The feverish pace of new discoveries still being made in the genome editing revolution can make it difficult to decipher a quotidian advance from a genuine paradigm shifter. As a result, additions to the CRISPR “toolbox” are sometimes met with complacency.
But in a paper published online today in Nature, David Liu, PhD, and colleagues at the Broad Institute lay out a new mechanism for genome editing called prime editing that does not make double-strand breaks in the target sequence or use a donor DNA template. “This is the beginning of an aspiration to make any DNA change in any position of a living cell or organism,” says Liu.
Described as “search-and-replace” genome editing technology, prime editing supplies the desired edit in an extension to the guide RNA, which is then converted to DNA using the enzyme reverse transcriptase. The technology can introduce targeted insertions, deletions, and all 12 possible base-to-base conversions (each of the four DNA nucleotides or bases can be converted to three other types). Liu notes that there are some 75,000 cataloged pathogenic mutations in human disease, but prime editing has the versatility and potential to correct the vast majority (89%) of them.
“While prime editing is new, the promise it shows is formidable,” declares Fyodor Urnov, PhD, scientific director at Innovative Genomics Institute and professor of molecular and cell biology at the University of California, Berkeley.
Liu’s latest tour de force is published in Nature and entitled, “Search-and-replace genome editing without double-strand breaks or donor DNA.” Liu is a professor in the department of chemistry at Harvard University, the Broad Institute, and a Howard Hughes Medical Institute Investigator. The work was led by Andrew Anzalone, MD, PhD, a postdoc in the lab.
Liu unveiled the technology at a genome editing conference ten days ago at the Cold Spring Harbor Laboratory, in front of a rapt audience of nearly 400 scientists. He closed his talk by saying, “I’m really looking forward to seeing what Andrew comes up with in the second year of his postdoc!”
Around the bases
Just three years ago, the Liu lab published a system called base editing, which took advantage of the CRISPR-Cas9 system to target a specific DNA sequence, but without cutting it. Base editing (see “All About that Base Editing”) utilizes a catalytically “dead” Cas9 (dCas9) fused to the base editing machinery, which can engineer a particular class of base substitution (transition) without introducing a double-stranded DNA break. Together with his colleagues Keith Joung, MD, PhD, and Feng Zhang, PhD, Liu co-founded Beam Therapeutics to commercialize the technology. The company recently filed to go public.
Liu tells GEN that a company has been formed around prime editing technology. The technology, he notes, has been licensed from the Broad Institute of MIT and Harvard and sub-licensed to Beam Therapeutics for certain fields. According to the Beam Therapeutics S-1 filing with the SEC on Sept 27, 2019, Beam has secured an exclusive license from Prime Medicine, to “pursue this new technology in certain fields and for certain applications similar to those we are already pursuing with base editing.” The license “does not cover all fields and applications of this new technology for gene editing and Prime Medicine retains broad rights to use this technology outside of the fields licensed to us.”
Prime editing, in similar fashion to base editing, does not require a DNA donor template and uses a single molecular complex to engineer a precise change at the target site in the genome. The process is “somewhat complicated,” Liu says, involving both an engineered protein and an engineered RNA.
Here, the dCas9 is fused to the enzyme reverse transcriptase, which can transcribe RNA to DNA. An extended guide RNA referred to as the prime editing guide RNA (pegRNA) is constructed to not only specify the target site but also encode the desired edit and prime reverse transcription. The edit from the pegRNA is transferred into the target site. In the process, a branched intermediate is formed with two single strand DNA flaps; an unedited 5′ flap and a 3′ flap with the edited sequence from the pegRNA. As the 5′ flap is the preferred target of the cell’s endogenous endonucleases, the 3′ edited flap will drive the incorporation of the edited DNA strand creating heteroduplex DNA. The cell’s repair machinery will then permanently replace the original DNA strand by the edited DNA.
If CRISPR-Cas9 and other programmable nucleases are like scissors and base editors are like pencils, Liu says, then prime editors are like word processors—capable of searching for target DNA sequences and precisely replacing them with edited DNA sequences. “Nucleases, base editors, and prime editors each have complementary strengths and weaknesses—just as scissors, pencils, and word processors each have useful roles.”
All three classes of genome editing, Liu adds, will have important roles going forward in research and clinical settings. Base editors, notes Liu, can offer higher editing efficiencies and fewer indel byproducts than prime editors, while prime editors offer more targeting flexibility and precision.
Get to the point
In the Nature article, Anzalone, Liu, and colleagues describe several iterations of the prime editing platform to improve efficiency and specificity, which they then test by performing more than 175 edits in human cells. These mutations were of varying types including all 12 possible point mutations, as well as insertions up to 44 basepairs (bp) in length and deletions up to 80 bp. Among the genes they have successfully edited are those mutated in some well-known genetic diseases that have been difficult to make previously, including sickle cell disease and Tay Sachs disease.
Anzalone et al. were also able to introduce prime editing into mouse cortical neurons—cells that do not divide—using a lentivirus vector. The team reports that the technique is more efficient, makes fewer byproducts, and has lower off-target editing than traditional CRISPR-Cas9 editing.
“I was already excited about the therapeutic promise of base editors,” says Ross Wilson, PhD, project scientist at the Innovative Genomics Institute. “Prime editors have nearly all the same strengths, with additional advantages.” As a result, Wilson predicts that prime editors will likely be more straightforward and widespread in their use. According to Liu, the reagents have already been deposited in Addgene, the nonprofit repository that is used for plasmid sharing.
Unlike base editors, which can currently perform 25% of possible nucleotide substitutions (including those for a sizeable proportion of genetic disorders), prime editors have the ability to change any nucleotide to any other nucleotide. Prime editing can also resolve frameshifts induced by small insertions or deletions—something that base editors cannot do.
Another potential advantage of prime editing is that it makes fewer off-target edits when compared with CRISPR-Cas9. Liu speculates that this higher DNA specificity is due to prime editing’s requirement for three separate DNA pairing events to occur before editing can take place, rather than just the single-guide interaction in CRISPR gene editing. Hence there are three opportunities “to reject an off-target sequence,” says Liu.
As with gene editing techniques that have preceded prime editing, delivery remains the universal hurdle for a number of reasons. Prime editing eliminates the need for co-delivery of a corrective DNA template, a factor that can magnify standard challenges in the delivery of genome editing machinery. However, prime editing introduces new challenges as the protein constructs used are too large to be packaged in the most commonly used viral vectors (AAV).
“We [people in the field] were surprised that prime editing worked at all,” Wilson tells GEN. Relying on an exogenous polymerase to sneak new genetic information into a cell’s genome is a wildly ambitious undertaking, he says. “It’s amazing that the resulting enzyme is so efficient.”
Will prime editing live up to the early promise? “We should find out soon whether this cognate of a college sports star can perform well on a bigger stage,” notes Urnov. “We all hope of course it will be like Alex Morgan or Aaron Rodgers in this regard, and we should know soon. Plain-vanilla editing has established all the key systems and assays that will determine whether prime editing has a future in experimental medicine.”
An overview of prime editing
A prime editing (PE) complex is made up of the dCas9 and the RT. The PE:pegRNA complex nicks a single strand of DNA. The resulting 3′ end primes reverse transcription of new DNA with the desired edit and the 3′ edited flap will drive the incorporation of the edited DNA strand creating heteroduplex DNA. The cell’s repair machinery will then permanently replace the original DNA strand by the edited DNA.
Slides courtesy of the Liu lab.