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. , 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. or 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)). 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 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.


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

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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]