Researchers Report New Strategy to Enhance CRISPR–Cas9-Mediated Homologous Recombination

CRISPR-Cas9-mediated homology-directed repair (HDR) can introduce desired mutations at targeted sites in the genome, but editing efficiencies remain poor in many cell types, including cells that are deficient in DNA repair activity.

In an attempt to augment the efficiency of CRISPR-Cas9-mediated HDR, researchers at ETH Zurich and Memorial Sloan Kettering Cancer Center in New York sought to identify suppressors of CRISPR-Cas9-mediated HDR by performing a genome-wide CRISPR screen in Fanconi anaemia (FA) patient lymphoblastic cell lines. FA is a rare disease that arises through loss-of-function mutations in any of > 20 different genes, and is characterised by bone marrow failure and increased risk of developing cancer later in life. The mutations underlying FA also greatly reduce HDR, which makes it very difficult to develop gene-editing therapies for the disease.

Following the CRISPR screen, the team, led by Jacob Corn at ETH Zurich, found that a single exonuclease, TREX1, reduces HDR efficiency when the repair template is a single-stranded or linearised double-stranded DNA. They furthermore show that TREX1 expression can be used as a biomarker for CRISPR-Cas9-mediated HDR because the high levels of TREX1 expression observed in many different cell types predicts poor HDR outcomes. Their findings, published yesterday in Nature Biotechnology, demonstrate rescue of HDR efficiency (with up to eight-fold improvements) either by knocking out TREX1 or by using single-stranded DNA templates chemically protected from TREX1 activity. They study offers an explanation as to why some cell types are more amenable to editing than others and highlights avenues for increasing CRISPR-Cas9-mediated HDR in TREX1-expressing contexts.

Read the full article in Nature Biotechnology here.