Ideation, Iteration, Innovation At the Frontier of Genome Editing (pt. 2 of 3)

Prime Editing is the latest breakthrough in genome editing and adds a potential tool to the CRISPR-Cas9 toolkit

From an article on gene editing from The Conversation

Contents

1. Introducing to the World: Prime Editing (pt. 1)
2. DNA and RNA: A Refresher (pt. 1)
3. Editing The Genome: Enter CRISPR-Cas9 (pt. 1)
4. A Complementary Technique to CRISPR-Cas9: Base Editing (pt. 2)
5. An Evolved Version of Base Editing: Prime Editing (pt. 2)
6. Why This Is Groundbreaking: Word Processors (pt. 3)
7. A Final Word On Ideation, Iteration and Innovation (pt. 3)
8. 🔑 Takeaways (pt. 3)

Continued from pt. 1. Or skip ahead to pt. 3.

4. A Complementary Technique to CRISPR-Cas9: Base Editing

or as the Liu Group titled their paper specifically:
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Because of the limitations listed above of ZFNs, TALENs and CRISPR-Cas9 (all require DSBs and HDR), people started looking for alternatives that do not rely on homology-directed repair.

One recent successful attempt to trigger genome modification without a full break of the DNA strand has been called ‘base editing.’ This technique was also explored and refined in Liu’s lab, with one of their papers published in 2017.

Base editing is achieved with the help of specific DNA base editors — either cytosine base editors (CBEs) or adenine base editors (ABEs). DNA base editors are comprised of:

• A modified Cas9 nickase that does not create a double strand break but can unfold the double helix and using the guide RNA to indicate a single strand DNA cut site that the Cas9 then cuts.
• An engineered-base modification enzyme (a deaminase) that remove a specific component of one of the bases, turning it into the chemical equivalent of the desired base. The cell’s natural repair and replication process will then change this into the desired base and then will change the complementary base it’s linked to on the other chain:

Here’s an example of a CBE and how it works, courtesy of the Addgene blog

Base Editors have been shown to efficiently and precisely edit point mutations in DNA relative to CRISPR-Cas9.

Limitations:

1. Base Editing is limited to four transition mutations (C → T, G → A, A → G, T → C).
2. Genome modifications requiring deletion or insertion are not possible.
3. Off-target modifications — although with less frequency than CRISPR Cas9 — still do happen.

5. An Evolved Version of Base Editing: Prime Editing

or as the Liu Group titled their paper specifically:
Search-and-replace genome editing without double-strand breaks or donor DNA

The limitation of base editing was an important trigger for the ideation, iteration, and innovation that led to prime editing.

Instead of connecting a modified Cas9 to a base modification enzyme, Professor Liu and co reasoned, what if we could instead combine the targeting abilities of Cas9 with the ability to directly write new strings of DNA that can replace the original DNA sequence? In order to achieve this, they fused a modified reverse transcriptase to Cas9 (called the Prime Editor) and engineered a new prime editing guide RNA (pegRNA) comprised of 1) a spacer sequence for targeting that would bind to complementary protospacer sequence in DNA like with classic CRISPR-Cas9 techniques and 2) a sequence complementary to the desired new DNA sequence serving the same role as repair templates in classic CRISPR-Cas9 genome editing techniques.

From the 2019 paper published in Nature linked above

Then to transfer information from pegRNAs to target DNA, they hypothesized and validated that genomic DNA nicked at the target site (to expose a 3’-hydroxyl group) could be used to prime the reverse transcription of the edit encoding sequence of the pegRNA directly into the target site. After the PAM strand is nicked, the primer binding site of pegRNA base pairs or hybridizes with the this strand. Connected to the the primer binding site on the RNA is a reverse transcriptase (RT) template sequence containing the desired edits that the reverse transcriptase uses to polymerize DNA directly into the target site.

This step results in a branched intermediate with two redundant single-stranded DNA flaps on the PAM strand:

• a 5’ flap containing the unedited DNA sequence and
• a 3’ flap containing the edited sequence synthesized from the pegRNA.

They chose this because although hybridizing of the complementary 5’ flap to the unedited (non-PAM) strand is favored thermodynamically, 5’ flaps are the preferred substrate on which endonucleases like FEN1 operate to excise 5’ flaps generated during lagging-strand DNA synthesis and long-patch base excision repair. Also, 5’ exconucleases like EXO1 can be used. Thus, they reasoned that the preferential 5’ flap excision and 3’ flap ligation could drive incorporation of the edited DNA strand, creating heteroduplex DNA containing an edited strand and an unedited strand. DNA repair to resolve this issue by copying the information in the edited strand to the complementary strand and would thus. Based on work with base editing to resolve heteroduplex DNA, they hypothesized that nicking the non-edited DNA strand could bias DNA repair to preferentially replace the non-edited strand as desired.

From the 2019 paper published in Nature linked above

The authors of the 2019 piece tested prime editing in four human cell lines in vitro, and specifically tested the ability to modify the primary genetic causes of sickle cell disease (requiring a transversion) and Tay-Sachs (requiring a deletion), and to install protective measures against prions (a different tranversion). Further, they tested in post-mitotic mouse neurons.

PrimeDesign has recently launched a tool that allows you to design your own pegRNA for Prime Editing. You can play with it for yourself — for instance, I looked at the single-base mutation associated with sickle-cell anemia, and you can then use that specific substitution to design a desired pegRNA in order to help make the desired edit. For instance, based on the sickle-cell substitution required, you would input: ATGGTGCACCTGACTCCTG(A/T)GGAGAAGTCTGCCGTTACT.

When compared to Base Editing, Prime Editing has shown the following advantages in vitro:

1. It can do insertions, deletions, and all 12 types of point mutations.
2. It may potentially be able to address up to 88.7% of known mutations, whereas Base Editing’s maximum potential is up to 50% of known mutations.
3. When very selective installation of the CG to TA edit is desired at any position or combination of positions. This is because:

• On the one hand, when multiple cytosines or adenines are present and bystander edits are undesirable, or when PAMs that position target nucleotides for base editing are not available (e.g., a PAM sequence that exists approximately 15 bases from target site), prime editors offer substantial advantages.
• On the other hand, when a single target nucleotide is present within the base editing window, or when bystander edits are acceptable, current base editors are typically more efficient and generate fewer indels than prime editors. Thus when comparing base and prime editors for targeted genome editing that both are capable of (e.g., transition mutations), there are nuanced considerations that will lead to deciding which is the better choice.

When compared to original CRISPR-Cas9 editing, Prime Editing has shown the following advantages in vitro:

1. It does not require DSBs or donor templates.
2. It can edit in non-dividing cells in vitro (tested on post-mitotic mouse cortical neurons).
3. It has higher or similar efficiency and fewer byproducts.
4. Editing versatility with ability to edit with equal efficiency whether the desired changes are 5 or 50 base pairs from the nick. Additionally, ability to insert up to 44 base pairs or delete up to 80 base pairs.

Limitations:

1. The combined Cas9 protein and reverse transcriptase is large, and may potentially limit delivery.
2. There is wide variance in efficiency and indel rates. Even though it has been shown to work in nondividing cells, the efficiency rates are low (only 7.1%) — leading to more areas for future exploration. It also must work in non-dividing human cells.
3. Will Prime Editing stand up in vivo and clinical trials on humans? Prime Editing does not have the same length of history of research and development relative to original CRISPR-Cas9 (since 2012) and base editing (since 2016). This article from 2016 captures a review of the extensive work, modifications and ecosystem of tools built around CRISPR-Cas9. It has taken 7–8 years before the initial research on CRISPR-Cas9 has been tested in real tests on humans for genetic blindness, severe hemoglobinopathies, and cancer (and even then, still with a very limited, small number of patients). A lot more research and development is required to see if initially promising results will work in real life.

Continued in pt. 3. Or return to pt. 1.