Prime Editing

Prime Editing

Developed by Andrew Anzalone and David Liu.

So building on what the two knew about base editing and CRISPR-Cas9, they began working on a new technique to cut just one strand of DNA, leaving the other intact. It uses the same Cas9 nuclease as frequently deployed in the CRISPR system but combines the enzyme with two new reagents: a guide RNA called pegRNA, which leads Cas9 to the desired spot in the genome, and a reverse transcriptase that initiates the addition of a new sequence or base into the genome. Once the new genetic material is incorporated into the cut strand of DNA, the prime editor nicks the unedited strand, signaling to the cell to rebuild it to match the edited strand.

<img src="/90fff2d3-bd6a-52fd-a613-0bd8809ca06d.png"/>       <img src="/261d2c83-40bb-5ef8-984f-478dd1e8a919.png"/>   Prime editing is more complex than CRISPR editing. It requires three separate steps in which the DNA must match up with parts of the prime editing system. Liu says he believes this might be the secret to its accuracy. CRISPR-Cas9 relies on one pairing step: the guide RNA must pair with the target DNA. Prime editing requires this first step, but also includes two more components, a part of the guide RNA called the primer must also bind to the target site and the newly introduced DNA must bind to the original site. “If any one of those three DNA pairing events fail, then prime editing can’t proceed,” says Liu. “We believe that those three independent pairing events each provide an opportunity to reject off-target sequences,” he adds.

Differences from Base Editing

The first difference between base editing and prime editing is that base editing has been widely used for the past 3 1/2 years in organisms ranging from bacteria to plants to mice to primates. Addgene tells me that the DNA blueprints for base editors from our laboratory have been distributed more than 7,500 times to more than 1,000 researchers around the world, and more than 100 research papers from many different laboratories have been published using base editors to achieve desired gene edits for a wide variety of applications. While we are very excited about prime editing, it’s brand-new and there has only been one paper published thus far. So there’s much to do before we can know if prime editing will prove to be as general and robust as base editing has proven to be.

We directly compared prime editors and base editors in our study, and found that current base editors can offer higher editing efficiency and fewer indel byproducts than prime editors, while prime editors offer more targeting flexibility and greater editing precision. So when the desired edit is a transition point mutation (C to T, T to C, A to G, or G to A), and the target base is well-positioned for base editing (that is, a PAM sequence exists approximately 15 bases from the target site), then base editing can result in higher editing efficiencies and fewer byproducts. When the target base is not well-positioned for base editing, or when other “bystander” C or A bases are nearby that must not be edited, then prime editing offers major advantages since it does not require a precisely positioned PAM sequence and is a true “search-and-replace” editing capability, with no possibility of unwanted bystander editing at neighboring bases.

Of course, for classes of mutations other than the four types of point mutations that base editors can make, such as insertions, deletions, and the eight other kinds of point mutations, to our knowledge prime editing is currently the only approach that can make these mutations in human cells without requiring double-stranded DNA cuts or separate DNA templates.

Prime editing can do multi-letter base-editing, which could tackle fatal genetic disorders such as Tay-Sachs, which is caused by a mutation of four DNA letters.

Prime editing is a gene editing method that can perform targeted small insertions, deletions, and base swapping in a precise way. This sounds very much like existing CRISPR methods, you might say.

The outcomes of prime editing have been achieved previously in different ways. The ability to delete bases is a hallmark of knockouts, while the ability to insert specific nucleotides in a precise manner is the premise underlying knock-ins. What sets prime editing apart from traditional CRISPR is that it enables targeted editing without generating double-stranded DNA breaks. Moreover, targeted insertions can be achieved without the need for donor DNA templates. Looking to just edit bases? No problem. Prime editing expands the limited scope of current base editing abilities (4 possible combinations C>T, G>A, T>C, and A>G) to all 12 combination swaps. 

In summary, prime editing is an all-in-one solution with small improvements in the CRISPR process that impact the final result—a classic sum-is-larger-than-the-parts scenario.

The guide RNA, called prime editing guide RNA (pegRNA), is substantially larger than standard sgRNAs commonly used for CRISPR gene editing (>100nt vs. 20nt). The pegRNA is a sgRNA with a primer binding sequence (PBS) and the template containing the desired RNA sequence added at the 3’ end. It is worth noting that currently, pegRNAs are created using plasmids and in-vitro transcription. Together, they form the PE:pegRNA complex, which is used to mediate genome editing within the cell.

Process of Prime Editing

The PE:pegRNA complex binds to the target DNA, and Cas9 nicks only one strand, generating a flap. The PBS, located on the pegRNA, binds to the DNA flap and the edited RNA sequence is reverse transcribed using the reverse transcriptase. The edited strand is incorporated into the DNA at the end of the nicked flap, and the target DNA is repaired with the new reverse transcribed DNA. The original DNA segment is removed by a cellular endonuclease. This leaves one strand edited, and one strand unedited. In the newest PE system, PE3 and PE3b, the unedited strand can be corrected to match the newly edited strand by using an additional standard guide RNA. In this case, the unedited strand is nicked by a Cas9 nickase and the newly edited strand is used as a template to repair the nick, thus completing the edit.

Prime Editing

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