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CRISPR: The End for Zinc Fingers?

By , MD, PhD

The past decade has brought rapid and significant innovations in genome-editing techniques. For the first time researchers have the opportunity to manipulate essentially any gene in a plethora of cells and organisms, using targeted nucleases that were designed for sequence-specific binding of the DNA.

One of the first breakthrough methods of gene targeting was the usage of chimeric proteins called zinc-fingers nucleases (ZFN) to create double-strand breaks. Still, the real revolution was the introduction of CRISPR and CRISPR associated (Cas) systems into the biomedical research arena.

One specific CRISPR nuclease – Cas9 – paired with short guide RNA has the ability to recognize the target DNA via Watson-Crick pairing. The guide sequence found within CRISPR RNAs typically correlates to bacteriophage sequences, constituting the mechanism for CRISPR antiviral defense, which can easily be substituted with a sequence of interest in order to retarget the Cas9 nuclease.

Why is CRISPR better than zinc fingers?

ZFN and CRISPR/Cas9 systems function on a similar concept: a nuclease is steered towards a specific sequence in the genome to create a double-strand break in the DNA. Once such a break is generated, the inherent DNA repair machinery of the cell is activated, and during the repair process of double-strand break the genome is modified.

First successful utilization of ZFN was in fruit flies as early as 2002, and since then it has been used to alter the genome of a myriad of different organisms – including those not considered as genetic model systems. The binding specificity of the designed zinc-finger domain points the ZFN to a specific genomic site.

Despite the advantages of genome editing with ZFN, there are several potential disadvantages in comparison with CRISPR/Cas9 system. For example, there is no straightforward and easy way to construct zinc finger domains to bind a comprehensive stretch of nucleotides with high affinity.

Furthermore, commercial ZFN modules are quite expensive, there are certain difficulties to perform replacement of large fragments (which is pivotal for inducible knockouts), and the technique necessitates screening in order to identify targeted events in animal models.

The CRISPR/Cas technology offers a myriad of advantages over ZFN, as it relies on a single targeting molecule (guide RNA) for DNA sequence recognition. This fact simplifies the construction of vectors with multiple guide RNAs for multiplexed gene targeting.

Another way in which construction of CRISPR plasmids is simplified in comparison to ZFN is that DNA recognition sites are composed of nucleic acid rather than protein. Cloning of the DNA-recognition component of guide RNA sequence is thus comparatively simple.

There is also a possibility of multiplexed targeting by Cas9 with the introduction of a series of short guide RNAs (rather than a set of bulky proteins). Such multiplexed modifications (alongside high efficiency and the ease of targeting) have opened a wide array of applications in biotechnology and medicine. All that does not mean that there is no use of ZFN anymore, but the tide is swiftly turning towards CRISPR/Cas technology.

Disadvantages of CRISPR/Cas

Naturally, CRISPR/Cas9 system also has certain disadvantages that have to be taken into account. One of them is a reported high incidence of nonspecific DNA cleavage; while this has cooled some of the initial enthusiasm about this method, a potential remedy is the expression of two CRISPR modules with nickase activity against two genomic sites that are closely adjacent to one another.

Then there is a problem of mosaicism, where mutant allele is produced in only some of the cells, as nucleases may not inevitably cut the DNA during one stage of embryonic development. The production of multiple mutations in one organism is also possible, which can create phenotyping bottlenecks in mouse models.

Regardless of those burning problems, CRISPR/Cas9 genome-editing technique presents staggering opportunities for addressing a number of illnesses beyond the reach of previous treatment modalities. Taking into account the accelerating rate of technological progress, as well as a wide range of research and clinical applications, the road ahead of us will certainly be a thrilling one.

Sources

  1. http://www.jci.org/articles/view/72992
  2. http://genesdev.cshlp.org/content/28/17/1859.full
  3. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694601/
  4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/
  5. http://zlab.mit.edu/assets/reprints/Cox_D_Nat_Med_2015.pdf
  6. Thakore PI, Gersbach CA. Genome Engineering for Therapeutic Applications. In: Laurence J, Franklin M, editors. Translating Gene Therapy to the Clinic: Techniques and Approaches. Academic Press, Elsevier, 2015; pp. 27-44.

Further Reading

Last Updated: Jan 13, 2016

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