The 2020 Nobel Prize in Chemistry was awarded to Drs. Emmanuelle Charpentier and Jennifer Doudna for their contributions in the development of CRISPR/Cas9. This transformative technology has revolutionized the gene targeting field. Yet to realize its clinical potential to cure genetic disease, many issues remain to be addressed. For example, large size gene knock-in efficacy rate is still low. With respect to safety, substantial off-target editing events occur, raising concerns of tumorigenicity associated with the technology. Equally alarming, the on-target insertion or deletion (indel) rates are often higher than those of the desired precise correction. Other challenges beyond the level of DNAs include the lack of a safe and effective in vivo delivery system as well as immunogenicity issues. Each of these will need to be adequately addressed to satisfy the tolerability, safety, and efficacy for the targeted clinical development for genetically defined diseases.
In our current report, to overcome efficacy and safety issues associated with Cas9 at the DNA level, we looked into the DNA repair mechanisms. Gene editing nucleases are efficient in generating DNA double-stranded breaks, which are repaired by either the error-prone non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway. The balance between NHEJ and HDR determines the outcome of gene editing applications, for which HDR is preferred in precise gene editing applications.
In 2016, our group reported the beneficial effects of a RAD51 agonist, RS-1, in Cas9 or TALEN mediated large size gene knock-in experiments (1). Following this rationale, in our current work (2), we engineered the spCas9 protein by fusing a thirty-six amino acid long peptide encoded by BRCA2 Exon 27 (Brex27), which has been reported to bind RAD51 to enhance HDR (3). We name this new variant meticulous integration Cas9 (miCas9), to reflect its extraordinary capacity to enable “maximum integration” yet with “minimal indels”, as well as to recognize its development at the University of Michigan.
In comparison to spCas9, miCas9 satisfactorily addresses the aforementioned efficacy and safety deficiencies: it increases large size gene knock-in rates by multiple folds; it reduces off-target indel rates; and importantly it reduces undesirable on-target indel rates, the first nuclease that can achieve this to the best of our knowledge.
It is further demonstrated that the fusion motif, Brex27, can be used as a plug-and-play module to improve other gene editing nucleases. This may benefit many “new” or “understudied” nucleases, to which a simple fusion of Brex27 is expected to improve both their efficacy and safety performances.
Lastly, we want to point out that the small size of Brex27 is advantageous. Unlike some other fusion motifs to Cas9, adding Brex27 only increases the size of spCas9 by 2%. This is an important aspect because when it comes to in vivo delivery of therapeutic biologics, “size matters” and any “room saving” helps. In this regard, Brex27 is the smallest effective HDR promoting motif to date.
In summary, we show that a rationally designed Cas9 variant miCas9 possesses a unique combination of desirable features including improving knock-in rates, reducing undesirable off-target events, and reducing undesirable on-target indel events, providing a “one small stone for three birds” tool in gene editing (Figure). miCas9 and the Brex27 module may find broad applications in gene editing research and therapeutics.
Figure 1. miCas9 in gene editing applications. Ds-KI: double-stranded donor mediated knock-in. Indel: insertions or deletions.
1: RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat Commun, 7:10548. doi: 10.1038/ncomms10548 (2016)
2: MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits. Nat Commun, 2020. https://www.nature.com/articles/s41467-020-19842-2
3: Interaction with the BRCA2 C terminus protects RAD51-DNA filaments from disassembly by BRC repeats. Nature structural & molecular biology, 14, 475-483 (2007).