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CRISPR Researchers Develop System to Catalog Wide Range of Gene Editing Outcomes

NEW YORK – Researchers from the Gene Editing Institute at the Christiana Care Health System have developed a CRISPR-based system to provide a more accurate and unbiased view of the outcomes of gene editing and DNA repair.

As CRISPR-Cas systems advance toward the clinic, it is important to identify all the outcomes of gene editing activity in human cells. In their study, published on Friday in the Nature journal Communications Biology, the researchers reported that they used an in vitro gene editing system in which a CRISPR-Cas complex provided the double-stranded cleavage and a mammalian cell-free extract provided the enzymatic activity to promote non-homologous end joining (NHEJ), micro-homology mediated end joining (MMEJ), and homology-directed repair (HDR). They then recorded the broad spectrum of gene editing outcomes using Cas9 and Cas12a in combination with single-stranded donor templates.

"These data will enable a more educated choice surrounding the types and amounts of genetic engineering tools to employ for the treatment of a genetic disease," the authors wrote. "For example, while high levels of single point mutation repair have been widely reported, the accompanying secondary genetic outcomes have not been completely described. Thus, a global view of gene editing activity is likely to be foundational as to whether to move forward with clinical application or not."

For this study, the researchers chose to target a plasmid containing the beta galactosidase gene, lacZ. Gene editing of lacZ shows as an alteration in colony color from blue to white. The two single-stranded DNA oligonucleotides that served as the donor fragments were named 1364-NS to designate a donor fragment that was complementary to the sense strand and 1364-S, which was complementary to the nonsense strand. In the absence of donor DNA, the researchers expected some form of NHEJ or MMEJ to reconnect the linear plasmid template. In the presence of donor DNA, they expected some form of HDR to take place, leading to precise or error-prone repair.

The first combination included a CRISPR-Cas9 complex and a single-stranded donor template complementary to the nonsense strand, 1364-S. From the 37 colonies sequenced, only two were found to contain a precisely inserted NotI site, which indicated precise HDR activity. The remaining colonies within the population exhibited small indels ranging from minus-5 bases to plus-1 base.

Next, gene editing reactions containing CRISPR-Cas9 and a single-stranded donor template complementary to the sense strand were tested. From this reaction, 34 bacterial colonies were analyzed, and indel formation was once again found to dominate the population of isolated molecules. The researchers also found that HDR activity was elevated threefold when the 1364-NS donor template was coupled to CRISPR-Cas9.

In the following experiments, they used Cas12a with the 1364-S donor template. While most products harbored deletions ranging from minus-7 to minus-13 bases, the researchers also found small and large insertions ranging from plus-2 to plus-26. Insertions and deletions of this size were not observed in either reaction catalyzed by Cas9.

"Six of the 32 colonies analyzed contained genetically modified templates with precise repair, thus specific HDR activity was found in 18.8 percent of the samples," the authors wrote. "It is important to note that this 18.8 percent compares favorably to the 5.4 percent precise HDR generated in Cas9 reactions using the 1364-S donor template."

In the final combination of Cas12a and the 1364-NS donor template, HDR was "highly enhanced," with precise repair found in more than 65 percent of the colonies, the researchers said. The remaining products showed a wide range of indels with deletions ranging from minus-4 to minus-28 bases and insertions from plus-1 to plus-17 bases.

"Not only is this the highest level of homology-directed repair observed in our study, this combination also generates the widest array of error-prone genetic modifications," the authors concluded. "From these data, we conclude the combination of CRISPR-Cas12a and the 1364-NS template exhibit the highest degree of precise HDR driven by gene-editing activity within this system."

The researchers noted that one important aspect of this in vitro system is that it enabled them to visualize the wide array of genetic modifications created through the process of CRISPR-based gene editing in a straightforward way. "Such information forms the basis for determining risk-benefit decisions surrounding the effectiveness of genetic engineering tools to treat human disease," they added.

The team also noted that it is developing a comparable in vivo system that recapitulates a simple and verifiable readout. However, transfection efficiencies and transport of the crRNA/Cas complex to the nucleus could add variability to all cell-based systems.