Strategy for unlimited cycles of scarless oligonucleotide directed gene editing inChlamydomonas reinhardtii

Abstract

AbstractCRISPR/Cas9 gene editing in the model green algaChlamydomonas reinhardtiirelies on the use of selective marker genes to enrich for non-selectable target mutations. This becomes challenging when many sequential modifications are required in a single cell line, as useful markers are limited. Here we demonstrate a cyclical selection process which only requires a single marker gene to identify an almost infinite sequential series of CRISPR-based target gene modifications. TheNIA1(Nit1, NR; nitrate reductase) gene was this selectable marker. In the forward stage of the cycle, a stop codon was engineered into theNIA1gene at the CRISPR target location. Cells retaining the wild typeNIA1gene were killed by chlorate, whileNIA1knockout mutants survived. In the reverse phase of the cycle, the stop codon engineered into theNIA1gene during the forward phase was edited back to the wild type sequence. Using nitrate as the sole nitrogen source, here only the reverted wild type cells survived. By using CRISPR to specifically deactivate and reactivate theNIA1gene, a marker system was established that flipped back and forth between chlorate- and auxotrophic (nitrate) based selection. This provided a scarless cyclical marker system that enabled an indefinite series of CRISPR edits in other, non-selectable genes. Here, we demonstrate that this ‘Sequential CRISPR via Recycling Endogenous Auxotrophic Markers(SCREAM)’ technology enables an essentially limitless series of genetic modifications to be introduced to a single cell lineage ofC. reinhardtiiin a fast and efficient manner to complete complex genetic engineering.