Overview of Genome Editing

An overview of genome editing, including the technologies used and gene therapy strategies employed

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Genome editing utilizes engineered or bacterial nucleases for targeted modifications of the genome to prevent or treat a disease1,2

GENOME EDITING

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Figure adapted from Wang D, Gao G. 2014.1

Genome-editing technologies have been developed rapidly over the past decade2

Genome-Editing Mechanisms

Genome-editing technologies utilize sequence-specific programmable nucleases to introduce targeted sequence changes in the DNA via natural cellular pathways3,4

  • Once the programmable nucleases recognize the target sequence, they bind to the DNA at the target site and induce a double-strand break (DSB)3–5
  • This DSB is recognized by endogenous DNA repair pathways that drive repair of the broken target DNA3–6
  • These repair mechanisms introduce the targeted change in the DNA to achieve genome editing5

Non-homologous End Joining (NHEJ)

  • NHEJ repairs DSBs in the absence of a donor template by directly ligating the broken ends of the DNA with little or no homology5,7
  • However, nucleotides can be lost or gained at the ends prior to ligation, making NHEJ an error-prone process7
  • In some cases, when the DSB generates overhangs, NHEJ can be used with an exogenous donor template that has compatible overhangs to repair DNA independently of homologous recombination6,8

Homology-Directed Repair (HDR)

  • HDR requires donor templates with regions homologous to the sequences surrounding the DSB6
  • The specificity afforded by this homology allows for targeted gene modifications, such as nucleotide substitutions or insertions6

Figure adapted from Bortesi L, Fischer R. 2015.6
DNA, deoxyribonucleic acid; DSB, double-strand break.

Genome-Editing Technologies

The key constituents most commonly used to facilitate genome editing are:

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)

  • CRISPR–Cas9 consists of a single RNA guide strand (gRNA) bound to the Cas9 endonuclease. The gRNA pairs with the sequence complementary to the target DNA sequence and directs Cas9 to the target site5,9

Figures adapted from Bortesi L, Fischer R. 20156 and Eid A, Mahfouz MM. 201612
Cas9, clustered regularly interspaced short palindromic repeats-associated protein 9; gRNA, ribonucleic acid guide strand; PAM, protospacer adjacent motif.

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Zinc-finger nucleases (ZFNs)

  • ZFNs are chimeric proteins that consist of a zinc-finger DNA-binding domain, which targets a specific site on the DNA, and the FokI endonuclease, which cleaves the target DNA5,10,11

Figures reproduced from Gaj T, et al. 2016.5
F, zinc-finger domain.

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Transcription activator-like effector nucleases (TALENs)

  • TALENs consist of the transcription activator-like effector (TALE) DNA-binding domain, which recognizes the target DNA sequence, and the FokI endonuclease, which cleaves the DNA5

Figures adapted from Bortesi L, Fischer R. 20156 and Eid A, Mahfouz MM. 201612
DNA, deoxyribonucleic acid; TALE, transcription activator-like effector.

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There are other approaches for genome editing that are in early stages of development11,13-15

CRISPR–Cas12 and Cas13

  • While CRISPR–Cas9 is a commonly used technology, other Cas proteins, including Cas12 and Cas13, are being investigated for genome editing13,15
  • A distinguishing feature of Cas12 in genome editing is its ability to target previously 
untargetable sequences15
  • CRISPR–Cas13 is an RNA-editing strategy capable of modifying transcripts that contain pathogenic variants13

Zinc-Finger Transcription Factors (ZFTFs)

  • ZFTFs differ from other genome-editing approaches, as they do not utilize nucleases to modify the target gene2,11,14
  • ZFTFs are designed to selectively regulate the expression of a target gene11,14
  • They are currently being explored as a novel therapeutic approach for diseases of the central nervous system14

Differences Between Genome-Editing Technologies

Although ZFNs and TALENs were the first genome-editing technologies developed, their use was limited. CRISPR–Cas9 rapidly replaced these approaches to become the more widely used genome-editing technology2,4,16

ZFNs and TALENs displayed:4,16

  • Low specificity resulting in off-target editing
  • Moderate-to-complex design

In contrast, the CRISPR–Cas9 system provided:4,16

  • Better efficiency
  • Simpler design
  • More diverse clinical applications

However, recent developments in design have improved the efficiency and specificity of ZFNs to minimize off-target editing17

The main differences between ZFNs, TALENs, and CRISPR–Cas9 are summarized in the table:

DNA binding Zinc-finger protein TALE protein gRNA
DNA cleavage FokI FokI Cas9
DNA recognition range 18–36 bp
(3 bp per zinc-finger module)
30–40 bp
(1 bp per TALE module)
22 bp
(DNA–RNA base pairing)
Recognition sequence Sequence containing G base as follows:
5’-GNNGNNGNN-3’
Sequence starting from
5’-T and ending with A-3’
Sequence immediately followed by a PAM 5’-NGG-3’
Advantages Sequence-based module engineering

Small protein size (<1 kb)
High specificity

Accurate recognition by
1 bp

Relatively easy selection of target region
Free selection of target region

Simple synthesis of gRNA

Multiplexing ability
Limitations Difficult sequence selection and zinc-finger engineering

Expensive and time consuming
Not applicable to methyl cytosine

Expensive and time consuming

Large protein size (>3 kb)
Large protein size (>4 kb)

Table adapted from Shim G, et al. 2017.18
A, adenine; bp, base pairs; CRISPR–Cas9, clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9; DNA, deoxyribonucleic acid; G, guanine; gRNA, ribonucleic acid guide strand; kb, kilobase; N, nucleotide; PAM, protospacer adjacent motif; T, thymine; TALE, transcription activator-like effector; TALEN, TALE nuclease; ZFN, zinc-finger nuclease.

Genome-Editing Strategies

Depending on the DNA repair pathway utilized and the availability of a donor template, different genome modifications can be achieved5,6

Gene Knockout

  • NHEJ can mediate disabling insertions or deletions of random base pairs, causing gene knockout5,6

Deletions

  • NHEJ can be used to repair two DSBs on the same gene resulting, for example, in the in-frame deletion of the intervening sections harboring disease-causing mutations5,19

Inversions

  • NHEJ can be used to induce inversions of DNA sections to disrupt gene function5,20

Insertions

  • In the presence of an exogenously delivered donor template that has compatible overhangs to the target DNA region, site-specific insertion of genomic DNA can be achieved by NHEJ6,8

Figure adapted from Bortesi L, Fischer R. 20156 and Gaj T, et al. 2016.5
DNA, deoxyribonucleic acid.

Insertions

  • Insertion of genomic DNA can be achieved by HDR if the template DNA 
has homologous regions flanking the sequence to be inserted6,19

Gene Modifications

  • HDR can be used to introduce specific nucleotide substitutions at a targeted region6

Figure adapted from Bortesi L, Fischer R. 2015.6
DNA , deoxyribonucleic acid.

Overview of Base Editing

Recently, tools have been developed that enable single base-pair conversions at targeted genomic sites independent of DNA DSBs and HDR — a process termed base editing21,22

Base editing is carried out by chimeric proteins known as base editors, which are capable of deaminating the cytidine or adenosine nucleosides21,23,24

When deaminated, cytidine, which normally pairs with guanine (G), is converted to uridine (U), and adenosine (A), which normally pairs with thymine (T), is converted to inosine (I)21

The resulting mismatches are resolved by DNA repair mechanisms21

  • The U–G mismatch is resolved to form U–A base pairs and finally T–A base pairs21
  • Inosine base pairs with cytosine (C) to form the resulting I–C base pairing; this is resolved to form G–C base pairs21

Cytidine Deamination

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Adenosine Deamination

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Figures adapted from Eid A, et al. 2018.21

Given that base editing is in early development, this process will not be discussed in detail in this module

Clinical Applications of Genome-Editing Technologies

While there are currently no approved therapies that utilize genome editing, this gene therapy strategy is actively being investigated across a range of diseases, including:2,25

CANCER

CANCER

  • TALENs and CRISPR–Cas9 genome-editing technologies have been investigated for their potential use in modifying T cells to improve their ability to identify and kill cancer cells2,26
  • Most of these clinical trials are in Phase 1 or 1/2 development2
  • Indications under investigation include treatment of cervical cancer, acute lymphoblastic leukemia, metastatic non-small cell lung cancer, prostate cancer, esophageal cancer, renal cell cancer, multiple myeloma, and melanoma2

MONOGENIC DISEASES

MONOGENIC DISEASES

  • ZFN-mediated in vivo genome editing therapies are currently in Phase 1/2 development for the treatment of mucopolysaccharidosis type I and II27
  • These therapies are designed to insert a correct copy of the α-L-iduronidase (IDUA) gene into liver cells27,28

HEREDITARY EYE DISEASES

HEREDITARY EYE DISEASES

  • A CRISPR–Cas9 gene therapy for the treatment of a hereditary blindness disorder, Leber congenital amaurosis type 10 (LCA10), is currently in Phase 1/2 development29,30
  • This genome-editing therapy is designed to delete the mutation-causing region of the gene29
  • It is the first in vivo CRISPR–Cas9-based genome-editing therapy to reach clinical development that allows for administration of the therapy directly into the eye29

HEMATOLOGIC DISEASES

HEMATOLOGIC DISEASES

  • ZFN and CRISPR–Cas9 are being investigated in a number of clinical trials for hematologic diseases, including hemophilia B (Phase 1), β-thalassemia (Phase 1/2), and sickle cell disease (Phase 1/2)2,31

Genome editing is also in preclinical development for cardiovascular diseases, metabolic diseases, and neurodegenerative diseases2

References

  1. Wang D, Gao G. Discov Med 2014;18(98):151–161.
  2. Li H, et al. Signal Transduct Target Ther 2020;5(1):1.
  3. Hoshijima K, et al. Methods Cell Biol 2016;135:121–147.
  4. Chandrasegaran S, Carroll D. J Mol Biol 2016;428(5 Pt B):963–989.
  5. Gaj T, et al. Cold Spring Harb Perspect Biol 2016;8(12):a023754.
  6. Bortesi L, Fischer R. Biotechnol Adv 2015;33(1):41–52.
  7. Symington LS. Crit Rev Biochem Mol Biol 2016;51(3):195–212.
  8. Maresca M, et al. Genome Res 2013;23(3):539–546.
  9. Farboud B, et al. Genetics 2019;211(2):431–457.
  10. Kim YG, et al. Proc Natl Acad Sci U S A 1996;93(3):1156–1160.
  11. Wilson KA, et al. Mol Ther Nucleic Acids 2013;2(4):e87.
  12. Eid A, Mahfouz MM. Exp Mol Med 2016;48(10):e265.
  13. Cox DBT, et al. Science 2017;358(6366):1019–1027.
  14. Pfizer. Press release. January 3, 2018. Available at: https://www.pfizer.com/news/press-release/press-release-detail/sangamo_and_pfizer_announce_collaboration_for_development_of_zinc_finger_protein_gene_therapy_for_als. Accessed October 12, 2020.
  15. Yan F, et al. Cell Biol Toxicol 2019;35(6):489–492.
  16. Khan SH. Mol Ther Nucleic Acids 2019;16:326–334.
  17. Miller JC, et al. Nat Biotechnol 2019;37(8):945–952.
  18. Shim G, et al. Acta Pharmacol Sin 2017;38(6):738–753.
  19. Wang D, et al. Cell 2020;181(1):136–150.
  20. Xiao A, et al. Nucleic Acids Res 2013;41(14):e141.
  21. Eid A, et al. Biochem J 2018;475(11):1955–1964.
  22. Gaudelli NM, et al. Nature 2017;551(7681):464–471.
  23. National Center for Biotechnology Information. PubChem. Cytidine. Available at: https://pubchem.ncbi.nlm.nih.gov/compound/Cytidine. Accessed October 19, 2020.
  24. National Center for Biotechnology Information. PubChem. Adenosine. Available at: https://pubchem.ncbi.nlm.nih.gov/compound/adenosine. Accessed October 19, 2020.
  25. Administration USFaD. Approved cellular and gene therapy products. Available at: https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products. Accessed February 9, 2021.
  26. National Cancer Institute. How CRISPR is changing cancer research and treatment. Available at: https://www.cancer.gov/news-events/cancer-currents-blog/2020/crispr-cancer-research-treatment. Accessed October 13, 2020.
  27. Harmatz P, et al. Mol Genet Metab 2018;123(2):S59–S60.
  28. ClinicalTrials.gov. NCT02702115. Available at: https://clinicaltrials.gov/ct2/show/NCT02702115. Accessed October 20, 2020.
  29. Ledford H. Nature 2020;579(7798):185.
  30. ClinicalTrials.gov. NCT03872479. Available at: https://clinicaltrials.gov/ct2/show/NCT03872479. Accessed October 19, 2020.
  31. ClinicalTrials.gov. NCT03745287. Available at: https://clinicaltrials.gov/ct2/show/NCT03745287. Accessed November 6, 2020.