Principles and Significance of Base Editing and Prime Editing

Alex Sobko^*
*Correspondence: Alex Sobko, Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel, Email:

Keywords

Crispr • Genome editing • Base editing • Prime editing

Introduction

Classical Crispr-Cas9 Editing

CRISPR-Cas9 relies on non-homologous end joining (NHEJ) or homology-directed repair (HDR) following generation of double-strand breaks by nucleases, such as Cas9. HDR allows precise editing by donor DNA. However, precision of this method is not perfect – sometimes, it leaves uncorrected sequences at double-strand breaks and can result in integration in off-target sites [1-17].

Base Editing

This method was originally developed by Alexis Komor, David Liu and colleagues at Liu lab. To date, two major classes of Base Editors have been described: cytosine base editors (CBEs), which change C•G base pairs to T•A (Komor, Kim, Packer, Zuris, & Liu, 2016) and adenosine base editors (ABEs), which convert A•T base pairs to G•C (Gaudelli et al. 2017).

Base editor is designed so that there is a fusion between deaminase and Cas9 nickase or catalytically inactive dead Cas9. Deaminase functions to deaminate cytosine (C) and adenine (A) bases. Other base conversions are not currently possible with this method, as well as targeted deletions, insertions or some other types of mutations.

This system is tethered to a single-stranded DNA (ssDNA)-modifying enzyme. Cas protein makes a complex with a user-programmed guide RNA (gRNA) and binds to a genomic locus (termed the “protospacer”) that is complementary to the sequence of the gRNA and harbors a protospaceradjacent motif (PAM, a short DNA sequence specific to the Cas protein used; Upon binding, the Cas protein locally denatures the target double-stranded DNA region to form an R-loop, exposing a small window ~5 nucleotides (nt) long of ssDNA (Jiang et al., 2016). Once bound, the ssDNA-modifying enzyme catalyzes the deamination of target nucleobases within this window. Subsequent DNA replication or repair of these modified base intermediates (uracil or inosine) results in permanent introduction of single-base substitutions. Base Editor system is being currently optimized by Alexis Komor lab at UC San Diego.

There are many advantages in this method. Sometimes it out-performs Prime Editing. Still there are certain limitations of this method that preclude applying base editing approach as a strategy to cure many genetic diseases. It does not allow certain genetic engineering applications, including molecular tagging.

Prime Editing

This method has been developed more recently in the laboratory of Professor David Liu and being optimized by David Liu’s and other laboratories. This method does not require double-stranded DNA breaks.

Prime Editors consist of Cas9 H840A nickase fused to M-MLV reverse transcriptase (RT) and prime editing guide RNA (pegRNA). pegRNA includes primer binding sequence (PBS) and RT template sequence with the desired edit.

Peg RNA 1) guides Cas9 H840A nickase to genomic DNA sequence to be edited; 2) determines the edited sequence to be inserted. Cas9 nicks one strand, RT fused to Cas9 copies edited sequence at the nicked target site, then cell uses DNA repair machinery to incorporate the edited sequence, making permanent change in genomic DNA.

The steps of editing by Prime Editor:

1) Upon target recognition, the protospacer adjacent motif (PAM)–containing strand is nicked by Cas9 H840A nickase that makes single-stranded break in one of the strands (non-target strand);

2) Primer binding sequence (PBS) of pegRNA hybridizes with 3’-region of the nicked strand;

3) Reverse transcription of the template target sequence (3’-flap edit);

4) Ligation and incorporation of newly synthesized sequence;

5) Edited sequence is incorporated into both strands by DNA repair.

Conclusion

The advantage of Prime Editor method is that it has broad range of applications including various types of mutations, insertions, deletions, combinations of several mutations. For example, insertions and deletions of up to 80 nucleotides could be useful for genetic engineering. It is also an advantage that this method does not involve generation of double-stranded breaks.

References

1. Koonin E. The Logic of Chance. The Nature and Origin of Biological Evolution. Pearson Education  (2012)
2. Jennifer Doudna, Kevin Doxzen and Martin Jinek. The Explorer’s Guide to Biology
3. Jennifer Doudna and Prashant Mali, A Laboratory Manual  (2016)
4. Deveau, Hélène, Josiane E. Garneau and Sylvain Moineau. "CRISPR/Cas system and its role in phage-bacteria interactions." Annu Rev Microbiol 64 (2010): 475-493.

Google Scholar, Crossref, Indexed at

5. Karginov, Fedor V. and Gregory J. Hannon. "The CRISPR system: small RNA-guided defense in bacteria and archaea." Mol Cell 37 (2010): 7-19.

Google Scholar, Crossref, Indexed at

6. Koonin, Eugene V. and Kira S. Makarova. "CRISPR-Cas: an adaptive immunity system in prokaryotes." "F1000 Biol Rep 1 (2009).

Google Scholar, Crossref, Indexed at

7. Van der Oost, John and Stan JJ Brouns. "RNAi: prokaryotes get in on the act." Cell 139 (2009): 863-865.

Google Scholar, Crossref, Indexed at

8. Doudna, Jennifer A. "The promise and challenge of therapeutic genome editing." Nature 578 (2020): 229-236.

Google Scholar, Crossref, Indexed at

9. Komor, Alexis C., Yongjoo B. Kim, Michael S. Packer and John A. Zuris, et al. "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage." Nature 533 (2016): 420-424.

Google Scholar, Crossref, Indexed at

10. Gaudelli, Nicole M., Alexis C. Komor, Holly A. Rees and Michael S. Packer, et al. "Programmable base editing of A• T to G• C in genomic DNA without DNA cleavage." Nature 551 (2017): 464-471.

Google Scholar, Crossref, Indexed at

11. Vasquez, Carlos A., Quinn T. Cowan and Alexis C. Komor. "Base Editing in Human Cells to Produce Single‐Nucleotide‐Variant Clonal Cell Lines." Curr Protoc Mol Biol 133 (2020): e129.

Google Scholar, Crossref, Indexed at

12. Mok, Beverly Y., Marcos H. de Moraes, Jun Zeng and Dustin E. Bosch, et al. "A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing." Nature 583 (2020): 631-637.

Google Scholar, Crossref, Indexed at

13. Zhang, Hao, Zijian Kang, Haiyi Gong and Da Xu, et al. "The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes." BioRxiv (2020).

Google Scholar, Crossref, Indexed at

14. Lapinaite, Audrone, Gavin J. Knott, Cody M. Palumbo and Enrique Lin-Shiao, et al. "DNA capture by a CRISPR-Cas9–guided adenine base editor." Science 369 (2020): 566-571.

Google Scholar, Crossref, Indexed at

15. Kim, Do Yon, Su Bin Moon, Jeong-Heon Ko and Yong-Sam Kim, et al. "Unbiased investigation of specificities of prime editing systems in human cells." Nucleic Acids Res 48 (2020): 10576-10589.

Google Scholar, Crossref, Indexed at

16. Gao, Pan, Qing Lyu, Amr R. Ghanam and Cicera R. Lazzarotto, et al. "Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression." Genome Biol 22 (2021): 1-21.

Google Scholar, Crossref, Indexed at

17. Geurts, Maarten H., Eyleen de Poel, Cayetano Pleguezuelos-Manzano and Rurika Oka, et al. "Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids." Life Sci Alliance 4 (2021).

Google Scholar, Crossref, Indexed at