EPICA.2 increased the activity of compact CasΦ2 in human cells by up to 70-fold

A new version of the compact CRISPR editor CasΦ2 edited DNA in human cells up to 70 times more efficiently than the original enzyme

On August 13, bioRxiv published a preprint on evoCasΦ2, a version of CasΦ2 containing six amino acid substitutions. The authors compared it with the original enzyme and two variants designed by selecting substitutions based on the known protein structure. They tested all four enzymes at four genomic sites in the human HEK293T cell line.

CasΦ2 is a CRISPR protein that uses a guide RNA to locate and cut a specified DNA sequence. It contains 757 amino acids, so it requires less space in a delivery system than the larger Cas9. The original CasΦ2 showed almost no DNA editing activity in mammalian cells, which made successful variants difficult to distinguish from random noise.

To address this problem, the authors developed EPICA.2, a two-stage directed evolution system. They generated many slightly modified CasΦ2 variants and first screened them in yeast. A DNA cut restored the function of a gene, allowing the cell to grow. Four rounds of mutation and repeated selection produced a library of more active variants.

The researchers then transferred the library into human cells containing a frameshifted and therefore inactive green fluorescent protein gene. CasΦ2 cleavage sometimes restored the correct reading frame, causing the protein to fluoresce, and the authors selected these fluorescent cells. Long-read sequencing of each individually tagged DNA molecule preserved the combination of substitutions within each variant. This approach identified evoCasΦ2, which contains six substitutions.

The effect depends on the combination of all six substitutions. At the control site, no individual substitution reproduced the full effect, and removing any substitution from the complete variant reduced its activity. At four endogenous genomic sites in HEK293T cells, evoCasΦ2 edited DNA up to 70 times more efficiently than the original CasΦ2 and outperformed two variants designed through rational selection of substitutions. At some target sites, the proportion of edited DNA remained below 5%.

EPICA.2 extends the original EPICA. In 2024, that system produced a variant of another compact nuclease, CjCas9, with up to 12 times the activity of the original enzyme at endogenous sites in human cells. In the new preprint, the researchers adapted this approach to CasΦ2, whose original signal was nearly indistinguishable from background. The selection proceeded from yeast growth to cellular fluorescence and finally to direct measurement of DNA editing.