New CRISPR Approach Enables “Seamless” Gene Insertions

Researchers in Switzerland have created a new method to generate knock-ins in Drosophila melanogaster, a common laboratory model.

The approach, named SEED/Harvest, combines CRISPR-Cas9 technology with the single-strand annealing (SSA pathway).

“Since we would like to introduce and analyze changes in the DNA throughout the genome for our research, the method must be both precise and efficient,” said Dr. Markus Affolter, professor at the the Biozentrum University of Basel and the study’s lead author. “The SEED/Harvest method is both. It combines the most robust screening of successful insertions and all the advantages of seamless tagging.”

SEED/Harvest enables proteins to be labeled in specific tissue and cell types

There are many reasons why scientists might want to generate knock-ins within laboratory organisms: to study the function of specific genes, model diseases, understand how proteins function and interact or simply to conduct basic research.

When CRISPR-Cas9 was established as a gene-editing tool several years ago, it transformed this line of research.

In the CRISPR-Cas9 system, a guide RNA (gRNA) guides the Cas9 enzyme – which can be likened to a pair of molecular scissors – to a specific location within the genome, where it binds to the targeted sequence. After binding, Cas9 cuts both strands of DNA triggering the cell’s DNA repair system into motion.

This repair process allows researchers to introduce targeted changes into the genome. The gold standard method for generating knock-ins typically takes advantage of homology directed repair (HDR). Here, a repair template carrying the desired genetic insertion is used by the cell’s molecular machinery to repair the break, and the repair template is incorporated into the genome. Et voila, you have inserted a new gene into the genome!

“However, HDR rates are often low in vivo, and thus, an efficient way for screening correct insertions is required,” Affolter and colleagues explained.

To support screening, researchers have explored the feasibility of two-step knock-in approaches, whereby a marker is also inserted into the targeted DNA site, before being removed after screening. But current process can introduce scars.

“Scarless editing is fundamental for many purposes, such as internal tagging of proteins or the generation of point mutants, cases in which scars could disrupt the protein sequence in unpredictable ways,” Affolter and colleagues said.

That’s why the researchers have developed SEED/Harvest, which they described as a “seamless” approach. It’s a method comprising two steps. First, scientists introduce a marker into the desired DNA site that sits within an exon, also known as a protein-coding region. Next, the marker is removed and the DNA breakpoints are repaired by the SSA pathway.

“After insertion of the SEED cassette and screening, the screening marker is seamlessly removed by a subsequent CRISPR-triggered repair event, resolved by SSA. In this step, the repeats anneal and the region in between is removed, resulting in the scarless removal of the 3xP3-dsRED marker and ultimately resulting in the desired gene editing,” the researchers explained.

SEED/Harvest enables proteins to be labeled in specific tissues and cell types. “We can now control and determine in various tissues and developmental stages when and where genes are activated or inactivated”, said Dr. Gustavo Aguilar, a post-doctoral researcher in Affolter’s group and the study’s first author. “This opens up new possibilities for research to investigate the dynamics of proteins systematically in living cells in real-time.”

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Reference: Aguilar G, Bauer M, Vigano MA, et al. Seamless knock-ins in Drosophila via CRISPR-triggered single-strand annealing. Dev Cell. 2024. doi:10.1016/j.devcel.2024.06.004

This article is a rework of a press release issued by the Biozentrum University of Basel. Material has been edited for length and content.