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Gene modification nucleases such as CRISPR / Cas9 can efficiently create double-stranded breaks at a target locus in the genome. Often, double-stranded breaks are subsequently repaired by non-homologous end-junction mechanisms, leading to insertions and deletions (indel). One strategy, called homology-directed repair (HDR), is used to obtain gene knock-in patterns (targeted gene insertion).
These gene editing techniques are relatively efficient in single strand annealing, as is common with Streptococcus pyogenes Cas9 (spCas9). Alternatively, the efficiency rates for gene editing nucleases for double-stranded DNA-mediated knock-in via homologous recombination are low and require improvement.
To improve the efficiency of spCas9, previous strategies involved merging the key modules needed for the HDR path to spCas9. While this has generally increased successful double-helix knock-in rates, it is accompanied by off-target indel, thereby reducing the accuracy of the system. Additionally, the fusion modules are often large (more than 100 amino acids), which poses a challenge to packaging and transduction efficiencies via adeno-associated virus (AAV) delivery.
During homologous recombination, RAD51 protein / single-stranded DNA nucleoprotein strands perform homology search and strand swapping. A motif containing 36 amino acids encoded by BRCA2 Exon 27 (Brex27) helps stabilize these filaments during the process.
University of Michigan researchers hypothesized that fusion of Brex27 with spCas9 will lead to local enrichment of RAD51 and stabilization of RAD51 single-stranded DNA nucleoprotein strands at induced on-target and off-target rupture sites. from Cas9. They believed this would be useful for reducing undesirable in-target and out-of-target indel events and improve double-stranded knock-in rates.
To explore this possibility, the scientists constructed a variant of plasmid DNA that expresses miCas9 (pDNA) that could be compared to the more traditional pDNA that expresses spCas9. They began by comparing different nucleases for the efficiency of beating a green fluorescent protein (GFP) gene at the AAV 1 (AAVS1) integration site locus in human cells. They found that the use of miCas9 consistently improved the GFP knock-in rate by two to three times in multiple cell types.
Indel damage on the target is a potential gene editing problem, as it can prevent targeted corrections from occurring. Compared to other Cas9 variants, the miCas9 variant always achieved the lowest target indel rates, which were up to 75% lower than those of spCas9. The miCas9 variant also effectively reduced indel rates in all off-target loci examined by the researchers, compared to spCas9.
The researchers also demonstrated that Brex27 (mini motif) can be used as a plug-and-play module to improve the efficiency of other gene-editing nucleases (miHiFiCas9, miCas9mSA). The addition of Brex27 led to a further 76.7% reduction in off-target indel rates compared to spCas9.
The researchers explained that Brex27’s miniature size may be advantageous due to its suitability for AAV-mediated delivery (4.7kb limited packaging capacity) for gene therapies. The mini motif also offers several synergistic interactions with pre-existing gene editing nuclease systems.
It provides a “one small stone for three birds” tool in gene editing, the authors said.
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