Scientists optimise genome editing procedures

Tuesday, 14 February, 2023

Scientists optimise genome editing procedures

In the course of optimising key procedures of genome editing, researchers from Heidelberg University have succeeded in improving the efficiency of molecular genetic methods such as CRISPR/Cas9 and related systems, and in broadening their areas of application.

Led by Prof Dr Joachim Wittbrodt, the scientists fine-tuned the genome editing tools to enable effective genetic screening for modelling specific gene mutations, as well as the modification of initially inaccessible DNA sequences. This opens up extensive new areas of work in basic research and therapeutic applications.

The most common methods of genome editing include the ‘gene scissors’ CRISPR/Cas9 and its variants, known as base editors. In both cases, enzymes have to be transported into the nucleus of the target cell. Upon arrival, the CRISPR/Cas9 system cuts the DNA at specific sites, which causes a double strand break; new DNA segments can then be inserted at that site. Base editors use a similar molecular mechanism but they do not cut the DNA double strand; instead, an enzyme coupled with the Cas9 protein performs a targeted exchange of nucleotides — the basic building blocks of the genome. In three successive studies, the Heidelberg researchers succeeded in enhancing the efficiency and applicability of these methods.

A challenge when using CRISPR/Cas9 involves the efficient delivery of the required Cas9 enzymes to the nucleus, with Heidelberg researcher Dr Tinatini Tavhelidse-Suck explaining, “The cell has an elaborate ‘bouncer’ mechanism — it distinguishes between proteins that are allowed to translocate into the nucleus and those that are supposed to stay in the cytoplasm.” Access is enabled here by a tag made up of a few amino acids that functions like an admission ticket.

The scientists have now come up with a kind of ‘VIP admission ticket’ that lets enzymes equipped with it into the nucleus very quickly. Described in the journal eLife, they have named it “high efficiency-tag”, or “hei-tag” for short.

“Other proteins that have to penetrate the cell nucleus are also more successful with hei-tag,” said Dr Thomas Thumberger, a researcher at Heidelberg’s Centre for Organismal Studies (COS). The team showed that Cas9 in connection with the hei-tag ticket can enable highly efficient, targeted genome alterations not only in the model organism medaka, also known as the Japanese rice fish, but also in mammalian cell cultures and mouse embryos.

In a further study, also published in eLife, the Heidelberg scientists showed that base editors operate highly efficiently in the living organism and are even suited to genetic screening. In an experiment with Japanese rice fish, they were able to show that these locally limited, targeted modifications in individual buildings blocks of the DNA achieve an outcome that is otherwise only obtained by the comparatively laborious breeding of organisms with altered genes.

The research team at COS, in cooperation with cardiologist Dr Jakob Gierten from Heidelberg University Hospital, focused on certain genetic mutations that were suspected of triggering congenital heart defects in humans. Through modifying individual building blocks of the DNA of the relevant genes in the model organism, the scientists were able to imitate and study fish embryos with the described heart defects. The targeted intervention led to visible changes in the heart already during early stages of fish embryonic development, which enabled the researchers to confirm the original suspicion and establish a causal connection between genetic alteration and clinical symptoms.

The precise intervention in the genome of the fish embryos was made possible through especially developed software ACEofBASEs, which is available online. It allows for identifying genetic locations that very efficiently lead to desired changes in the target genes and the resultant proteins. The scientists say that the Japanese rice fish is an excellent genetic model organism for modelling mutations like those identified from the respective patients, with Gierten stating, “Our method enables an efficient screening analysis and could therefore offer a starting point for developing individualised medical treatment.”

Following successful genome editing of the oca2 gene, the ratio of originally pigmented to unpigmented cells in the embryonic eye of the Japanese rice fish serves as a readout for Cas9 efficiency; this was used to optimise the CRISPR/Cas9 system. Top shows low knock-out efficiency of standard Cas9 enzymes and bottom shows increased knock-out rate using the improved heiCas9. Image ©Thomas Thumberger (COS).

A third study, published in the journal Development, deals with the limitations of base editors. For such an editor to bind the DNA of a target cell, there has to be a certain sequence motif. This is called Protospacer Adjacent Motif, or PAM.

“If this motif is lacking near the DNA building block to be changed, it is impossible to exchange nucleotides,” Thumberger said. A team under his direction has now found a way to get around this limitation.

Two base editors in a single cell are used in succession. In an initial step, a new DNA binding motif for a further base editor is generated, upon which this second editor, which is applied simultaneously, can edit a site that was inaccessible before. This staggered use turned out to be highly efficient. With this trick, the scientists were able to increase the number of possible application sites of established base editors by 65%. Now DNA sequences that were initially inaccessible can also be modified.

“Optimising the existing tools for genome editing and their fine-tuned application results in enormously varied possibilities for basic research and, potentially, novel therapeutic approaches,” Wittbrodt concluded.

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