Prime Editing: A New Twist in the CRISPR Tale

By Ruchi Jhonsa, Ph.D.

Modifying the genome for correcting disease-causing genetic mutations or making designed babies seemed like a far-fetched idea a decade ago. But with technology like CRISPR, it became possible to edit the genome in the desired fashion. Although, the conventional CRISPR technology failed when it was found to introduce unwanted changes in the genome, ongoing attempts to identify the pain points in the CRISPR technology brought new improvements to the gene editing table. In the recent article published in Nature, Dr. David Liu and coworkers (1) discuss a prime editing system that is versatile and may be able to address ~89% of the human genetic variants that causes diseases.

Evolution of the Prime Editing Technology

1) Conventional CRISPR Technology

The original CRISPR edited the gene by making double stranded breaks in it that would undergo repair by the virtue of cell autonomous DNA repair machinery. This process, however, introduced complicated rearrangements of genetic pieces thereby causing bystander effects.

2) Base Editing Technology

To overcome shortcomings of conventional CRISPR technology, Dr. Liu and his colleagues at the Broad Institute of MIT and Harvard introduced an improvement on the existing CRISPR technique that had the potential to change as small as a single base in a gene. Popularly known as Base editing (2, 3), the technology couples guide RNA to a modified cas9 that only cuts one DNA strand along with a deaminase enzyme that can chemically convert C to T, T to C, A to G and G to A. Although, this technique greatly reduced the off target errors caused by conventional CRISPR, it did not completely remove them. In an independent study conducted on rice plants (4), the adenine to guanine base editors worked perfectly without introducing off target errors but cytosine to thymine base editors roughly doubled the background level of off target mutations. Base editing offers a lot of potential for correcting genetic diseases caused by single letter mutations. However, it faced major difficulty attacking sickle cell mutation, as it would require switching T to A on the mutated gene.

3) ‘Search and Replace’ Prime Editing Technology

An improvement on traditional CRISPR and base editing technique came out this year that made precision editing possible with greater target flexibility (1). Another brainchild of Dr. Liu, Prime editing, in principle is a true ‘search and replace’ technique that finds the target sequence using primer RNA sequence and replaces the mutant sequence for a normal one using reverse transcriptase. The main ingredients of the primer editor complex include Cas9, reverse transcriptase, and pegRNA. This is how prime editor works. At first, it looks for the target sequence by virtue of prime editing guide RNA or pegRNA; the pegRNA contains a guide sequence that finds and binds to a specific target. Following which modified Cas9 makes a nick on the strand of DNA containing the mutation. This nicked strand is recognized by the primer sequence on pegRNA that allows hybridization to occur between the 3’end of the nicked DNA strand and the pegRNA. The double stranded DNA-pegRNA complex so formed by hybridization is recognized by the reverse transcriptase component of the prime editor and the information from the prime template is copied into the host cells genome. Following this in a few steps single stranded DNA flaps generated during the process are cleaved and newly synthesized DNA is joined with the original DNA strand by the cell’s endogenous repair machinery.

Which is better? Prime Editing or Base Editing

Both prime and base editors have their limitations. Base editing is more efficient in targeting single nucleotide present within the base editing window devoid of multiple cytosines or adenines, or when PAM is positioned 15+2 nucleotides from target nucleotide. Whereas prime editing works well in the scenarios where multiple cytosines or adenines are present near the target site or when protospacer-adjacent motif (PAMs) are not positioned for base editing. It was shown that base editing could successfully repair target base present within ~5 base pair window about 13-15 base pairs from a PAM sequence whereas prime editing could edit as low as three base pairs upstream and as high as 29 base pairs downstream of a PAM (1). This indicates that both prime and base editing can complement each other’s limitations and work together to edit genetic mutations.

Prime Editor: Will it Change the Future of Gene Editing?

Pros: Since prime editor, doesn’t rely on non-homologous end joining or homology directed repair to fix the break, it is much more efficient than conventional CRISPR technology in editing a gene in terms of off target effects. Additionally, it can edit four single-base transition mutations targeted by base editors along with all the eight possible single-base transversion mutations (purine to pyrimidine or vice versa), as well as precisely targeted insertions and deletions thereby providing greater target flexibility.

Cons: So far prime editing is limited to smaller genetic alterations. As reported by Dr. Liu, genetic insertions of 44bp and deletions of 80bp were so far made possible by prime editors. Moreover, this technology has been tested in only four human cancer cell lines and postmitotic primary mouse cortical neurons and the efficiency of prime editing varied quite a bit amongst each of the cell lines. Therefore, further analysis is warranted on other cell types in the future to determine the efficiency of prime editors.

Commercial interests

Both prime editing and base editing technologies have formed the basis of Cambridge, Massachusetts-based startups Prime Medicine and Beam Therapeutics respectively. Beam Therapeutics was formed in 2016 with the vision of providing life long cures to patients suffering from diseases caused by point mutations in the gene. Now with the Prime editing technology, Prime Medicine, and Beam Therapeutics will jointly develop therapies for human genetic diseases while focusing on different types of edits and distinct disease targets.

References

1. Anzalone et al., 2019, Nature, 10.1038/s41586-019-1711-4
2. Gaudelli et al., 2017, Nature, 551, 464-471
3. Komor et al., 2016, Nature, 533 (7603), 420
4. Li et al., 2018, Genome Biol. 19:59

Author

Ruchi Jhonsa