Beyond CRISPR: Emerging Genetic Editing Technologies You Should Know

Since its discovery, CRISPR-Cas9 has swept the world over as a transformational gene-editing technology and has provided a unique ability of editing DNA that is relatively easy and precise to scientists. However, as much as CRISPR will continue to form the core of contemporary genetic engineering, there are other next generation technologies that are quickly making their way to the fore and stretching the edge of precision, safety, and versatility even further. It is also offered to overcome some of these limitations of CRISPR, namely, tools such as base editing, prime editing, and zinc finger nucleases (ZFNs), which open up new therapeutic potential.

In this paper, we are going to discuss these new technologies and how they compare with each other in terms of their peculiar mechanisms, advantages, and other possible areas of usage. We will also examine what is potentially ahead in the technology of gene-editing other than CRISPR.

CRISPR: The Root on the Modern Gene Editing

And before exploring newer technologies, it makes sense to have a clear picture of why CRISPR has been nearly revolutionary. CRISPR-Cas9 is based on the immune systems of bacteria, and it contains a guide protein (gRNA), which directs the Cas9 protein to a specific area in the genome, in which it develops a cut in both of the strands. This lesion can be subsequently repaired by the cell endogenous machineries, so that researchers can add or delete sequences of gene.

CRISPR-Cas9 has democratized gene editing since its adaptation to work on eukaryotic cells in 2012 because it is simple, cost effective and flexible to apply. Nonetheless, CRISPR has its flaws: the problem of off-target effects and difficulties with more accurate editing as well as the lack of available tools to edit in particular cell types have led to the creation of the next generation.

Base Editing Rewrapping Single Letters of the DNA

The next thing on the horizon that bears the greatest promise of going beyond CRISPR-Cas9 is base editing, a technology that was initially reported in 2016. In contrast to CRISPR, base editors chemically actuate one nucleotide, effectively exchanging one letter of the DNA code (A, T, C, G), without breaking both DNA strands and makes just single-stranded cuts.

As an example, cytosine base editors (CBEs) may be used to edit C-G base pairs to T-A whereas adenine base editors (ABEs) could be used to edit A-T to G-C. Such exactness will transform therapy of point mutations: one base alterations in the DNA that cause much genetic disease, such sickle cell anemia and cystic fibrosis.

Benefits of Base Editing

• Improved accuracy: Base editing does not involve induction of the double stranded break that increases the chances of introducing a large insertion or deletion (indel) which would have happened in conventional CRISPR-Cas9.
• Less off-targeting: Minimal modifications occurring to the genome as compared to the classical CRISPR.
• Efficiency in therapeutic context: Single-base changes also enable the possible remedying of a large amount of disease-causing mutations leaving behind a small degree of interference to the genome.

Limitations

• Limited to point mutations: The insertions and deletion of longer sequences of DNA not permitted.
• Editing window: Base editors have limited range of action to a narrow window of a few nucleotides and therefore are quite specific to target.

Prime editing: A Widely applicable genetic word processor

Although base editing works great on correcting single-letter errors, what happens when you want to perform some small insertion, deletion, or a combination of edits? Consider prime editing, which was initially proposed in the year 2019 and which in its structure is similar to base editing because of its ability to be as precise but also has the added feature of being able to edit more significant portions of DNA.

Prime editing involves a combination protein of Cas9 nickase (a mutant form of Cas9 that snips solely one strand of a DNA step) and a reverse transcriptase. This is directed by a prime editing guide RNA (pegRNA), which both determines a target site and what should be the edit. This allows insertions and deletions, and all of the 12 possible base-base substitutions.

 The benefits of Editing Prime

• Flexibility: Has the capability to introduce small insertions, deletions and any possible point mutations.
• Precision: Produces fewer double-stranded breaks, which minimises undesirable large-scale genomic rearrangement.
• Wide possible consumption: May become applicable as a disease modeling tool in many cell lines, generating animal models, or developing the treatment to treat various genetic diseases.

Limitations

• Efficiency issues: Based on some cell types, editing levels are a bit low as compared to the CRISPR-Cas9.
• Delivery complicatedness: The system is bigger and more complicated, thereby making it difficult to package into vehicles of delivery such as adeno-associated viruses (AAVs).

The First Programmable Editors are Zinc Finger Nucleases (ZFNs)

Prior to the discovery, which attracted attention globally, CRISPR, zinc finger nucleases (ZFNs) were used to edit genes in a programmed manner first. Invented in the 1990s, ZFNs are heterodimers of engineered proteins, one with zinc finger domains which bind a specific DNA sequence and one the FokI nuclease which nicks and cuts DNA.

Although ZFNs are more ancient and less malleable than CRISPR they formed the foundation of contemporary gene editing and are still used in therapeutic settings. Indeed, ZFNs are used as the initial in vivo gene-editing clinical trials in humans (involving Hunter syndrome and hemophilia B).

Outer benefits of ZFNs

• DNA specificity: In principle, Zinc fingers may be customized to recognize almost any DNA sequence.
• Small size: ZFN system is less bulky than CRISPR system hence it is easier to deliver using a viral vector.
• Clinically supported: ZFNs have already been applied to some treatments that have passed in a human trial stage.

Limitations

• Technically sophisticated program: The design of new ZFNs towards every target is labor intensive and technically complex.
• Reduced accuracy: ZFNs are at higher risks of off-target effects and unintentionally created mutations in comparison with CRISPR.
• Poor scalability: In comparison to the gRNA reprogramming in CRISPR, ZFN personalisation has to be carried out on a whole new protein.

The comparison between the Precision and Efficiency

So as to get a clearer idea of the relative comparisons of these technologies, here is a side-by-side comparison:

Non CRISPR Applications

Genetic disease Corrections

Base editing was also already used to correct point mutations in sickle cell disease and progeria laboratory models.

Prime editing Prime editing is a promising technology that can restore diseases involving specific insertions or deletions, like some forms of muscular dystrophy.

ZFNs Therapeutic applications ZFNs are actively being pursued in therapeutic applications in monogenic disease areas such as hemophilia.

Agricultural Innovation

Outside medicine, these tools had the potential to transform agriculture, such as the creation of crops that bear better traits, such as drought resistance, disease resistance, or enhanced vitamin content without having to introduce foreign genes that are one of the more contentious aspects of GMOs due to the regulatory restrictions that they face.

Synthetic Biology and Studies

Also with the development of new editing technologies, researchers are able to model the intricate diseases in greater detail, create synthetic lifeforms and test some of the fundamental questions in the field of genomics by making subtle alterations to the DNA code.

Future of editing genes

The future of gene editing is bright as genetic technologies are becoming more advanced:

• Combination therapies: Scientists are finding out the possibility of coupling base or prime editors with CRIPS-Cas systems to have the best of the two worlds of speed, precision, and versatility.
• Better delivery mechanisms: Nanoparticles, lipid-based carriers and engineered viral vectors are being developed to deliver these complicated editors to their target cells safely and effectively.
• Less off-target: Creation of high quality enzymes and advances in computational designs are making the off-targets effects less likely, improving safety profiles.
• Wider editing toolkits: As well as ZFNs, other editing systems are being developed more widely such as transcription activator-like effector nucleases (TALENs) or new variants of CRISPR such as Cas12, CasX which have additional editing possibilities and may provide even more precise editing.

Conclusion

Although CRISPR is already an effective method of editing genes, other novel technologies, such as base editing, prime editing, and zinc finger nucleases, are increasing the options of safe and precise genome editing guided by specificity and safety. There are different strengths and weaknesses of each technology, but all of them combined become a useful set of tools helping in solving genetic diseases, progressing agricultural innovation, and pushing further boundaries of synthetic biology.

As the research persists, even more sophisticated and streamlined editing approaches promising to achieve even higher results towards the actualization of the full potential of genomic science will come into existence. The post-CRISPR future is only starting to emerge, but the future holds a promise of a world that will benefit greatly with precision medicine and precision biotechnology changing how we perceive the genetic diseases.

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