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Paper Outlines Scheme to Create Transmissible CRISPR/Cas9 Gene Edits


NEW YORK (GenomeWeb) – A research duo from the University of California at San Diego has developed an approach for making "contagious" genome edits that occur at the same site in both partners of a chromosome pair and persist in subsequent generations.

This so-called "mutagenic chain reaction" (MCR), which was described online today in Science, makes it possible to introduce a recessive mutation at a given site using CRISPR/Cas9 — components of the bacterial immune system that are increasingly being relied on for genome editing applications.

But the genome editing doesn't end there. The MCR construct itself then converts the same site in the corresponding chromosome in a diploid organism with the help of homology-directed repair, the study's authors explained, making it possible to swap out a heterozygous mutation for a homozygous change within a single generation.

Because MCR alterations have the ability to convert matching sequences at the same target site, the engineered alterations aren't thinned out by wild type alleles segregating independently during the breeding process, the study's authors explained. Instead, the engineered sequences routinely replace the original sequences at that site.

"The big difference between this mutation and a traditional mutation is that traditional mutations, you can say, are passive," senior author Ethan Bier, a cell and developmental biology researcher at UCSD, told GenomeWeb.

On the other hand, an alteration that's added in with the help of MCR "spreads autocatalytically from one chromosome to the other," he explained, "and converts the germline to 100 percent mutant."

The combination of CRISPR (clustered regularly interspaced short palindromic repeat) elements and Cas9 nuclease enzymes — deployed against specific target sequences using guide RNAs — have been used for a wide range of genome editing applications.

For instance, a team led by investigators at the Broad Institute published a study in Cell earlier this month that used the CRISPR/Cas9 system as the basis for a mouse knockout screen for genes involved in cancer development and metastasis.

Others have applied CRISPR/Cas9-based gene editing to studies of aging and age-related disease in the short-lived African turquoise killifish. And still others have proposed using CRISPR-Cas9 to fine-tune members of microbial communities found in and on the human body.

The UCSD researchers began exploring strategies for introducing genetic modifications in a rapid and transmissible manner. To achieve this, the team made seemingly modest tweaks to the CRISPR/Cas9 gene editing cassette, creating a construct that contains the Cas9 nuclease enzyme gene, guide RNA targeting a sequence of interest, and sequences homologous to those found on either side of the targeted cut site.

Each of the components needs to be carefully designed so that MCR will proceed efficiently, noted first author Valentino Gantz, who developed the approach while pursuing his PhD in Bier's UCSD lab.

"[Gantz's] very clever idea was to have the place where the homology arms come up to the edge of the internal cassette that has the Cas9 and the guide RNA," Bier said.

For their current study, the researchers used MCR constructs with homology arms spanning roughly one kilobase of sequence on either side of the cut site. Gantz noted that results from past studies hint that there would be no efficiency benefit associated with stretching arm lengths out further than that.

"This is relevant only during the first insertion of the cassette," he said. "Once the cassette is inserted, the homology arm is pretty much the whole chromosome."

When they tested MCR in the fruit fly Drosophila melanogaster, focusing on the so-called "yellow" locus on the X chromosome, the researchers found they could introduce heterozygous tweaks to a fruit fly embryo that spread to the same target site on the complementary chromosome.

The resulting homozygous changes not only carried over into germline and somatic cells in adult flies produced by MCR, but also appeared to influence the phenotypes of their offspring.

After injecting wild type fruit fly embryos with the MCR construct, the team crossed these flies with wild type flies, producing far more yellow offspring than would be predicted by straightforward Mendelian inheritance.

When the female MCR offspring were again crossed to wild type flies, the researchers found that the yellow phenotype was transmitted with more than 95 percent efficiency.

The yellow coloring was slightly less common in fruit flies generated by crossing MCR male offspring with wild type females. There, all but one of the 41 females in the second generation had the MCR-related yellow coloring, while all 50 male offspring had wild type coloring.

The team used PCR and targeted sequencing to verify that the color changes had, indeed, stemmed from the MCR cassette and to search for explanations when alleles at the locus weren't converted by MCR.

The researchers noted that the same general approach is expected to work for any site that can already be targeted for CRISPR/Cas9 gene editing, regardless of the features of composition of sequences comprising the homology arms.

"It should work, in principle, for anywhere you can direct [guide RNA], which is just about anywhere," Bier said.

The researchers are confident that MCR can act on the germline in ways that are carried forward in germline and somatic cells during development. But it remains to be seen whether the same type of conversion can occur directly in the somatic cells and, if so, whether specific tissues are more apt to MCR conversion than others.

"We know that the animals that carry a construct like this have a full body mutant phenotype," Bier explained. "But what we don't know at the moment is whether the somatic cells are homozygous for the MCR element — in which case, it would be working in somatic cells exactly as it is in germ cells."

"Alternatively," he noted, "it could be that you have one copy that's the MCR [in somatic cells] and the other copy that's just been cut by the Cas9-gRNA and has been repaired by non-homologous end joining to give you a standard mutation."

The team has not yet explored the possibility of performing MCR directly in adults fruit flies, which Bier said would likely require additional tweaks to the method to conditionally activate the Cas9 nuclease.

Based on their findings so far, the researchers speculated that MCR may ultimately provide an avenue for inserting transgenes in plant or insect pests or deterring the spread of disease-causing pathogens by vector species.

The researchers are looking at the possibility of applying similar strategies to the mosquito, for example, with an eye to curtailing processes involved in the spread of malaria-causing parasites.

They noted that it may be beneficial to look at the possibility of applying MCR to other model organisms — particularly since genetic tools are lacking for many sequenced animals — or even in the context of human disease.

If scientists get a clear idea of how such constructs behave in mammals, for instance, MCR could theoretically be used to target deleterious viruses or cancer DNA. Still, Bier noted that that would "require many, many more steps of development than the mosquito application, which is one that can be done immediately based on the existing technology."

One area that will likely need to be addressed when moving to more complex genomes, for instance, is off-target CRISPR/Cas9 activity. Whereas off-target events seem to be relatively rare in the modest-sized fruit fly genome, Gantz said, they may be more of a concern when dealing with larger mammalian genomes.

Likewise, Bier pointed out that it will be important to ensure that the MCR alterations aren't capable of acting as mutagens after multiple generations. If they do, additional steps will likely be needed to create a system for temporarily applying MCR and then removing the cassette, he predicted.

Despite their optimism about the approach, Bier and Gantz also cautioned that continued discussion is needed to hammer out appropriate applications of MCR and other genome editing approaches.

The team noted that strong containment measures are warranted to ensure that organisms altered with MCR aren't involuntarily released into the wild. Similarly, they argued that researchers may want to physically separate the Cas9 from the guide RNA source when it's not imperative to use the full MCR construct.

"In addition to … positive applications of MCR technology, we are also keenly aware of the substantial risks associated with this highly invasive method, since the failure to take stringent precautions could lead to the unintentional release of MRC organisms in to the environment," they wrote. "We therefore concur with others that a dialogue on this topic should become an immediate high-priority issue."