Genome Editing without Double-Stranded DNA Breaks

Bianca Kojin, Ph.D. is a Post-doctoral Research Associate in the Department of Entomology,exas A&M University and is interested in the genetic transmission of engineered transgenes. More About the Author

Komor et al. reviewed two strategies for genome editing that do not rely on the Double Strand Breaks (DSB) in a recent issue ACS Chemical Biology. The authors specifically address oligonucleotide-directed mutagenesis and base pairing.

Possibly the most harmful type of lesion the DNA may undergo is the breakage of the two strands (DSB) and the repair of this damage is of great importance to maintain genome stability. The repair may occur through a few different but complementary pathways like homology directed repair (HDR), nonhomologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA). DSB are frequently repair by these pathways and in a less extent by HDR; however, they often generate stochastic insertions and deletions (indels).

The general strategy of oligo-directed mutagenesis (ODM).  This image is from Bertoni (2014) https://doi.org/10.3389/fphys.2014.00148

Unfortunately, genome modification using HDR remains inefficient, leading researchers to look for alternative strategies that do not involve DSB.

The authors provide an overview of oligonucleotide-directed mutagenesis stating this methodology as a powerful and enabling molecular cloning strategy. This method employs the use of oligonucleotides homologous to a region of interest that encompasses a desired mutation enabling DNA modification in a locus-specific manner. This strategy has been extensively used in plant genome editing like tobacco, corn, rice, wheat and rapeseed, however this technique has several limitations compromising its efficiency thus unsuitable for some research applications and most therapeutic uses.

Another genome editing methodology explored by the authors is base editing. On that section of the paper, Komor et al. explain how they engineered the Cas9 protein to achieve precise, irreversible conversion of one base pair to another in a programmable manner without generating DSB; they named the modified protein BE3. They highlighted how the development of BE3 induced branches of distinct improvements to overcome the inherent limitations of the system to better suit specific experiments.

Image result for base editing be3

Overview of how BEs (Base Editors) function. This image is from another recent review of base editing by Lee et al (2017) DOI: 10.12972/jabng.20170010

In looking to the future, Komor et al. considered exploring RNA editing rather than DNA editing. They mentioned that in the dearth of naturally DNA-modifying catalysts, RNA has a variety of enzymatic options for modulating its biological activity with a temporal resolution without the risk of permanently modifying the genome therefore a safer option for gene therapies, for example.

In conclusion, methods such as base editing that exclude the need for cytotoxic and mutagenic DSBs offer safer, controlled and precise genome editing alternatives to current DSB-based strategies and insect scientists are likely to find them of great interest.

Alexis C. Komor, Ahmed H. Badran, and David R. Liu (2018), Editing the Genome Without Double-Stranded DNA Breaks, ACS Chemical Biology 2018 13 (2), 383-388, DOI: 10.1021/acschembio.7b00710

 

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