ABSTRACT Over a thousand diseases are caused by mutations that alter gene expression levels. The potential of nuclease-deficient zinc fingers, TALEs or CRISPR fusion systems to treat these

diseases by modulating gene expression has recently emerged. These systems can be applied to modify the activity of gene-regulatory elements — promoters, enhancers, silencers and insulators,

subsequently changing their target gene expression levels to achieve therapeutic benefits — an approach termed _cis_-regulation therapy (CRT). Here, we review emerging CRT technologies and

assess their therapeutic potential for treating a wide range of diseases caused by abnormal gene dosage. The challenges facing the translation of CRT into the clinic are discussed. Access

access $209.00 per year only $17.42 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which

are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS

TRANSCRIPTIONAL CO-ACTIVATORS: EMERGING ROLES IN SIGNALING PATHWAYS AND POTENTIAL THERAPEUTIC TARGETS FOR DISEASES Article Open access 13 November 2023 GENE THERAPY AND GENE CORRECTION:

TARGETS, PROGRESS, AND CHALLENGES FOR TREATING HUMAN DISEASES Article 09 October 2020 PROGRAMMABLE MRNA THERAPEUTICS FOR CONTROLLED EPIGENOMIC MODULATION OF SINGLE AND MULTIPLEXED GENE

EXPRESSION IN DIVERSE DISEASES Article Open access 13 March 2025 REFERENCES * Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. _Nucleic Acids

Res._ 44, D862–D868 (2016). Article  CAS  PubMed  Google Scholar  * Matharu, N. et al. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency.

_Science_ 363, eaau0629 (2019). THIS STUDY IS THE FIRST TO HIGHLIGHT THE UTILITY OF CRISPRA UPREGULATION AS A THERAPEUTIC APPROACH TO RESCUE A HAPLOINSUFFICIENT DISEASE USING BOTH TRANSGENIC

AND AAV TARGETING OF EITHER A PROMOTER OR ENHANCER IN POSTNATAL MOUSE MODELS OF OBESITY. Article  CAS  PubMed  Google Scholar  * Rehm, H. L. et al. ClinGen—the Clinical Genome Resource. _N.

Engl. J. Med._ 372, 2235–2242 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Elkon, R. & Agami, R. Characterization of noncoding regulatory DNA in the human genome.

_Nat. Biotechnol._ 35, 732–746 (2017). Article  CAS  PubMed  Google Scholar  * Chatterjee, S. & Ahituv, N. Gene regulatory elements, major drivers of human disease. _Annu. Rev. Genomics

Hum. Genet._ 18, 45–63 (2017). Article  CAS  PubMed  Google Scholar  * Gasperini, M., Tome, J. M. & Shendure, J. Towards a comprehensive catalogue of validated and target-linked human

enhancers. _Nat. Rev. Genet._ 21, 292–310 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR

epigenetic editing. _Nat. Commun._ 11, 485 (2020). Article  PubMed  PubMed Central  CAS  Google Scholar  * Giardine, B. et al. Systematic documentation and analysis of human genetic

variation in hemoglobinopathies using the microattribution approach. _Nat. Genet._ 43, 295–301 (2011). Article  CAS  PubMed  Google Scholar  * VanderMeer, J. E. & Ahituv, N.

_cis_-Regulatory mutations are a genetic cause of human limb malformations. _Dev. Dyn._ 240, 920–930 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sankaran, V. G. et al. A

functional element necessary for fetal hemoglobin silencing. _N. Engl. J. Med._ 365, 807–814 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Matharu, N. K. & Ahanger, S.

H. Chromatin insulators and topological domains: adding new dimensions to 3D genome architecture. _Genes_ 6, 790–811 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Matharu,

N. & Ahituv, N. Minor loops in major folds: enhancer–promoter looping, chromatin restructuring, and their association with transcriptional regulation and disease. _PLoS Genet._ 11,

e1005640 (2015). Article  PubMed  PubMed Central  CAS  Google Scholar  * Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. _Nat.

Rev. Drug Discov._ 18, 358–378 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang, J., Lu, Z., Wientjes, M. G. & Au, J. L. Delivery of siRNA therapeutics: barriers and

carriers. _AAPS J._ 12, 492–503 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mercuri, E. et al. Nusinersen versus sham control in later-onset spinal muscular atrophy. _N.

Engl. J. Med._ 378, 625–635 (2018). Article  CAS  PubMed  Google Scholar  * van Deutekom, J. C. et al. Local dystrophin restoration with antisense oligonucleotide PRO051. _N. Engl. J. Med._

357, 2677–2686 (2007). Article  PubMed  Google Scholar  * Li, H. et al. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and

prospects. _Signal. Transduct. Target. Ther._ 5, 1 (2020). Article  PubMed  PubMed Central  Google Scholar  * Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing

CRISPR–Cas9 for precision genome regulation and interrogation. _Nat. Rev. Mol. Cell Biol._ 17, 5–15 (2016). Article  CAS  PubMed  Google Scholar  * Dever, D. P. et al. CRISPR/Cas9 β-globin

gene targeting in human haematopoietic stem cells. _Nature_ 539, 384–389 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Khosravi, M. A. et al. Targeted deletion of _BCL11A_

gene by CRISPR–Cas9 system for fetal hemoglobin reactivation: a promising approach for gene therapy of β-thalassemia disease. _Eur. J. Pharmacol._ 854, 398–405 (2019). Article  CAS  PubMed 

Google Scholar  * Weber, L. et al. Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype. _Sci. Adv._ 6, eaay9392

(2020). Article  PubMed  PubMed Central  CAS  Google Scholar  * Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. _Nat. Rev.

Genet._ 19, 770–788 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA.

_Nature_ 576, 149–157 (2019). CAS  PubMed  PubMed Central  Google Scholar  * Lek, M. et al. Analysis of protein-coding genetic variation in 60,706 humans. _Nature_ 536, 285–291 (2016).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Karczewski, K. J. et al. The mutational constraint spectrum quantified from variation in 141,456 humans. _Nature_ 581, 434–443 (2020).

Article  CAS  PubMed  PubMed Central  Google Scholar  * van der Klaauw, A. A. & Farooqi, I. S. The hunger genes: pathways to obesity. _Cell_ 161, 119–132 (2015). Article  PubMed  CAS 

Google Scholar  * Michaud, J. L. et al. _Sim1_ haploinsufficiency causes hyperphagia, obesity and reduction of the paraventricular nucleus of the hypothalamus. _Hum. Mol. Genet._ 10,

1465–1473 (2001). Article  CAS  PubMed  Google Scholar  * Huszar, D. et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. _Cell_ 88, 131–141 (1997). Article

  CAS  PubMed  Google Scholar  * Yarrington, R. M., Verma, S., Schwartz, S., Trautman, J. K. & Carroll, D. Nucleosomes inhibit target cleavage by CRISPR–Cas9 in vivo. _Proc. Natl Acad.

Sci. USA_ 115, 9351–9358 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Horlbeck, M. A. et al. Nucleosomes impede Cas9 access to DNA in vivo and in vitro. _eLife_ 5, e12677

(2016). Article  PubMed  PubMed Central  CAS  Google Scholar  * Colasante, G. et al. dCas9-based _Scn1a_ gene activation restores inhibitory interneuron excitability and attenuates seizures

in Dravet syndrome mice. _Mol. Ther._ 28, 235–253 (2020). THIS REPORT SHOWS HOW CRISPRA UPREGULATION OF SODIUM VOLTAGE-GATED CHANNEL 1 CAN IMPROVE THE DISEASE PHENOTYPES IN A MOUSE MODEL FOR

DRAVET SYNDROME. Article  CAS  PubMed  Google Scholar  * Ogiwara, I. et al. Nav1.1 localizes to axons of parvalbumin-positive inhibitory interneurons: a circuit basis for epileptic seizures

in mice carrying an _Scn1a_ gene mutation. _J. Neurosci._ 27, 5903–5914 (2007). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, X. T. et al. tCRISPRi: tunable and reversible,

one-step control of gene expression. _Sci. Rep._ 6, 39076 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Savell, K. E. et al. A neuron-optimized CRISPR/dCas9 activation

system for robust and specific gene regulation. _eNeuro_ https://doi.org/10.1523/ENEURO.0495-18.2019 (2019). * Jost, M. et al. Titrating gene expression using libraries of systematically

attenuated CRISPR guide RNAs. _Nat. Biotechnol._ 38, 355–364 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kemaladewi, D. U. et al. A mutation-independent approach for

muscular dystrophy via upregulation of a modifier gene. _Nature_ 572, 125–130 (2019). Article  CAS  PubMed  Google Scholar  * Liao, H. K. et al. In vivo target gene activation via

CRISPR/Cas9-mediated trans-epigenetic modulation. _Cell_ 171, 1495–1507.e1415 (2017). TOGETHER WITH KEMALADEWI ET AL. (2019), THIS PAPER SHOWS THAT UPREGULATION OF AN ALTERNATIVE GENE CAN

AMELIORATE THE DISEASE-ASSOCIATED PHENOTYPE IN MOUSE MODELS OF TWO DIFFERENT MUSCULAR DYSTROPHIES, MDC1A AND DMD. Article  CAS  PubMed  PubMed Central  Google Scholar  * Gawlik, K.,

Miyagoe-Suzuki, Y., Ekblom, P., Takeda, S. & Durbeej, M. Laminin α1 chain reduces muscular dystrophy in laminin α2 chain deficient mice. _Hum. Mol. Genet._ 13, 1775–1784 (2004). Article

  CAS  PubMed  Google Scholar  * Sunada, Y., Bernier, S. M., Utani, A., Yamada, Y. & Campbell, K. P. Identification of a novel mutant transcript of laminin α2 chain gene responsible for

muscular dystrophy and dysmyelination in _dy_ 2J mice. _Hum. Mol. Genet._ 4, 1055–1061 (1995). Article  CAS  PubMed  Google Scholar  * US National Library of Medicine. _ClinicalTrials.gov_

https://clinicaltrials.gov/ct2/show/NCT00428935 (2007). * US National Library of Medicine. _ClinicalTrials.gov_ https://clinicaltrials.gov/ct2/show/NCT02376816 (2015). * US National Library

of Medicine. _ClinicalTrials.gov_ https://clinicaltrials.gov/ct2/show/NCT03362502 (2017). * US National Library of Medicine. _ClinicalTrials.gov_

https://clinicaltrials.gov/ct2/show/NCT03375164 (2017). * US National Library of Medicine. _ClinicalTrials.gov_ https://clinicaltrials.gov/ct2/show/NCT01519349 (2012). * US National Library

of Medicine. _ClinicalTrials.gov_ https://clinicaltrials.gov/ct2/show/NCT03333590 (2017). * Haidet, A. M. et al. Long-term enhancement of skeletal muscle mass and strength by single gene

administration of myostatin inhibitors. _Proc. Natl Acad. Sci. USA_ 105, 4318–4322 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Torres, L. F. & Duchen, L. W. The

mutant _mdx_: inherited myopathy in the mouse. Morphological studies of nerves, muscles and end-plates. _Brain_ 110, 269–299 (1987). Article  PubMed  Google Scholar  * Rafael, J. A.,

Tinsley, J. M., Potter, A. C., Deconinck, A. E. & Davies, K. E. Skeletal muscle-specific expression of a utrophin transgene rescues utrophin-dystrophin deficient mice. _Nat. Genet._ 19,

79–82 (1998). Article  CAS  PubMed  Google Scholar  * Kennedy, T. L. et al. Micro-utrophin improves cardiac and skeletal muscle function of severely affected D2/_mdx_ mice. _Mol. Ther.

Methods Clin. Dev._ 11, 92–105 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Song, Y. et al. Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy

animal models. _Nat. Med._ 25, 1505–1511 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wojtal, D. et al. Spell checking nature: versatility of CRISPR/Cas9 for developing

treatments for inherited disorders. _Am. J. Hum. Genet._ 98, 90–101 (2016). Article  CAS  PubMed  Google Scholar  * Wehling-Henricks, M. et al. _Klotho_ gene silencing promotes pathology in

the _mdx_ mouse model of Duchenne muscular dystrophy. _Hum. Mol. Genet._ 25, 2465–2482 (2016). CAS  PubMed  PubMed Central  Google Scholar  * Kuro-o, M. et al. Mutation of the mouse _klotho_

gene leads to a syndrome resembling ageing. _Nature_ 390, 45–51 (1997). Article  CAS  PubMed  Google Scholar  * Chen, C. D., Zeldich, E., Li, Y., Yuste, A. & Abraham, C. R. Activation

of the anti-aging and cognition-enhancing gene _klotho_ by CRISPR–dCas9 transcriptional effector complex. _J. Mol. Neurosci._ 64, 175–184 (2018). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Chen, B. & Altman, R. B. Opportunities for developing therapies for rare genetic diseases: focus on gain-of-function and allostery. _Orphanet J. Rare Dis._ 12, 61 (2017).

Article  CAS  PubMed  PubMed Central  Google Scholar  * A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington‘s disease chromosomes. The Huntington‘s

Disease Collaborative Research Group. _Cell_ 72, 971–983 (1993). Article  Google Scholar  * Dabrowska, M., Juzwa, W., Krzyzosiak, W. J. & Olejniczak, M. Precise excision of the CAG tract

from the huntingtin gene by Cas9 nickases. _Front. Neurosci._ 12, 75 (2018). Article  PubMed  PubMed Central  Google Scholar  * Kaemmerer, W. F. & Grondin, R. C. The effects of

huntingtin-lowering: what do we know so far? _Degener. Neurol. Neuromuscul. Dis._ 9, 3–17 (2019). CAS  PubMed  PubMed Central  Google Scholar  * Fink, K. D. et al. Allele-specific reduction

of the mutant huntingtin allele using transcription activator-like effectors in human Huntington’s disease fibroblasts. _Cell Transpl._ 25, 677–686 (2016). Article  Google Scholar  *

Garriga-Canut, M. et al. Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice. _Proc. Natl Acad. Sci. USA_ 109, E3136–E3145 (2012). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Ehrnhoefer, D. E., Butland, S. L., Pouladi, M. A. & Hayden, M. R. Mouse models of Huntington disease: variations on a theme. _Dis. Model. Mech._

2, 123–129 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Slow, E. J. et al. Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease. _Hum. Mol.

Genet._ 12, 1555–1567 (2003). Article  CAS  PubMed  Google Scholar  * Bosnakovski, D. et al. Muscle pathology from stochastic low level DUX4 expression in an FSHD mouse model. _Nat. Commun._

8, 550 (2017). Article  PubMed  PubMed Central  CAS  Google Scholar  * Jones, T. I. et al. Facioscapulohumeral muscular dystrophy family studies of DUX4 expression: evidence for disease

modifiers and a quantitative model of pathogenesis. _Hum. Mol. Genet._ 21, 4419–4430 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Himeda, C. L. et al. Identification of

epigenetic regulators of DUX4-fl for targeted therapy of facioscapulohumeral muscular dystrophy. _Mol. Ther._ 26, 1797–1807 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Furuhashi, M., Saitoh, S., Shimamoto, K. & Miura, T. Fatty acid-binding protein 4 (FABP4): pathophysiological insights and potent clinical biomarker of metabolic and cardiovascular

diseases. _Clin. Med. Insights Cardiol._ 8, 23–33 (2014). PubMed  Google Scholar  * Chung, J. Y., Ain, Q. U., Song, Y., Yong, S. B. & Kim, Y. H. Targeted delivery of CRISPR interference

system against _Fabp4_ to white adipocytes ameliorates obesity, inflammation, hepatic steatosis, and insulin resistance. _Genome Res._ 29, 1442–1452 (2019). THIS REPORT UTILIZES CRISPRI

TARGETED DELIVERY IN ADIPOCYTES TO DOWNREGULATE A BIOMARKER OF HIGH-FAT DIET-INDUCED OBESITY. Article  CAS  PubMed  PubMed Central  Google Scholar  * Won, Y. W. et al. Oligopeptide complex

for targeted non-viral gene delivery to adipocytes. _Nat. Mater._ 13, 1157–1164 (2014). Article  CAS  PubMed  Google Scholar  * Robertson, K. D. DNA methylation and human disease. _Nat. Rev.

Genet._ 6, 597–610 (2005). Article  CAS  PubMed  Google Scholar  * Liu, X. S. et al. Rescue of Fragile X syndrome neurons by DNA methylation editing of the _FMR1_ gene. _Cell_ 172,

979–992.e976 (2018). THIS REPORT DEMONSTRATES THAT MODULATION OF DNA METHYLATION IN THE FRAGILE X-ASSOCIATED TRINUCLEOTIDE REPEAT COULD INCREASE _FMR1_ GENE EXPRESSION. Article  CAS  PubMed

  PubMed Central  Google Scholar  * Chiurazzi, P. & Neri, G. Pharmacological reactivation of inactive genes: the Fragile X experience. _Brain Res. Bull._ 56, 383–387 (2001). Article  CAS

  PubMed  Google Scholar  * Tabolacci, E. & Chiurazzi, P. Epigenetics, Fragile X syndrome and transcriptional therapy. _Am. J. Med. Genet. A_ 161A, 2797–2808 (2013). Article  PubMed  CAS

  Google Scholar  * Tabolacci, E., Palumbo, F., Nobile, V. & Neri, G. Transcriptional reactivation of the _FMR1_ gene. A possible approach to the treatment of the Fragile X syndrome.

_Genes_ 7, 49 (2016). Article  PubMed Central  CAS  Google Scholar  * Liu, X. S. et al. Editing DNA methylation in the mammalian genome. _Cell_ 167, e217 (2016). THIS REPORT USES TET1 OR

DNMT3A FUSED TO DCAS9 TO SHOW HOW DNA METHYLATION COULD BE EDITED IN BOTH CELLS AND MICE. Article  CAS  Google Scholar  * Amabile, A. et al. Inheritable silencing of endogenous genes by

hit-and-run targeted epigenetic editing. _Cell_ 167, 219–232.e14 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Saunderson, E. A. et al. Hit-and-run epigenetic editing

prevents senescence entry in primary breast cells from healthy donors. _Nat. Commun._ 8, 1450 (2017). TOGETHER WITH AMABILE ET AL. (2016), THIS PAPER USES A ‘HIT-AND-RUN’ EPIGENETIC EDITING

STRATEGY THAT METHYLATES DNA TO SILENCE GENE EXPRESSION. Article  PubMed  PubMed Central  CAS  Google Scholar  * Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing

strategies for the functional annotation of CTCF insulators. _Nat. Commun._ 10, 4258 (2019). Article  PubMed  PubMed Central  CAS  Google Scholar  * Rossi, D. et al. β2-Microglobulin is an

independent predictor of progression in asymptomatic multiple myeloma. _Cancer_ 116, 2188–2200 (2010). CAS  PubMed  Google Scholar  * Margueron, R. & Reinberg, D. Chromatin structure and

the inheritance of epigenetic information. _Nat. Rev. Genet._ 11, 285–296 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Lupianez, D. G., Spielmann, M. & Mundlos, S.

Breaking TADs: how alterations of chromatin domains result in disease. _Trends Genet._ 32, 225–237 (2016). Article  CAS  PubMed  Google Scholar  * Lawrence, M., Daujat, S. & Schneider,

R. Lateral thinking: how histone modifications regulate gene expression. _Trends Genet._ 32, 42–56 (2016). Article  CAS  PubMed  Google Scholar  * Baffert, F. et al. Cellular changes in

normal blood capillaries undergoing regression after inhibition of VEGF signaling. _Am. J. Physiol. Heart Circ. Physiol._ 290, H547–H559 (2006). Article  CAS  PubMed  Google Scholar  *

Benjamin, L. E., Golijanin, D., Itin, A., Pode, D. & Keshet, E. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor

withdrawal. _J. Clin. Invest._ 103, 159–165 (1999). Article  CAS  PubMed  PubMed Central  Google Scholar  * Snowden, A. W., Gregory, P. D., Case, C. C. & Pabo, C. O. Gene-specific

targeting of H3K9 methylation is sufficient for initiating repression in vivo. _Curr. Biol._ 12, 2159–2166 (2002). Article  CAS  PubMed  Google Scholar  * Mendenhall, E. M. et al.

Locus-specific editing of histone modifications at endogenous enhancers. _Nat. Biotechnol._ 31, 1133–1136 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hilton, I. B. et al.

Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. _Nat. Biotechnol._ 33, 510–517 (2015). Article  CAS  PubMed  PubMed Central  Google

Scholar  * O’Geen, H. et al. Ezh2–dCas9 and KRAB–dCas9 enable engineering of epigenetic memory in a context-dependent manner. _Epigenetics Chromatin_ 12, 26 (2019). Article  PubMed  PubMed

Central  Google Scholar  * Stamatoyannopoulos, G. Control of globin gene expression during development and erythroid differentiation. _Exp. Hematol._ 33, 259–271 (2005). Article  CAS  PubMed

  PubMed Central  Google Scholar  * Lettre, G. et al. DNA polymorphisms at the BCL11A, HBS1L-MYB, and β-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell

disease. _Proc. Natl Acad. Sci. USA_ 105, 11869–11874 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Uda, M. et al. Genome-wide association study shows BCL11A associated

with persistent fetal hemoglobin and amelioration of the phenotype of β-thalassemia. _Proc. Natl Acad. Sci. USA_ 105, 1620–1625 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar 

* Deng, W. et al. Reactivation of developmentally silenced globin genes by forced chromatin looping. _Cell_ 158, 849–860 (2014). THIS REPORT IS THE FIRST TO HIGHLIGHT THE POTENTIAL TO TREAT

SICKLE CELL DISEASE OR Β-THALASSAEMIA BY ALTERING CHROMATIN LOOPING OF THE Β-GLOBIN LOCUS. Article  CAS  PubMed  PubMed Central  Google Scholar  * Breda, L. et al. Forced chromatin looping

raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers. _Blood_ 128, 1139–1143 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Liang, F.

S., Ho, W. Q. & Crabtree, G. R. Engineering the ABA plant stress pathway for regulation of induced proximity. _Sci. Signal._ 4, rs2 (2011). Article  PubMed  PubMed Central  Google

Scholar  * Morgan, S. L. et al. Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping. _Nat. Commun._ 8, 15993 (2017). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Hao, N., Shearwin, K. E. & Dodd, I. B. Programmable DNA looping using engineered bivalent dCas9 complexes. _Nat. Commun._ 8, 1628 (2017). Article  PubMed  PubMed Central  CAS

  Google Scholar  * Halmai, J. et al. Artificial escape from XCI by DNA methylation editing of the _CDKL5_ gene. _Nucleic Acids Res._ 48, 2372–2387 (2020). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Finer, M. & Glorioso, J. A brief account of viral vectors and their promise for gene therapy. _Gene Ther._ 24, 1–2 (2017). Article  CAS  PubMed  Google Scholar

  * Burton, E. A., Fink, D. J. & Glorioso, J. C. Gene delivery using herpes simplex virus vectors. _DNA Cell Biol._ 21, 915–936 (2002). Article  CAS  PubMed  Google Scholar  * Collins,

M. & Thrasher, A. Gene therapy: progress and predictions. _Proc. Biol. Sci._ 282, 20143003 (2015). PubMed  PubMed Central  Google Scholar  * Dunbar, C. E. et al. Gene therapy comes of

age. _Science_ 359, eaan4672 (2018). Article  PubMed  CAS  Google Scholar  * Fischer, A. Gene therapy: from birth to maturity requires commitment to science and ethics. _Hum. Gene Ther._ 28,

958 (2017). Article  CAS  PubMed  Google Scholar  * Kuo, C. Y. & Kohn, D. B. Gene therapy for the treatment of primary immune deficiencies. _Curr. Allergy Asthma Rep._ 16, 39 (2016).

Article  PubMed  PubMed Central  CAS  Google Scholar  * Alton, E. W. et al. Non-invasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice.

_Nat. Genet._ 5, 135–142 (1993). Article  CAS  PubMed  Google Scholar  * Zhu, N., Liggitt, D., Liu, Y. & Debs, R. Systemic gene expression after intravenous DNA delivery into adult mice.

_Science_ 261, 209–211 (1993). Article  CAS  PubMed  Google Scholar  * Alton, E. et al. Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised,

double-blind, placebo-controlled, phase 2b trial. _Lancet Respir. Med._ 3, 684–691 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Alton, E. W. et al. Cationic lipid-mediated

CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebo-controlled trial. _Lancet_ 353, 947–954 (1999). Article  CAS  PubMed  Google Scholar  *

Kulkarni, J. A., Cullis, P. R. & van der Meel, R. Lipid nanoparticles enabling gene therapies: from concepts to clinical utility. _Nucleic Acid. Ther._ 28, 146–157 (2018). Article  CAS 

PubMed  Google Scholar  * Zhang, L. et al. Lipid nanoparticle-mediated efficient delivery of CRISPR/Cas9 for tumor therapy. _NPG Asia Mater._ 9, e441 (2017). Article  CAS  Google Scholar  *

Finn, J. D. et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. _Cell Rep._ 22, 2227–2235 (2018). Article  CAS  PubMed 

Google Scholar  * Yin, H. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. _Nat. Biotechnol._ 35, 1179–1187 (2017). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Gangopadhyay, S. A. et al. Precision control of CRISPR–Cas9 using small molecules and light. _Biochemistry_ 58, 234–244 (2019). Article  CAS  PubMed

  Google Scholar  * Zhang, J., Chen, L., Zhang, J. & Wang, Y. Drug inducible CRISPR/Cas systems. _Comput. Struct. Biotechnol. J._ 17, 1171–1177 (2019). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Hynes, A. P. et al. Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins. _Nat. Commun._ 9, 2919 (2018). Article  PubMed 

PubMed Central  CAS  Google Scholar  * Landsberger, M. et al. Anti-CRISPR phages cooperate to overcome CRISPR–Cas immunity. _Cell_ 174, 908–916.e12 (2018). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Pawluk, A., Davidson, A. R. & Maxwell, K. L. Anti-CRISPR: discovery, mechanism and function. _Nat. Rev. Microbiol._ 16, 12–17 (2018). Article  CAS  PubMed 

Google Scholar  * Kent, W. J. et al. The human genome browser at UCSC. _Genome Res._ 12, 996–1006 (2002). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gilbert, L. A. et al.

Genome-scale CRISPR-mediated control of gene repression and activation. _Cell_ 159, 647–661 (2014). THIS STUDY SHOWS THE POTENTIAL OF CRISPRA OR CRISPRI LARGE-SCALE GENOMIC SCREENS TO

UPREGULATE OR DOWNREGULATE GENE EXPRESSION, RESPECTIVELY. Article  CAS  PubMed  PubMed Central  Google Scholar  * Landry, J. R., Mager, D. L. & Wilhelm, B. T. Complex controls: the role

of alternative promoters in mammalian genomes. _Trends Genet._ 19, 640–648 (2003). Article  CAS  PubMed  Google Scholar  * Andersson, R. et al. An atlas of active enhancers across human cell

types and tissues. _Nature_ 507, 455–461 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Consortium, E. P. et al. An integrated encyclopedia of DNA elements in the human

genome. _Nature_ 489, 57–74 (2012). Article  CAS  Google Scholar  * Fulco, C. P. et al. Systematic mapping of functional enhancer–promoter connections with CRISPR interference. _Science_

354, 769–773 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Roadmap Epigenomics, C. et al. Integrative analysis of 111 reference human epigenomes. _Nature_ 518, 317–330

(2015). Article  CAS  Google Scholar  * Schoenfelder, S. & Fraser, P. Long-range enhancer-promoter contacts in gene expression control. _Nat. Rev. Genet._ 20, 437–455 (2019). Article 

CAS  PubMed  Google Scholar  * Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. _Cell_ 176, 377–390.e19 (2019). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Xie, S., Duan, J., Li, B., Zhou, P. & Hon, G. C. Multiplexed engineering and analysis of combinatorial enhancer activity in single cells. _Mol. Cell_

66, 285–299.e5 (2017). Article  CAS  PubMed  Google Scholar  * Doni Jayavelu, N., Jajodia, A., Mishra, A. & Hawkins, R. D. Candidate silencer elements for the human and mouse genomes.

_Nat. Commun._ 11, 1061 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Pang, B. & Snyder, M. P. Systematic identification of silencers in human cells. _Nat. Genet._ 52,

254–263 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cuddapah, S. et al. Global analysis of the insulator binding protein CTCF in chromatin barrier regions reveals

demarcation of active and repressive domains. _Genome Res._ 19, 24–32 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Phillips-Cremins, J. E. & Corces, V. G. Chromatin

insulators: linking genome organization to cellular function. _Mol. Cell_ 50, 461–474 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Khoury, A. et al. Constitutively bound

CTCF sites maintain 3D chromatin architecture and long-range epigenetically regulated domains. _Nat. Commun._ 11, 54 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Simeonov,

D. R. et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. _Nature_ 549, 111–115 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Duan, D.

Systemic AAV micro-dystrophin gene therapy for Duchenne muscular dystrophy. _Mol. Ther._ 26, 2337–2356 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fang, R. H., Kroll, A.

V., Gao, W. & Zhang, L. Cell membrane coating nanotechnology. _Adv. Mater._ 30, e1706759 (2018). Article  PubMed  PubMed Central  CAS  Google Scholar  * Veiga, N. et al. Cell specific

delivery of modified mRNA expressing therapeutic proteins to leukocytes. _Nat. Commun._ 9, 4493 (2018). Article  PubMed  PubMed Central  CAS  Google Scholar  * Zylberberg, C., Gaskill, K.,

Pasley, S. & Matosevic, S. Engineering liposomal nanoparticles for targeted gene therapy. _Gene Ther._ 24, 441–452 (2017). Article  CAS  PubMed  Google Scholar  * Guilinger, J. P. et al.

Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. _Nat. Methods_ 11, 429–435 (2014). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Jantz, D. & Berg, J. M. Probing the DNA-binding affinity and specificity of designed zinc finger proteins. _Biophys. J._ 98, 852–860 (2010). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Kocak, D. D. et al. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. _Nat. Biotechnol._ 37, 657–666 (2019). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Pattanayak, V., Guilinger, J. P. & Liu, D. R. Determining the specificities of TALENs, Cas9, and other genome-editing enzymes. _Methods

Enzymol._ 546, 47–78 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Liu, H. et al. CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and

activation. _Bioinformatics_ 31, 3676–3678 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sherry, S. T., Ward, M. & Sirotkin, K. dbSNP-database for single nucleotide

polymorphisms and other classes of minor genetic variation. _Genome Res._ 9, 677–679 (1999). Article  CAS  PubMed  Google Scholar  * Adan, A., Kiraz, Y. & Baran, Y. Cell proliferation

and cytotoxicity assays. _Curr. Pharm. Biotechnol._ 17, 1213–1221 (2016). Article  CAS  PubMed  Google Scholar  * Corrigan-Curay, J. et al. Genome editing technologies: defining a path to

clinic. _Mol. Ther._ 23, 796–806 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * de Bono, J. S., Tolcher, A. W. & Rowinsky, E. K. The future of cytotoxic therapy:

selective cytotoxicity based on biology is the key. _Breast Cancer Res._ 5, 154–159 (2003). Article  PubMed  PubMed Central  CAS  Google Scholar  * Ferdosi, S. R. et al. Multifunctional

CRISPR–Cas9 with engineered immunosilenced human T cell epitopes. _Nat. Commun._ 10, 1842 (2019). Article  PubMed  PubMed Central  CAS  Google Scholar  * Simhadri, V. L. et al. Prevalence of

pre-existing antibodies to CRISPR-associated nuclease Cas9 in the USA population. _Mol. Ther. Methods Clin. Dev._ 10, 105–112 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Mehta, A. & Merkel, O. M. Immunogenicity of Cas9 protein. _J. Pharm. Sci._ 109, 62–67 (2020). Article  CAS  PubMed  Google Scholar  * Wagner, D. L. et al. High prevalence of

_Streptococcus pyogenes_ Cas9-reactive T cells within the adult human population. _Nat. Med._ 25, 242–248 (2019). Article  CAS  PubMed  Google Scholar  * Esvelt, K. M. et al. Orthogonal Cas9

proteins for RNA-guided gene regulation and editing. _Nat. Methods_ 10, 1116–1121 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fonfara, I. et al. Phylogeny of Cas9

determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR–Cas systems. _Nucleic Acids Res._ 42, 2577–2590 (2014). Article  CAS  PubMed  Google Scholar  *

Hirano, H. et al. Structure and engineering of _Francisella novicida_ Cas9. _Cell_ 164, 950–961 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kostyushev, D. et al.

Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus. _Cell Mol. Life Sci._ 76, 1779–1794 (2019). Article  CAS 

PubMed  Google Scholar  * Najm, F. J. et al. Orthologous CRISPR–Cas9 enzymes for combinatorial genetic screens. _Nat. Biotechnol._ 36, 179–189 (2018). Article  CAS  PubMed  Google Scholar  *

Agustin-Pavon, C., Mielcarek, M., Garriga-Canut, M. & Isalan, M. Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin

repression in mice. _Mol. Neurodegener._ 11, 64 (2016). Article  PubMed  PubMed Central  CAS  Google Scholar  * Nelson, C. E. et al. Long-term evaluation of AAV–CRISPR genome editing for

Duchenne muscular dystrophy. _Nat. Med._ 25, 427–432 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ertl, H. C. J. Preclinical models to assess the immunogenicity of AAV

vectors. _Cell Immunol._ 342, 103722 (2019). Article  PubMed  CAS  Google Scholar  * Mingozzi, F. & High, K. A. Immune responses to AAV vectors: overcoming barriers to successful gene

therapy. _Blood_ 122, 23–36 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Vandamme, C., Adjali, O. & Mingozzi, F. Unraveling the complex story of immune responses to

AAV vectors trial after trial. _Hum. Gene Ther._ 28, 1061–1074 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Barnes, C., Scheideler, O. & Schaffer, D. Engineering the

AAV capsid to evade immune responses. _Curr. Opin. Biotechnol._ 60, 99–103 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hareendran, S. et al. Adeno-associated virus (AAV)

vectors in gene therapy: immune challenges and strategies to circumvent them. _Rev. Med. Virol._ 23, 399–413 (2013). Article  CAS  PubMed  Google Scholar  * Hardcastle, N., Boulis, N. M.

& Federici, T. AAV gene delivery to the spinal cord: serotypes, methods, candidate diseases, and clinical trials. _Expert Opin. Biol. Ther._ 18, 293–307 (2018). Article  CAS  PubMed 

Google Scholar  * Cukras, C. et al. Retinal AAV8-RS1 gene therapy for X-linked retinoschisis: initial findings from a phase I/IIa trial by intravitreal delivery. _Mol. Ther._ 26, 2282–2294

(2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Chandler, R. J., LaFave, M. C., Varshney, G. K., Burgess, S. M. & Venditti, C. P. Genotoxicity in mice following AAV gene

delivery: a safety concern for human gene therapy? _Mol. Ther._ 24, 198–201 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Chandler, R. J. et al. Vector design influences

hepatic genotoxicity after adeno-associated virus gene therapy. _J. Clin. Invest._ 125, 870–880 (2015). Article  PubMed  PubMed Central  Google Scholar  * Torchilin, V. P. Recent advances

with liposomes as pharmaceutical carriers. _Nat. Rev. Drug Discov._ 4, 145–160 (2005). Article  CAS  PubMed  Google Scholar  * Lee, E. J., Guenther, C. M. & Suh, J. Adeno-associated

virus (AAV) vectors: rational design strategies for capsid engineering. _Curr. Opin. Biomed. Eng._ 7, 58–63 (2018). Article  PubMed  PubMed Central  Google Scholar  * Ogden, P. J., Kelsic,

E. D., Sinai, S. & Church, G. M. Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design. _Science_ 366, 1139–1143 (2019). Article  CAS  PubMed

  PubMed Central  Google Scholar  * Yee, J. K. Off-target effects of engineered nucleases. _FEBS J._ 283, 3239–3248 (2016). Article  CAS  PubMed  Google Scholar  * Urnov, F. D., Rebar, E.

J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Genome editing with engineered zinc finger nucleases. _Nat. Rev. Genet._ 11, 636–646 (2010). Article  CAS  PubMed  Google Scholar  *

Palpant, N. J. & Dudzinski, D. Zinc finger nucleases: looking toward translation. _Gene Ther._ 20, 121–127 (2013). Article  CAS  PubMed  Google Scholar  * Joung, J. K. & Sander, J.

D. TALENs: a widely applicable technology for targeted genome editing. _Nat. Rev. Mol. Cell Biol._ 14, 49–55 (2013). Article  CAS  PubMed  Google Scholar  * Wright, D. A., Li, T., Yang, B.

& Spalding, M. H. TALEN-mediated genome editing: prospects and perspectives. _Biochem. J._ 462, 15–24 (2014). Article  CAS  PubMed  Google Scholar  * Jiang, F. & Doudna, J. A.

CRISPR–Cas9 structures and mechanisms. _Annu. Rev. Biophys._ 46, 505–529 (2017). Article  CAS  PubMed  Google Scholar  * Adli, M. The CRISPR tool kit for genome editing and beyond. _Nat.

Commun._ 9, 1911 (2018). Article  PubMed  PubMed Central  CAS  Google Scholar  * Flint, J. & Shenk, T. Viral transactivating proteins. _Annu. Rev. Genet._ 31, 177–212 (1997). Article 

CAS  PubMed  Google Scholar  * Cheng, A. W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. _Cell Res._ 23, 1163–1171 (2013).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. _Nat. Methods_ 12, 326–328 (2015). Article  CAS  PubMed

  PubMed Central  Google Scholar  * Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. _Cell_ 154, 442–451 (2013). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Yeo, N. C. et al. An enhanced CRISPR repressor for targeted mammalian gene regulation. _Nat. Methods_ 15, 611–616 (2018). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Li, F. et al. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes. _Nucleic Acids Res._ 35,

100–112 (2007). Article  PubMed  CAS  Google Scholar  * Siddique, A. N. et al. Targeted methylation and gene silencing of VEGF-A in human cells by using a designed Dnmt3a–Dnmt3L single-chain

fusion protein with increased DNA methylation activity. _J. Mol. Biol._ 425, 479–491 (2013). Article  CAS  PubMed  Google Scholar  * Chen, H. et al. Induced DNA demethylation by targeting

ten–eleven translocation 2 to the human ICAM-1 promoter. _Nucleic Acids Res._ 42, 1563–1574 (2014). Article  CAS  PubMed  Google Scholar  * Konermann, S. et al. Genome-scale transcriptional

activation by an engineered CRISPR–Cas9 complex. _Nature_ 517, 583–588 (2014). THIS REPORT DEVELOPED THE DCAS9-BASED SAM (TABLE 2) AND DEMONSTRATES ITS ABILITY TO SIMULTANEOUSLY UPREGULATE

SEVERAL GENES AND BE USED FOR A LARGE-SCALE DRUG RESPONSE SCREEN. Article  PubMed  PubMed Central  CAS  Google Scholar  * Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. &

Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. _Cell_ 159, 635–646 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references ACKNOWLEDGEMENTS This article was supported in part by grants 1R01DK090382 and 1R01DK124769 from the National Institute of Diabetes and Digestive and Kidney Diseases

(NIDDK), the Simons Foundation Autism Research Initiative grants 629287 and 564256, the University of California, San Francisco (UCSF) School of Pharmacy 2017 Mary Anne Koda-Kimble Seed

Award for Innovation and the Innovative Genomics Institute RIDER award 2019. The authors regret they could not include and highlight the comprehensive list of citations of their fellow

scientists due to space limitations. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San

Francisco, CA, USA Navneet Matharu & Nadav Ahituv * Institute for Human Genetics, University of California San Francisco, San Francisco, CA, USA Navneet Matharu & Nadav Ahituv *

Innovative Genomics Institute, University of California San Francisco, San Francisco, CA, USA Navneet Matharu Authors * Navneet Matharu View author publications You can also search for this

author inPubMed Google Scholar * Nadav Ahituv View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS N.M. and N.A. conceptualized, reviewed the

literature and wrote the manuscript. CORRESPONDING AUTHORS Correspondence to Navneet Matharu or Nadav Ahituv. ETHICS DECLARATIONS COMPETING INTERESTS N.A. is an equity holder of, and a

scientific advisor for Encoded Therapeutics, a gene regulation therapeutics company. N.A. and N.M. are cofounders of Enhancer Therapeutics Inc. and co-inventors on a related patent

(Publication number WO/2018/148256). N.M. and N.A. are co-inventors on a patent (US Patent US2018017186) submitted by the University of California, San Francisco, that covers gene therapy

for haploinsufficiency. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Reviews Drug Discovery_ thanks the anonymous reviewers for their contribution to the peer review of this work.

PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. GLOSSARY * _Cis_-Regulatory elements (CREs). DNA

sequences that regulate the transcription of a neighbouring gene. * Packaging Assembly of the nucleic acids and capsid during virus generation. * DNA scars Irreversible and unintended DNA

changes caused mainly due to off-targeting by DNA targeting modules with functional nucleases. * DNA looping Physical DNA–DNA interaction in the genome within 3D nuclear space. *

Nanoparticles Particles that are between 1 and 100 nm in diameter. * Intracerebroventricular A route of delivery via injection into the cerebrospinal fluid in cerebral ventricles. *

Trinucleotide repeat expansion A specific 3-bp DNA sequence that has more copies than normal in the genome. * Bioavailability The proportion of the therapeutic agent upon administration that

has an active effect. * Off-targeting The effects arising due to non-specific and unintended targeting of DNA targeting modules such as zinc fingers, transcription activator-like effector

(TALE) and CRISPR in the genome. * Delivery routes The methods of administration of a therapeutic agent based on the site of action. * Capsid (Also known as a viral envelope). The

proteinaceous shell that packages the genetic material of the virus. Its structure is important in determining viral stability, delivery and host interactions. * Pre-existing immunity The

adaptive immune response of the body due to pre-exposure to an antigen. * AAV serotypes (Adeno-associated virus serotypes). The variations in the capsid surface proteins of an

adeno-associated virus that can define its transduction efficiency in different tissue or cell types. * Blood–brain barrier The blood–brain barrier is the membrane made from endothelial

cells surrounding the blood vessels that selectively allows solutes to transfer from the blood to the central nervous system. * Intrathecal A route of delivery via injection into the spinal

canal in order to avoid the blood–brain barrier selective permeability. * Intravitreal A route of delivery into the vitreous humour of the eye. * Episomes Circular DNA that is not integrated

in the genome. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Matharu, N., Ahituv, N. Modulating gene regulation to treat genetic disorders. _Nat Rev

Drug Discov_ 19, 757–775 (2020). https://doi.org/10.1038/s41573-020-0083-7 Download citation * Accepted: 19 August 2020 * Published: 05 October 2020 * Issue Date: November 2020 * DOI:

https://doi.org/10.1038/s41573-020-0083-7 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative