CRISPR, the gene-editing tool that has been all the rage the past few years, does not modify the genome as neatly or predictably as a word processer might alter a document. Wherever the CRISPR cursor lands in a mammalian genome, the outcome—a short deletion or, possibly, a copy-paste—depends on the relative efficiencies of two different DNA repair mechanisms. Most often, a CRISPR-induced double-strand break is repaired via a mechanism that tends to favor short deletions, limiting CRISPR’s utility as gene-insertion tool. This repair mechanism, however, can be suppressed, effectively increasing the efficiency of the other mechanism, the one that may be more desirable—if the idea is to insert preplanned genetic modifications.

A means of favoring CRISPR’s “copy-paste” functionality was achieved by scientists at the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch. They described their technique March 24 in Nature Biotechnology, in an article entitled, “Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.”

The homology-directed repair (HDR) pathway enables the insertion of preplanned genetic modifications using engineered DNA molecules that share identical sequence regions with the targeted gene and which are recognized as a repair template. Thus, HDR repair is very precise but occurs only at low frequency in mammalian cells.

The other repair system, called non-homologous end-joining (NHEJ) is more efficient in nature but less precise, since it readily reconnects free DNA ends without repair template, thereby frequently deleting short sequences from the genome. Therefore, NHEJ repair can only be used to create short genomic deletions, but does not support precise gene modification or the insertion and replacement of gene segments.

The MDC researchers succeeded in increasing the efficiency of the more precisely working HDR repair system by temporarily inhibiting the most dominant repair protein of NHEJ, the enzyme DNA Ligase IV. In their approach, they used various inhibitors such as proteins and small molecules. They also used what MDC researcher Ralf Kühn called a “trick of nature”—the suppression of Ligase IV with the proteins of adeno viruses.

“We suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems,” wrote the authors of the Nature Genetics article. “Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR four- to fivefold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines.”

CRISPR technology is already used in the laboratory to correct genetic defects in mice. Researchers also plan to modify the genetic set up of induced pluripotent stem cells (iPS), which can be differentiated into specialized cell types or tissues. That is, researchers are able to use the new tool to introduce patient-derived mutations into the genome of iPS cells for studying the onset of human diseases. “Another future goal, however, is to use CRISPR for somatic gene therapy in humans with severe diseases,” MDC researcher Klaus Rajewsky pointed out.

Rajewsky added that the new capabilities to precisely edit the genome have sparked off an intense debate in the USA and elsewhere, since the new precision tools could also be applied to modifying the genome in human germ cells or embryos. Although manipulation of the human germline is prohibited by law in many countries, including Germany, a global ban is not in effect. The MDC researchers are fascinated by the new opportunities the CRISPR-Cas9 system offers for biomedical research, but strictly reject genetic modification of the human germline.

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