Prime Editing & CRISPR-Cas9: Advancing Gene Editing

Gene editing has brought much promise for improving patient health and well-being. Much of this promise lies in its ability to correct disease-causing mutations through genome editing at the single base pair level. 

To harness genome editing for gene therapy, researchers developed tools to edit genes at specific target sites precisely. Among these editing tools is the CRISPR-Cas9 editing tool, derived from the CRISPR-Cas system in bacteria. Its discovery has led researchers to harness the CRISPR-Cas system for base editing in many genomes.

Over time, researchers have also developed other programmable editing technologies by modifying at least one component of the CRISPR-Cas system. One such approach is called prime editing. It improves upon CRISPR-Cas9 editing by reducing the risk of off-target gene modifications. While such methods have yet to be regularly used in the clinic, David Liu, the pioneer of prime editing, expressed hope that these methods will enter clinical trials as soon as next year.

Even so, incorporating prime editing and other gene editing approaches into the clinic requires researchers to ensure that they can edit genes correctly and safely. To address these concerns, we will discuss how CRISPR-Cas9 works, how prime editing improves CRISPR-Cas9 gene editing, and how prime editing can advance gene editing for diverse cell lines. 

Then, we will illustrate how Excedr’s leasing program helps researchers obtain the equipment needed to conduct a prime editing assay.

CRISPR-Cas9 & The Rise of Gene Editing

Gene editing began in earnest when researchers first discovered DNA in the 1960s. These efforts began with restriction enzymes that cut and spliced DNA. While researchers have harnessed other gene editing methods such as zinc finger nucleases, they come with other weaknesses that tempered the dream of gene editing. Since CRISPR-Cas9 editing emerged, the dream of gene editing came back in earnest.

Expressed in most bacteria and archaea, the CRISPR-Cas9 system protects microorganisms from foreign DNA, whether from a plasmid or a virus. CRISPR-Cas9 comprises four components that coordinate the cutting of double-stranded DNA: CRISPRs, Cas endonucleases, CRISPR-associated RNA (crRNA), and Trans-activating CRISPR RNA (TracrRNA). (insert link to Excedr article on CRISPR-Cas9 gene delivery). When combined, a ribonucleoprotein (RNP) complex forms. This complex recognizes the target sequence encoded in the crRNA-tracrRNA sequence, locally unwinds the DNA strand, and cuts the DNA at that site, forming a double-stranded break (DSB).

After discovering the CRISPR-Cas9 system in bacteria, researchers subsequently harnessed the system to fine-tune the editing of specific alleles through gene editing. This system comprises the following reagents:

• Single guide RNA (sgRNA): sgRNA is a sequence of single-stranded nucleotides that combine the CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA) elements of a CRISPR-Cas system into a single sequence. crRNA is the CRISPR array sequence transcribed, while the tracrRNA interacts with the crRNA to form the guide RNA (gRNA) in sgRNA.
• Cas9 protein: The cas9 protein is a member of the Cas9 family of enzymes. The sgRNA guides the cas9 protein to target genomic regions, cut the genomic region of interest, and replace it with the edited DNA sequence. In gene therapy, this process is known as Cas9-mediated insertional mutagenesis.
• Repair template: The DNA molecule contains the new DNA sequence to be integrated after CRISPR-Cas9-mediated cleavage.

Although CRISPR-Cas9 technologies have driven interest in gene editing tools, the DSBs they form encourage the generation of insertions and deletions (indels). Indels arise as spontaneous changes to individual nucleic acids, such as cytosine and adenine, and are called point mutations. One way indels arise is when cells use non-homologous end joining (NHEJ) to repair DSBs instead of homology-directed repair (HDR). The resulting mutations modify the gene sequence at the CRISPR/Cas site after gene editing and act as undesired genetic byproducts. These byproducts increase the risk of side effects and other deleterious patient outcomes.

Genome Editing with Prime Editors: Taking Gene Editing to the Next Level

Andrew Anzalone, Peyton Randolph, and other colleagues in David Liu’s lab at Harvard University recognized the problems that off-target effects had on implementing CRISPR-Cas9 gene editing. To minimize the risk of DSBs that CRISPR-Cas9 can incur during gene therapy, the research group developed an alternative gene-editing tool: prime editing. Published in Nat. Biotechnol. (Nature Biotechnology), prime editing introduces the desired base-to-base conversions and small indels without producing DSBs. Although the system is based on the CRISPR-Cas9 editing system, prime editing distinguishes itself from CRISPR-Cas9 in multiple ways:

• Prime editors (PEs): Instead of using a fully functional Cas9 endonuclease, prime editing uses PEs. All PEs contain the Cas9 nickase, the domain of the RNA-guided Cas9 protein that cuts DNA. The Cas9 nickase domain is next fused to a reverse transcriptase (RT) domain, which reencodes RNA back into DNA.
• PegRNA: The two domains combine and form a complex with a prime editing guide RNA (pegRNA). PegRNA provides the template for the desired edits to be integrated into target cells. All pegRNA sequences comprise three components. First, the pegRNA contains a spacer sequence that indicates to the PE the target DNA sequence to be modified. Second, the sgRNA scaffold guides the PE to the target region. Lastly, the sgRNA is extended with a 3’ extension sequence. This sequence contains a primer binding sequence (PBS) complementary to a portion of the PAM sequence, the protospacer, and an RT template that contains the desired edit.

When the two components join, they form the prime editing system and edit the genome as follows (see Figure 1 for a schematic):

• Nicking of the PAM-containing strand: Precise genome editing begins with the PE complex coming to its binding site and binding the target sequence. Once bound, the Cas9 nickase domain cuts the PAM-containing strand at the region indicated by the pegRNA. The double-stranded DNA then becomes single-stranded at the target region.
• PegRNA hybridization to the target sequence: One of the single strands hybridizes with the PegRNA’s spacer sequence. The 3’ end of the PegRNA binds the PBS sequence with the PAM strand. This creates the DNA complex that enables the edited DNA to be reverse-transcribed.
• Reverse transcription: The RT domain of the PE then reverse transcribes the RNA sequence containing the gene edit into the target DNA sequence. This yields a DNA flap from the old DNA sequence that equilibrates to ultimately allow the reverse-transcribed DNA sequence to ligate into the DNA template.

Despite the potential for precise gene editing that PEs provide, prime editing is still in its infancy. Researchers must address several challenges to make PEs more commonplace for gene editing therapies:

• Low editing efficiency: PE has highly variable editing efficiencies between human cell types, partly because low RNA stability limits the efficacy of RNA to enable DNA recognition. Researchers are currently expanding efforts to make prime editing more consistent across diverse cell types.
• Delivering prime editing components: Much like CRISPR-Cas delivery systems (insert link to Excedr article about CRISPR-Cas delivery systems), delivering prime editing components into target cells is a challenge, particularly since vectors like an adenovirus cannot hold the PEs and other components all at once. This requires coinfections by multiple AAVs to infect all the components concurrently.
• Precise pegRNA design: Designing an optimal pegRNA sequence requires researchers to consider several factors that can hamper prime editing. For instance, modifying the 3’ end of the RNA sequence affects how stable the pegRNA sequence is. Lower editing efficiency is also associated with low PBS melting temperatures. Hence, much care must be taken to design robust pegRNA sequences in targeting genomic DNA.

Applications of Genome Engineering with Prime Editing

While prime editing is a continually developing technology, researchers have begun implementing the editing technology for biotechnological applications. In fact, several industries have already started generating edited sequences for various practical applications:

• Agriculture: Researchers have begun developing prime editors for editing rice and wheat crops, allowing precise single-nucleotide mutations for advancing plant breeding and functional genomics research. As the technology continues developing, researchers are also improving editing efficiency with prime editors by modifying the pegRNA or enzyme for editing rice genomes.
• Animals: David Liu’s lab, through Jessie R. Davis, recently employed prime editing on mouse animal models. They demonstrated that they could conduct prime editing in mouse brains, liver, and heart with up to 46% efficiency. While the editing efficiency leaves room for further improvement, stabilizing RNA and improving expression could enhance PE efficiency.
• Bacteria: Researchers are also applying prime editing to E. coli, expanding the repertoire of gene editing tools to the prokaryotic realm.

In the clinic, researchers also see the potential of prime editors for treating genetic diseases with high specificity in vitro and in vivo. For instance, scientists have applied prime editing to correct mutations associated with liver and eye diseases in animal models. Preclinical trials for prime editing to treat human liver diseases are also underway for human gene therapy.

Excedr leases the equipment to conduct prime editing

As gene editing emerges as a viable therapeutic option, the biotech industry seeks to minimize mismatches and maximize editing efficiency. To this end, scientists must consider multiple advances such as PEs and ensure that the best reagents are employed. Maximizing editing efficiency with PEs hence requires conducting robust quality-control experiments. These experiments ensure that the edits are done correctly on multiple cell lines to treat diverse diseases.

Aware of the growth in the gene editing sector, Excedr has established a leasing program to accelerate molecular genetics research. Although we do not hold an equipment inventory, we can acquire the instruments you need from your manufacturer of choice. Through our leasing program, we can also supply you with the equipment you need to ensure maximum editing efficiency:

• PCR thermocyclers: PCR thermocyclers amplify target DNA through the polymerase chain reaction (PCR). As you select a thermal cycler, consider several technical factors to ensure accurate genome editing with PEs. Once you consider these factors, you can then move towards selecting the thermocycler that best meets your gene editing needs. That said, here are some of the factors to consider as you select one of our many PCR thermocyclers:‍
• Temperature accuracy and control: Researchers must ensure that the reaction temperatures are accurate to conduct, not just for each tube reaction, but also for the uniformity of temperatures within the block. This is especially important for primer annealing because that will affect how well the primer binds to the DNA target that you are seeking to amplify.‍
• Ramp rate: Ramp rate determines how fast it takes for a thermal cycler to reach a specified temperature, with higher ramp rates resulting in higher throughput for PCR assays.‍
• How many samples can the PCR thermocycler process? Depending on how many CRISPR-Cas guides you want to test or how many CRISPR genome edits you’re doing, you’ll want to decide how many you’ll need to do to get your desired results.‍
• DNA synthesizers: DNA synthesizers are machines that produce custom-built DNA molecules to produce oligonucleotides of a specific sequence. The typical DNA synthesis protocol, known as the phosphoramidite method, features a four-step process that adds individual nucleotides to a growing DNA chain with 5’-protected dimethoxytrityl (DMT) nucleotide phosphoramidites. However, the phosphoramidites have poor stability and must be incorporated quickly, and require substantial amounts of organic solvents to perform. As such, different vendors are adopting other approaches to make DNA synthesis more efficient and cost-effective for gene editing. Here are just some of the vendors adopting novel DNA synthesis methods where we lease from:‍
• DNA Script: DNA Script has developed the Enzymatic DNA Synthesis (EDS) Technology to facilitate benchtop DNA synthesis. EDS adopts an inkjet platform that employs a terminal deoxynucleotidyl transferase enzyme and a blocked nucleotide to make sure that nucleotides are added one at a time. The approach reduces the use of toxic reagents and makes DNA synthesis easier.‍
• Kilobaser: The Kilobaser DNA and RNA Synthesizer also provides a benchtop method for synthesizing nucleic acids. The machine is capable of producing nucleic acids in just one hour and can generate total yields of 300 pmols.

Lease Essential Equipment for Prime Editing Experiments

Researchers inspired by CRISPR-Cas9 editing have pursued the development of new gene editing methods. Most recently, researchers have begun using PEs to fine-tune the editing process. 

As the protocol continues to be refined, researchers must use many kinds of equipment to assess the quality of their nucleic acids and the efficacy of their gene edits. From PCR thermocyclers to DNA synthesizers, researchers must also find affordable ways to own and operate the equipment. 

Excedr’s leasing program can help you acquire the lab equipment you need to employ prime editing and precisely edit genes. Speak with our team today to learn how we can support your lab’s needs for developing gene therapies.