Prime Editing Vs. CRISPR-Cas9

The most well-known gene editing technique is CRISPR-Cas9 but a new player in the game is emerging. Although prime editing is still in its early stages, its potential uses are wide-ranging. Below we take a short, top-line look at both editing techniques before going on to summarize their differences.

CRISPR-Cas9

CRISPR-Cas9 is a genome editing technique first used successfully in 2013. The acronym CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats; while Cas9 stands for CRISPR-associated protein 9.

How Does CRISPR-Cas9 work?

The CRISPR-Cas9 system basically mimics a naturally occurring system in cells. In bacterial cells, snippets of DNA are taken from invading viruses and are then used to create DNA sequences called CRISPR arrays. In short, these arrays help the cells to ‘remember’ those viruses or other similar ones. Therefore, if a similar virus attacks the cell again, it produces RNA segments from the CRISPR arrays to specifically target the viral DNA. Then Cas9 proteins are used to cut the DNA.

In laboratory settings, a small piece of RNA containing a guide sequence is created and then binds to a specific section of DNA. It also binds with the Cas9 enzyme, cutting DNA at the target location. The cell’s internal repair machinery is then used to make changes to the DNA, whether that is removing sections or incorporating a new sequence.

Advantages of CRISPR-Cas9

The CRISPR-Cas9 system has applications in a wide variety of organisms, including both humans and plants. It is a faster as well as cheaper method for genomic editing than prime editing is and it generally achieves accurate results. A plethora of research has been concluded using CRISPR-Cas9, further refining the technique and embedding it into scientific usage.

Disadvantages of CRISPR-Cas9

There are disadvantages to genetic editing in general. As demonstrated by the “CRISPR babies” experiments, the human body is made up of a huge number of cells that need to be targeted. Leaving a small number of non-edited cells could still cause disease progression or other mosaic effects. Off-target mutations (where editing occurs at a point that is not actively targeted) can also cause problems; depending on where the mutations are located.

Prime Editing

The first thing to point out is that prime editing is a relatively new technique, only published in late 2019. Since it is a targeted genetic editing technique it can focus on specific DNA sites to replace genetic information. Therefore, it can facilitate insertions, deletions and conversions without breaking both strands of DNA or using DNA templates.

Components Of Prime Editing

Prime editing has three major components:

• A prime editing guide RNA (pegRNA) identifies the sequence of bases to be edited and then encodes the new sequence to replace the target. pegRNA is made up of an extended single guide RNA (sgRNA) with a primer binding site (PBS) and a reverse transcriptase template sequence.

During prime editing, the PBS allows the DNA strand to hybridise to the pegRNA, while the reverse transcriptase template sequence serves as the template for the edited genetic sequence.

• A fusion protein made up of two enzymes:

The first enzyme is a Cas9 H840A nickase, which specifically nicks a single strand (“nickase”). The Cas9 enzyme section of the nickase has a substitution in it to cause a single-strand break, rather than a double-stranded cut.

The second enzyme is an M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase which synthesises DNA from a template of single-stranded RNA.

• Another second sgRNA that directs the fusion protein to the DNA strand to be edited.

How Does Prime Editing Work?

Prime editing works by introducing the pegRNA and fusion protein to the target cell, then once inside the cell, the fusion protein nicks the cell’s DNA at the target sequence, initiating reverse transcription of the template sequence found in the pegRNA. This then creates an edited strand and an unedited strand of DNA. First, the unedited strand is removed then the newly edited strand is annealed back to form double-stranded DNA.

This then creates a mismatch in the base pairs between the two strands, which can have two outcomes. On one hand, using the mismatch repair mechanism of the cell, the edited strand is copied to the complementary strand, which incorporates the edited strand into the DNA of the cell.  Conversely, the original base sequence is reincorporated into the edited strand from the complementary strand, removing the edit from the DNA of the cell.

Advantages of Prime Editing

Since prime editing uses the cells’ intrinsic DNA mismatch system to incorporate changes in the nucleotide order into the cell, this reduces the number of unwanted or random by-products of genome editing.  There are potentially fewer off-target effects than with the CRISPR-Cas9 system; potentially marking it as a future technology for human therapeutic uses.

Prime editing is also highly precise and can be used flexibly along the DNA genome due to the single-stranded pegRNA, which allows for all types of insertions into the gene sequence, such as substituting or transitioning bases.

Disadvantages of Prime Editing

Undeniably, one notable disadvantage to prime editing is its relative youth – it came to the forefront in late 2019. It is still at proof-of-concept stage, with no therapeutic uses as of yet.

Overall

CRISPR-Cas9 is an older and much more embedded technique than prime editing. The two systems also differ in how they ‘cut’ into DNA strands. CRISPR-Cas9 removes both strands at the place they make an edit, while prime editing only nicks one of the two strands of DNA.

While CRISPR has been used in many settings, prime editing is still at an early stage of proof of principle. Nevertheless, it will be interesting to see what role prime editing plays in the future of genomic editing.

See Also:

Genome editing: CRISPR-Cas9

Search-and-replace genome editing without double-strand breaks or donor DNA (Nature)

Bioinformatics and the Pharmaceutical Industry

The Future of Genomics

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