NEW YORK — Two research groups have tweaked gene-editing tools to improve their editing efficiencies or their ease of delivery, respectively.
In papers appearing in Nature Biotechnology on Monday, two research groups tackled the problems of editing mitochondrial DNA with a base editor and delivering bulky prime editors. Both base editors and prime editors can be applied to introduce precise changes in a target DNA sequence, but both have drawbacks.
In one study, a Broad Institute-led team updated an all-protein cytosine base editor they developed, dubbed DdCBE. This base editor relies on TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) and allows for targeted C-G to T-A conversions within mitochondrial DNA.
But they found that the efficiency of DdCBE varied based on where the targeted C fell. To boost this efficiency, the researchers applied phage-assisted noncontinuous and continuous evolution approaches to develop DddA variants.
As they reported in Nature Biotechnology, two evolved variants — DddA6 and DddA11 — led to about a 4.3-fold improvement in mtDNA base editing at TC targets, as compared to the wild-type DddA. Additionally, DddA11 also improved bulk editing at AC and CC targets to about 15 percent to 30 percent average editing efficiency, as compared to less than 10 percent with DddA11. These variants further have similar high on-target to off-target editing ratios.
The Broad's David Liu and colleagues thus recommended that if scientists have TC targets, they should begin by using the canonical DdCBE, but if efficiency is low, to switch to DddA6. For non-TC targets, though, they suggested that scientists rely on DddA11 as it allows for more efficient editing of AC and CC targets. They cautioned that because DddA11 is active at TC, AC, and CC sites, bystander editing is more likely.
"Additional protein evolution or engineering could further improve the editing efficiency of DddA variants, especially at GC targets," Liu and colleagues wrote in their paper.
Meanwhile, researchers from the University of Massachusetts Chan Medical School have developed a split prime editor in which the Cas9 nickase is untethered from the reverse transcriptase. The use of prime editors, the researchers noted, has been affected by their bulky size and complexity, but splitting them into smaller components may enable them to be more easily delivered.
As they report in Nature Biotechnology, the researchers compared their split prime editor to unsplit versions of the same editor to find that it had similar editing efficiencies and did not lead to increased byproduct indels. When they further assessed the approach in vivo in mouse livers, the researchers noted that a split prime editor targeting a codon in the beta-catenin-encoding gene — the disruption of which drives tumor formation — led to an increased number of tumors per mouse, indicating that the approach is precise and efficient in vivo, according to the researchers.
This system can further be packaged into adeno-associated viral (AAV) vectors. The researchers were able to deliver a split primer system aimed at correcting a loss-of-function G-to-A mutation in the fumarylacetoacetate hydrolase gene, associated with tyrosinemia type I, in a mouse model using two different AAV vectors. However, they noted a low editing efficiency.
They further modularized the system by splitting the prime editing guide RNA in two: a sgRNA and a separate RT template (RTT) and the primer-binding site (PBS) sequence. They additionally engineered RTT-PBS to circularize to improve its stability, dubbing it a prime editing template RNA (petRNA).
"By separating the PBS-RTT from the editing guide, petRNAs may not only provide superior stability but also enable combinatorial or tiling approaches to identify highly efficient editing designs," UMass's Erik Sontheimer and colleagues wrote, adding that "modular PEs promise to facilitate effective and versatile in vivo delivery of PE for precise genome editing."