CRISPR‐Cas9: A New Tool for Gene Therapy


Programmable RNA (ribonucleic acid)‐guided genome engineering using the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)‐associated (CRISPR Cas) system has revolutionised the field of gene editing owing its adaptability and versatility in addressing a wide range of biological questions. It relies on harnessing critical components of the bacterial acquired immune system, namely the Cas9 protein and its associated guide ribonucleic acid (gRNA), to selectively target and edit a desired gene, thereby underscoring its potential as a promising gene therapy agent. Its application, however, encompasses several critical parameters, ranging from safe delivery of the agent to tackling issues of unintended off‐targeting in the genome. Similar to its predecessor molecular scissors, namely zinc finger nucleases (ZFNs) and transcription activator‐like nucleases (TALENs), CRISPR‐Cas9 has been studied extensively along these lines to facilitate its use in therapeutic interventions and has shown some early promise in taking benchtop discoveries to the clinic.

Key Concepts

  • CRISPR‐Cas9 is the most recent addition to the repertoire of ‘molecular scissors’ for genome editing.
  • The system relies on an RNA‐guided protein that can make desirable changes in the genome.
  • CRISPR‐Cas9 has shown a lot of promise for in vivo and in vitro editing, which can be translatable for disorders, particularly monogenic ones.
  • Components of the system have been used for correcting several disease‐relevant mutations in mice through zygotic, somatic or ex vivo gene‐editing strategies.
  • The success of CRISPR as a therapeutic tool depends on its inherent properties of efficiency and specificity.
  • Protein and sgRNA engineering combined with improved delivery strategies have led to the discovery of potent CRISPR‐based therapy products.
  • The first clinical trials using CRISPR are currently under process.

Keywords: molecular scissors; nonhomologous end joining; homology‐directed repair; delivery vectors; CRISPR; Cas9; sgRNA; iPSCs

Figure 1. Targeted genome editing by CRISPR‐Cas9 system. The Cas9 protein, guided by sgRNA (synthetic guide ribonucleic acid) to the target genomic loci, makes a cut at about 3 bp upstream to the protospacer adjacent motif (PAM), hybridising with the strand complementary to the PAM sequence‐bearing strand. In vivo, the cleaved DNA (deoxyribonucleic acid) can undergo repair with the help of either NHEJ (nonhomologous DNA end joining) pathway wherein insertions or deletions are introduced (represented by the yellow asterisk). Alternatively, in the presence of a homologous end‐containing donor fragments (as represented by green rectangles), recombination can take place. These two repair pathways of CRISPR‐Cas9 system have a therapeutic potential as an unhealthy gene can be silenced (via NHEJ pathway) or can be replaced with a healthy copy (via HDR (homology‐directed repair) pathway).
Figure 2. Ex vivo or in vivo approaches of targeted genome editing. The therapeutic potential of CRISPR‐Cas9 system can be explored to derive either adult stem cells or the more easily accessible somatic cells from patients (which can be reprogrammed into iPSCs (induced pluripotent cells)). Ex vivo CRISPR‐Cas9‐mediated genome editing comprises selection of cells that have acquired the desired changes, specifying the lineage to the iPSCs and expanding the corrected and differentiated cells or the corrected adult stem cells, followed by introduction of the cells into the patient. On the other hand, direct delivery of CRISPR components (in the form of mRNA (messenger ribonucleic acid) or ribonucleoprotein complex) encapsulated within suitable delivery vehicles into a patient constitutes in vivo approach of gene therapy.
Figure 3. Delivery vehicles for CRISPR‐Cas9 system. CRISPR‐Cas9 system can be delivered into the patient with the help of virus or nonvirus‐based vehicles. The viral vectors majorly involve lentiviruses, adenoviruses and adeno‐ associated viruses. Lentiviral vectors contain the CRISPR‐Cas9 cassette within their RNA genome that is reverse transcribed and integrated into the host genome, whereas the adenoviral vectors have the CRISPR‐Cas9 cassette within their double‐stranded DNA genome and are present episomally within the infected host cells. The adeno‐associated vectors bear the CRISPR‐Cas9 system in their single‐stranded DNA genome and can target dividing cells as well as nondividing cells. The nonviral delivery vehicles of the CRISPR‐Cas9 system involves nanoparticles that cargo the CRISPR components into cells, microinjection‐mediated direct delivery or electroporation‐mediated introduction of the CRISPR‐Cas9 complex.


Barrangou R, Fremaux C, Deveau H, et al. (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315: 1709–1712.

Bassuk AG, Zheng A, Li Y, et al (2016) Precision medicine: genetic repair of retinitis pigmentosa in patient‐derived stem cells. Scientific Reports 6: 19969.

Bolotin A, Quinquis B, Sorokin A, et al (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151: 2551–2561.

Brouns SJ, Jore MM, Lundgren M, et al (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321: 960–964.

Chen ZH, Yu YP, Zuo ZH, et al (2017) Targeting genomic rearrangements in tumor cells through Cas9‐mediated insertion of a suicide gene. Nature Biotechnology 35: 543–550.

Cong L, Ran FA, Cox D, et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819–823.

Dever DP, Bak RO, Reinisch A, et al. (2016) CRISPR/Cas9 beta‐globin gene targeting in human haematopoietic stem cells. Nature 539: 384–389.

Ding Q, Strong A, Patel KM, et al (2014) Permanent alteration of PCSK9 with in vivo CRISPR‐Cas9 genome editing. Circulation Research 115: 488–492.

Gao Y, Wu H, Wang Y, et al (2017) Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off‐target effects. Genome Biology 18: 13.

Garneau JE, Dupuis ME, Villion M, et al (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468: 67–71.

Gasiunas G, Barrangou R, Horvath P, et al (2012) Cas9‐crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America 109: E2579–E2586.

Hou P, Chen S, Wang S, et al. (2015) Genome editing of CXCR4 by CRISPR/cas9 confers cells resistant to HIV‐1 infection. Scientific Reports 5: 15577.

Jinek M, Chylinski K, Fonfara I, et al (2012) A programmable dual‐RNA‐guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816–821.

Li HL, Fujimoto N, Sasakawa N, et al (2015) Precise correction of the dystrophin gene in Duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR‐Cas9. Stem Cell Reports 4: 143–154.

Liang P, Xu Y, Zhang X, et al (2015) CRISPR/Cas9‐mediated gene editing in human tripronuclear zygotes. Protein & Cell 6: 363–372.

Liao H‐K, Gu Y, Diaz A, et al (2015) Use of the CRISPR/Cas9 system as an intracellular defense against HIV‐1 infection in human cells. Nature Communications 6: 6413.

Lin SR, Yang HC, Kuo YT, et al (2014) The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo. Molecular Therapy – Nucleic Acids 3: e186.

Long C, McAnally JR, Shelton JM, et al (2014) Prevention of muscular dystrophy in mice by CRISPR/Cas9‐mediated editing of germline DNA. Science 345: 1184–1188.

Mali P, Yang L, Esvelt KM, et al (2013) RNA‐guided human genome engineering via Cas9. Science 339: 823–826.

Mojica FJ, Diez‐Villasenor C, Garcia‐Martinez J, et al (2005) Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. Journal of Molecular Evolution 60: 174–182.

Nelson CE, Hakim CH, Ousterout DG, et al (2016) In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351: 403–407.

Pourcel C, Salvignol G and Vergnaud G (2005) CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151: 653–663.

Ramanan V, Shlomai A, Cox DB, et al (2015) CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Scientific Reports 5: 10833.

Schwank G, Koo BK, Sasselli V, et al (2013) Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13: 653–658.

Tabebordbar M, Zhu K, Cheng JK, et al (2016) In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351: 407–411.

Traxler EA, Yao Y, Wang Y‐D, et al (2016) A genome‐editing strategy to treat [beta]‐hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nature Medicine 22: 987–990.

Wang J and Quake SR (2014) RNA‐guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection. Proceedings of the National Academy of Sciences of the United States of America 111: 13157–13162.

Wang X, Raghavan A, Chen T, et al (2016) CRISPR‐Cas9 targeting of PCSK9 in human hepatocytes in vivo‐brief report. Arteriosclerosis, Thrombosis, and Vascular Biology 36: 783–786.

Xie F, Ye L, Chang JC, et al (2014) Seamless gene correction of β‐thalassemia mutations in patient‐specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Research 24: 1526–1533.

Yin H, Xue W, Chen S, et al (2014) Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature Biotechnology 32: 551–553.

Yin C, Zhang T, Qu X, et al (2017) In vivo excision of HIV‐1 provirus by saCas9 and multiplex single‐guide RNAs in animal models. Molecular Therapy 25: 1168–1186.

Further Reading

Cox DBT, Platt RJ and Zhang F (2015) Therapeutic genome editing: prospects and challenges. Nature Medicine 21: 121–131.

Dai WJ, Zhu LY, Yan ZY, et al (2016) CRISPR‐Cas9 for in vivo gene therapy: promise and hurdles. Molecular Therapy Nucleic Acids 5: e349.

Dever DP and Porteus MH (2017) The changing landscape of gene editing in hematopoietic stem cells: a step towards Cas9 clinical translation. Current Opinion in Hematology. DOI: 10.1097/MOH.0000000000000385.

Fellmann C, Gowen BG, Lin PC, et al (2017) Cornerstones of CRISPR‐Cas in drug discovery and therapy. Nature Reviews Drug Discovery 16: 89–100.

Maeder ML and Gersbach CA (2016) Genome‐editing technologies for gene and cell therapy. Molecular Therapy 24: 430–446.

Savic N and Schwank G (2016) Advances in therapeutic CRISPR/Cas9 genome editing. Translational Research 168: 15–21.

Wang HX, Li M, Lee CM, et al (2017) CRISPR/Cas9‐based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery. Chemical Reviews 117: 9874–9906.

Xue HY, Zhang X, Wang Y, et al (2016) In vivo gene therapy potentials of CRISPR‐Cas9. Gene Therapy 23: 557–559.

Yin H, Kauffman KJ and Anderson DG (2017) Delivery technologies for genome editing. Nature Reviews Drug Discovery 16: 387–399.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Sharma, Saumya, Maiti, Souvik, and Chakraborty, Debojyoti(Nov 2017) CRISPR‐Cas9: A New Tool for Gene Therapy. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0026629]