26S Proteasome. Annu Rev Biochem. 2018 Jun 20;87:697-724. doi:
10.1146/annurev-biochem-062917-011931. Epub 2018 Apr 13. PMID: 29652515;
PMCID: PMC6422034.
● Belle, Archana, et al. “Quantification of protein half-lives in the budding yeast proteome.”
Proceedings of the National Academy of Sciences, vol. 103, no. 35, 2006, pp. 13004–
13009, https://doi.org/10.1073/pnas.0605420103.
● Berndt J, Kovacs P, Ruschke K, Klöting N, Fasshauer M, Schön MR, Körner A, Stumvoll
M, Blüher M. Fatty acid synthase gene expression in human adipose tissue: association
with obesity and type 2 diabetes. Diabetologia. 2007 Jul;50(7):1472-80. doi:
10.1007/s00125-007-0689-x. Epub 2007 May 11. PMID: 17492427.
● Casas-Mollano JA, Zinselmeier MH, Erickson SE, Smanski MJ. CRISPR-Cas Activators
for Engineering Gene Expression in Higher Eukaryotes. CRISPR J. 2020 Oct;3(5):350-
364. doi: 10.1089/crispr.2020.0064. PMID: 33095045; PMCID: PMC7580621.
● Charis L Himeda, Takako I Jones, Peter L Jones, CRISPR/dCas9-mediated Transcriptional
Inhibition Ameliorates the Epigenetic Dysregulation at D4Z4 and Represses DUX4-fl in
FSH Muscular Dystrophy, Molecular Therapy, Volume 24, Issue 3, 2016, Pages 527-535,
ISSN 1525-0016, https://doi.org/10.1038/mt.2015.200.
● Clute, Paul and Julian G. Pines. “Temporal and Spatial Control of Cyclin B1 Destruction
in Metaphase.” Nature Cell Biology, vol. 1, no. 2, 1999, pp. 82-87.
● Duan J, Sun L, Huang H, Wu Z, Wang L, Liao W. Overexpression of fatty acid synthase
predicts a poor prognosis for human gastric cancer. Mol Med Rep. 2016 Apr;13(4):3027-
35. doi: 10.3892/mmr.2016.4902. Epub 2016 Feb 17. PMID: 26936091; PMCID:
● Eagle LR, Yin X, Brothman AR, Williams BJ, Atkin NB, Prochownik EV. Mutation of the
MXI1 gene in prostate cancer. Nat Genet. 1995 Mar;9(3):249-55. doi: 10.1038/ng0395-
249. PMID: 7773287.
● Finley, Daniel. "Recognition and processing of ubiquitin-protein conjugates by the
proteasome." Annual Review of Biochemistry, vol. 78, 2009, pp. 477-513.
● Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N,
Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. CRISPRmediated
modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013 Jul
18;154(2):442-51. doi: 10.1016/j.cell.2013.06.044. Epub 2013 Jul 11. PMID: 23849981;
PMCID: PMC3770145.
● Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., ... & Qi, L. S.
(2014). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.
Cell, 154(2), 442-451.
● Goldberg, A. L. (2003). Protein degradation and protection against misfolded or damaged
proteins. Nature, 426(6968), 895–899.
● Groll, Michael, et al. "A gated channel into the proteasome core particle." Nature Structural
Biology, vol. 7, no. 11, 1997, pp. 1062-1067.
● Haugwitz, M., Garachtchenko, T., Nourzaie, O. et al. Rapid, on-demand protein
stabilization and destabilization using the ProteoTuner™ systems. Nat Methods 5, iii–iv
(2008). https://doi.org/10.1038/nmeth.f.223
● Hershko, A., Ciechanover, A. (1998). The ubiquitin system. Annual Review of
Biochemistry, 67, 425–479.
● Hole K, Van Damme P, Dalva M, Aksnes H, Glomnes N, Varhaug JE, Lillehaug JR,
Gevaert K, Arnesen T. The human N-alpha-acetyltransferase 40 (hNaa40p/hNatD) is
conserved from yeast and N-terminally acetylates histones H2A and H4. PLoS One.
2011;6(9):e24713. doi: 10.1371/journal.pone.0024713. Epub 2011 Sep 15. PMID:
21935442; PMCID: PMC3174195
● Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of
cholesterol and fatty acid synthesis in the liver. J Clin Invest. 2002 May;109(9):1125-31.
doi: 10.1172/JCI15593. PMID: 11994399; PMCID: PMC150968.
● Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., ... &
Zhang, F. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nature
biotechnology, 31(9), 827-832.
● Hwang, C. S., Shemorry, A., & Varshavsky, A. (2003). N-terminal acetylation of cellular
proteins creates specific degradation signals. Science, 327(5968), 973-977.
● Javaid N, Pham TLH, Choi S. Functional Comparison between VP64-dCas9-VP64 and
dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells. Int J Mol Sci. 2021
Jan 1;22(1):397. doi: 10.3390/ijms22010397. PMID: 33401508; PMCID: PMC7795359.
● 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 Aug
17;337(6096):816-21. doi: 10.1126/science.1225829. Epub 2012 Jun 28. PMID:
22745249; PMCID: PMC6286148.
● Kapuscinski J. DAPI: a DNA-specific fluorescent probe. Biotech Histochem. 1995
Sep;70(5):220-33. doi: 10.3109/10520299509108199. PMID: 8580206.
● Karlson CKS, Mohd-Noor SN, Nolte N, Tan BC. CRISPR/dCas9-Based Systems:
Mechanisms and Applications in Plant Sciences. Plants (Basel). 2021 Sep 29;10(10):2055.
doi: 10.3390/plants10102055. PMID: 34685863; PMCID: PMC8540305.
● Khmelinskii A, Meurer M, Ho CT, Besenbeck B, Füller J, Lemberg MK, Bukau B, Mogk
A, Knop M. Incomplete proteasomal degradation of green fluorescent proteins in the
context of tandem fluorescent protein timers. Mol Biol Cell. 2016 Jan 15;27(2):360-70.
doi: 10.1091/mbc.E15-07-0525. Epub 2015 Nov 25. PMID: 26609072; PMCID:
● Khosrow-Khavar F, Fang NN, Ng AH, Winget JM, Comyn SA, Mayor T. The yeast ubr1
ubiquitin ligase participates in a prominent pathway that targets cytosolic thermosensitive
mutants for degradation. G3 (Bethesda). 2012 May;2(5):619-28. doi:
10.1534/g3.111.001933. Epub 2012 May 1. PMID: 22670231; PMCID: PMC3362944.
● Komander, D., Rape, M. (2012). The ubiquitin code. Annual Review of Biochemistry, 81,
● Kondratov, R. V., Kondratova, A. A., Lee, C., Gorbacheva, V. Y., Chernov, M. V., Antoch,
M. P., & Gudkov, A. V. (2006). Post-translational regulation of circadian transcriptional
CLOCK(NPAS2)/BMAL1 complex by CRYPTOCHROMES. Cell Cycle, 5(8), 890-895.
● Korepanova A, Douglas C, Leyngold I, Logan TM. N-terminal extension changes the
folding mechanism of the FK506-binding protein. Protein Sci. 2001 Sep;10(9):1905-10.
doi: 10.1110/ps.14801. PMID: 11514681; PMCID: PMC2253207.
● Kuo HP, Hung MC. Arrest-defective-1 protein (ARD1): tumor suppressor or oncoprotein?
Am J Transl Res. 2010 Jan 1;2(1):56-64. PMID: 20182582; PMCID: PMC2826822.
● Kuo J, Yuan R, Sánchez C, Paulsson J, Silver PA. Toward a translationally independent
RNA-based synthetic oscillator using deactivated CRISPR-Cas. Nucleic Acids Res. 2020
Aug 20;48(14):8165-8177. doi: 10.1093/nar/gkaa557. PMID: 32609820; PMCID:
● Kuo J, Yuan R, Sánchez C, Paulsson J, Silver PA. Toward a translationally independent
RNA-based synthetic oscillator using deactivated CRISPR-Cas. Nucleic Acids Res. 2020
Aug 20;48(14):8165-8177. doi: 10.1093/nar/gkaa557. PMID: 32609820; PMCID:
● Kuo J, Yuan R, Sánchez C, Paulsson J, Silver PA. Toward a translationally independent
RNA-based synthetic oscillator using deactivated CRISPR-Cas. Nucleic Acids Res. 2020
Aug 20;48(14):8165-8177. doi: 10.1093/nar/gkaa557. PMID: 32609820; PMCID:
● Laptenko, Oleg et al. “The p53 C Terminus Controls Site-Specific DNA Binding and
Promotes Structural Changes within the Central DNA Binding Domain.” Molecular and
Cellular Biology, vol. 31, no. 8, 2011, pp. 1739-1752.
● Lei, Y., Huang, Y., Lin, J. et al. Mxi1 participates in the progression of lung cancer via the
microRNA-300/KLF9/GADD34 Axis. Cell Death Dis 13, 425 (2022).
● Li Z, Rinas U. Recombinant protein production associated growth inhibition results mainly
from transcription and not from translation. Microb Cell Fact. 2020 Apr 6;19(1):83. doi:
10.1186/s12934-020-01343-y. PMID: 32252765; PMCID: PMC7137236.
● Naumann C, Mot AC, Dissmeyer N. Generation of Artificial N-end Rule Substrate Proteins
In Vivo and In Vitro. Methods Mol Biol. 2016;1450:55-83. doi: 10.1007/978-1-4939-
3759-2_6. PMID: 27424746.
● Neklesa TK, Winkler JD, Crews CM. Targeted protein degradation by PROTACs.
Pharmacol Ther. 2017 Jun;174:138-144. doi: 10.1016/j.pharmthera.2017.02.027. Epub
2017 Feb 14. PMID: 28223226.
● Nguyen KT, Lee CS, Mun SH, Truong NT, Park SK, Hwang CS. N-terminal acetylation
and the N-end rule pathway control degradation of the lipid droplet protein PLIN2. J Biol
Chem. 2019 Jan 4;294(1):379-388. doi: 10.1074/jbc.RA118.005556. Epub 2018 Nov 13.
PMID: 30425097; PMCID: PMC6322874.
● Nguyen, K.T., Mun, SH., Lee, CS. et al. Control of protein degradation by N-terminal
acetylation and the N-end rule pathway. Exp Mol Med 50, 1–8 (2018).
● Ole K Tørresen, Bastiaan Star, Pablo Mier, Miguel A Andrade-Navarro, Alex Bateman,
Patryk Jarnot, Aleksandra Gruca, Marcin Grynberg, Andrey V Kajava, Vasilis J
Promponas, Maria Anisimova, Kjetill S Jakobsen, Dirk Linke, Tandem repeats lead to
sequence assembly errors and impose multi-level challenges for genome and protein
databases, Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Pages
10994–11006, https://doi.org/10.1093/nar/gkz841
● Omachi K, Miner JH. Comparative analysis of dCas9-VP64 variants and multiplexed guide
RNAs mediating CRISPR activation. PLoS One. 2022 Jun 28;17(6):e0270008. doi:
10.1371/journal.pone.0270008. PMID: 35763517; PMCID: PMC9239446.
● Partch, C. L., et al. (2014). Molecular architecture of the mammalian circadian clock.
Trends in Cell Biology, 24(2), 90-99.
● Peters, J. M., Franke, W. W. (2012). Proteasomes: Protein degradation machines of the
cell. Trends in Biochemical Sciences, 37(12), 633–640.
● Pickart, Cecile M., and Robert E. Cohen. "Proteasomes and their kin: proteases in the
machine age." Nature Reviews Molecular Cell Biology, vol. 5, no. 3, 2004, pp. 177-187.
● Polevoda B, Brown S, Cardillo TS, Rigby S, Sherman F. Yeast N(alpha)-terminal
acetyltransferases are associated with ribosomes. J Cell Biochem. 2008 Feb 1;103(2):492-
508. doi: 10.1002/jcb.21418. PMID: 17541948.
● Polevoda, B., & Sherman, F. (2001). N-terminal acetyltransferases and sequence
requirements for N-terminal acetylation of eukaryotic proteins. Journal of Molecular
Biology, 31(3), 1005-1016.
● Ponugoti B, Kim DH, Xiao Z, Smith Z, Miao J, Zang M, Wu SY, Chiang CM, Veenstra
TD, Kemper JK. SIRT1 deacetylates and inhibits SREBP-1C activity in regulation of
hepatic lipid metabolism. J Biol Chem. 2010 Oct 29;285(44):33959-70. doi:
10.1074/jbc.M110.122978. Epub 2010 Sep 3. PMID: 20817729; PMCID: PMC2962496.
● Samarasinghe KTG, Jaime-Figueroa S, Burgess M, Nalawansha DA, Dai K, Hu Z,
Bebenek A, Holley SA, Crews CM. Targeted degradation of transcription factors by
TRAFTACs: TRAnscription Factor TArgeting Chimeras. Cell Chem Biol. 2021 May
20;28(5):648-661.e5. doi: 10.1016/j.chembiol.2021.03.011. Epub 2021 Apr 8. PMID:
33836141; PMCID: PMC8524358.
● Schneider-Poetsch, T., Ju, J., Eyler, D. et al. Inhibition of eukaryotic translation elongation
by cycloheximide and lactimidomycin. Nat Chem Biol 6, 209–217 (2010).
● Senturk S, Shirole NH, Nowak DG, Corbo V, Pal D, Vaughan A, Tuveson DA, Trotman
LC, Kinney JB, Sordella R. Rapid and tunable method to temporally control gene editing
based on conditional Cas9 stabilization. Nat Commun. 2017 Feb 22;8:14370. doi:
10.1038/ncomms14370. PMID: 28224990; PMCID: PMC5322564.
● Shin HY, Wang C, Lee HK, Yoo KH, Zeng X, Kuhns T, Yang CM, Mohr T, Liu C,
Hennighausen L. CRISPR/Cas9 targeting events cause complex deletions and insertions at
17 sites in the mouse genome. Nat Commun. 2017 May 31;8:15464. doi:
10.1038/ncomms15464. PMID: 28561021; PMCID: PMC5460021.
● Sreekanth V, Zhou Q, Kokkonda P, Bermudez-Cabrera HC, Lim D, Law BK, Holmes BR,
Chaudhary SK, Pergu R, Leger BS, Walker JA, Gifford DK, Sherwood RI, Choudhary A.
Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing
Outcomes. ACS Cent Sci. 2020 Dec 23;6(12):2228-2237. doi:
10.1021/acscentsci.0c00129. Epub 2020 Nov 18. PMID: 33376784; PMCID:
● Starheim, K.K., Gromyko, D., Velde, R. et al. Composition and biological significance of
the human Nα-terminal acetyltransferases. BMC Proc 3 (Suppl 6), S3 (2009).
● Sunbin Deng, Ronen Marmorstein, Protein N-Terminal Acetylation: Structural Basis,
Mechanism, Versatility, and Regulation, Trends in Biochemical Sciences, Volume 46,
Issue 1, 2021, Pages 15-27, ISSN 0968-0004, https://doi.org/10.1016/j.tibs.2020.08.005.
● Swanson, Robert, et al. "A conserved ubiquitin ligase of the nuclear envelope/endoplasmic
reticulum that functions in both ER-associated and Matalpha2 repressor degradation."
Genes & Development, vol. 15, no. 20, 2001, pp. 2660-2674.
● Synthetic Control of Protein Degradation during Cell Proliferation and Developmental
Processes, Jonathan Trauth, Johannes Scheffer, Sophia Hasenjäger, and Christof Taxis,
ACS Omega 2019 4 (2), 2766-2778, DOI: 10.1021/acsomega.8b03011
● Takahashi JS, Hong HK, Ko CH, McDearmon EL. The genetics of mammalian circadian
order and disorder: implications for physiology and disease. Nat Rev Genet. 2008
Oct;9(10):764-75. doi: 10.1038/nrg2430. PMID: 18802415; PMCID: PMC3758473.
● Tanaka K. The proteasome: from basic mechanisms to emerging roles. Keio J Med.
2013;62(1):1-12. doi: 10.2302/kjm.2012-0006-re. PMID: 23563787.
● Tasaki, T., Sriram, S. M., Park, K. S., & Kwon, Y. T. (2012). The N-end rule pathway.
Annual Review of Biochemistry, 81, 261-289.
● Van Damme P, Hole K, Gevaert K, Arnesen T. N-terminal acetylome analysis reveals the
specificity of Naa50 (Nat5) and suggests a kinetic competition between N-terminal
acetyltransferases and methionine aminopeptidases. Proteomics. 2015 Jul;15(14):2436-46.
doi: 10.1002/pmic.201400575. Epub 2015 Jun 5. PMID: 25886145.
● Van Damme, Petra, et al. "N-terminal acetylome analyses and functional insights of the Nterminal
acetyltransferase NatB." Proceedings of the National Academy of Sciences, vol.
109, no. 31, 2012, pp. 12449-12454.
● Varshavsky, A. (1991). Naming a targeting signal. Cell, 64(1), 13-15.
● Varshavsky, Alexander. "The N-end rule pathway and regulation by proteolysis."
Proceedings of the National Academy of Sciences of the United States of America, vol. 93,
no. 8, 1996, pp. 3219-3222.
● Voges, Dietrich, et al. "Ubiquitin-proteasome system." European Journal of Biochemistry,
vol. 268, no. 19, 1999, pp. 6065-6083.
● Vousden, K. H., & Prives, C. (2009). Blinded by the Light: The Growing Complexity of
p53. Cell, 137(3), 413-431.
● Zhuo, Chenya et al. "Spatiotemporal control of CRISPR/Cas9 gene editing." Nature
Communications, vol. 8, no. 1, 2017, p. 2102.
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