New Enzymes Allow for More Precise CRISPR Gene Editing Techniques

Bryn Williams 23′

A space filling model of a CRISPR protein (blue) with CRISPR RNA (green) bound to a single stranded DNA target (red).
Source: (Wikimedia Commons)

The gene editing tool CRISPR has garnered widespread popularity in recent years. The tool allows researchers to alter gene sequences, allowing for a medical experience more tailored to an individual’s unique genetic composition. CRISPR can be extremely useful, yet it still struggles in its precision and accuracy.2 Researchers have discovered an innovative new version of the CRISPR-Cas9 system called base editing that can improve the accuracy of targeting specific gene sequences with the help of new enzymes1. Base editing is proving to be a more accurate way than the traditional CRISPR-Cas9 system to edit the genome because it alters a single base pair at a time and is less prone to off-target mutations.

The CRISPR-Cas9 system utilizes a unique RNA strand that interacts with a particular sequence of DNA and directs the Cas9 enzyme to cut it. Cas9 breaks both strands of DNA, leaving a break in the DNA that the cell’s DNA repair mechanism replaces; but with this repair, some nucleotides in the DNA sequence are lost. While the technology can effectively change the genome, the technique is difficult to control, potentially causing damaging mutations at other, unintended locations in the genome. In an effort to better control the technique, scientists have searched for and discovered new enzymes that may be used to target DNA more specifically.2

These enzymes make the new CRISPR gene editing technique, base editing, possible. The base editing technique was originally proposed in 2016 and offers a more controlled way to edit DNA sequences.2 Researchers using base editing can target specific base pairs, changing individual nucleotides, as opposed to CRISPR-Cas9 which changes larger DNA segments. Base editing by altering a single letter in the DNA sequence offers significantly more control than the original CRISPR-Cas9 system, and in addition, the technique is safer because it mitigates the off-target mutations that can cause damage to the genome.2 With base editing, it is now possible to cure human diseases caused by a single nucleotide mutation such as sickle cell anemia.2 Across the globe, companies that specialize in gene editing like Addgene and Beam are exploring the technique’s potential.2

Although it is a promising new technique that solves many of CRISPR-Cas9’s problems, base editing has its own limitations. Researchers have identified problems involving the enzyme used to mutate Cytosine to Thymine, noting that it tends to alter other nucleotides in the genome regardless of location2. Though these off-target mutations can be dangerous to the subject and difficult to locate, they occur less frequently using the base editing technique than when using CRISPR.2 It’s hard to know exactly how accurate the technique is, because to identify an off-target mutation made by the enzyme, scientists would have to sequence the entire genome and search for the mutation which can be expensive and time intensive.2

In response to this problem, a research team led by David Liu, a chemical biologist at the Broad Institute of MIT and Harvard, proposed various novel methods to identify damaging mutations that require less time and resources.2 One of these methods involves testing increasing bacterial antibiotic resistance. The higher the frequency of resistance the more mutations the bacterial genome underwent.2

Liu expanded on his idea and used methods like the bacterial resistance technique to determine which enzymes produced the least amount of off-target base pair mutations. Results of the research could improve the accuracy of mutation, which is extremely important to the efficacy of gene editing in the medical field.2 If scientists discover base-editing enzymes that result in no off-target mutations, the technique can be used to treat various genetic diseases. Liu and his team are close to reaching this goal. They recently discovered enzymes that can exchange Cytosine to Thymine with minimal off-target mutations. The enzymes are still not perfect, but they have greater predictability and accuracy than the previous CRISPR-Cas9 system2.

Research is ongoing to find more accurate enzymes. Researchers like Liu and the geneticist Hui Yang at the CAS Institute of Neuroscience in Shanghai continue to improve the base editing technique.2 For instance, Liu and his team have developed a new gene editing process called prime editing that builds upon the base editing technique and offers a wider range of nucleotide alterations and even more precision.3 Yang’s team continues to discover new enzymes for the base editing technique that result in no identifiable off-target mutations.2 The possibilities of this new technology are provide hope for more accurate gene editing, and consequently, more focused and successful medical treatments.

References:

[1] Doman, J. L., Raguram, A., Newby, G. A. & Liu, D.R. (2020). Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nature Biotechnology. https://doi.org/10.1038/s41587-020-0414-6

[2] Heidi Ledford. (2020). Super-precise CRISPR tool enhanced by enzyme engineering. Nature.

[3] Heidi Ledford. (2020). Super-precise new CRISPR tool could tackle a plethora of genetic diseases. Nature. https://doi.org/1038/d41586-019-03164-5

 

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