Silencing Two Genes Improved the Performance of a DNA Editor Delivered to Cells in Lipid Nanoparticles

Silencing Two Genes Improved the Performance of a DNA Editor Delivered to Cells in Lipid Nanoparticles

On August 13, a team at the University of Wisconsin in Madison published a genetic screen in which they individually knocked out 19 114 human genes and measured how each knockout affected a DNA editor delivered in lipid nanoparticles. Six genes passed follow-up validation. Knocking out GJB2 or BET1L increased editing in several cellular models.

The researchers delivered the Cas9 DNA editor in a lipid nanoparticle, which is a fatty shell containing the protein and a guide RNA. After entering a cell, the cargo must escape from an intracellular vesicle and reach the nucleus, where the DNA is located. In this screen, the researchers knocked out one gene in each cell. Sequencing, which reads the DNA sequence, then identified the disrupted gene and showed whether the editor had modified the intended site.

The authors separately tested 26 candidates, and the effect was reproduced for six of them. Knocking out GJB2 or BET1L increased the efficiency of editors that change individual DNA “letters” without cutting both strands. In cells carrying a model KCNJ13 mutation, GJB2 silencing produced 19.3% correction, which was 6.7 times the rate in control cells. BET1L silencing produced 14.5% correction, which was five times the control rate.

The team then examined where along the delivery pathway the difference emerged. When Cas9 was introduced by electroporation, which uses an electrical pulse to carry material across the cell membrane, the advantage of cells with the genes knocked out disappeared in most candidate cell lines. In cells lacking BET1L, the fluorescent Cas9 signal in the nucleus was approximately one and a half times higher. Taken together, these experiments link part of the effect to the intracellular delivery of the editor.

The authors next used retinal pigment epithelial cells grown from cells obtained from a patient with a KCNJ13 mutation. The proportion of DNA carrying the corrected sequence increased from 5.8% to 46.6% after GJB2 silencing and to 20.9% after BET1L silencing. Some edited cells regained Kir7.1 protein activity. Kir7.1 is an ion channel impaired by this mutation, and the instrument detected the characteristic electrical currents produced by its activity.

This screen can identify factors that affect every stage of delivery, from lipid nanoparticle entry into the cell to editor activity in the nucleus.