CRISPR technique allows for gene splicing without introducing foreign DNA bits

October 23, 2015 by Bob Yirka, Phys.org report

A depiction of the double helical structure of DNA. Its four coding units (A, T, C, G) are color-coded in pink, orange, purple and yellow. Credit: NHGRI
(Phys.org)—A team of researchers affiliated with several institutions in South Korea has found a way to introduce an enzyme into a cell using the CRISPR technique without having to use a bacterial carrier—the result, the group claims, is a possible way to bypass laws meant to outlaw genetically modified organisms (GMOs) from being used in the human food chain. In their paper published in the journal Nature, the team outlines their technique and explains why it works.

CRISPR is, of course, a means of guiding a gene editing platform to a particular place within a using RNA—the idea is to remove parts of a genome that are undesirable or to replace them with parts that are, to produce a plant or animal with desirable characteristics. The traditional approach is to get a protein called Cas9 working in a plant cell and then to use a bacterial agent to carry the result to a desired genome part using RNA strands. The problem with this is that sometimes bits of the bacteria DNA can end up in the final product or the Cas9 itself winding up in the genome of the final plant.

While technically speaking these problems do not prevent the resulting plant from behaving as desired, they do cause the plant to be classified as a GMO, which some people have taken exception to—so much so that laws have been passed preventing their use in consumer food products. In this new work, the team has developed a modified CRISPR technique that does not rely on bacterial agents and in fact do not require gene shuttling at all. Instead, they have found a way to assemble Cas9 with RNA outside of a plant and then use solvents to get them inside and to the genome they are meant to modify. The result is a plant that does not get any new DNA. And that, the team suggests, may be enough to keep foods that are grown using the technique from being banned under GMO laws.

It is not clear at this time if this new technique or others like it that do not involve the transfer of DNA in developing new , will still have their result be deemed as GMOs or not—that will be up to public perception and legislators reacting to them.

Explore further: New techniques improve specificity of CRISPR/Cas9 genome editing tools

More information: Je Wook Woo et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins, Nature Biotechnology (2015). DOI: 10.1038/nbt.3389

Abstract
Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.

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