First evidence for TIGR-Tas activity in a photosynthetic eukaryote

Abstract

TIGR–Tas systems are a recently described class of RNA-guided DNA-targeting systems whose activity has been demonstrated in bacterial and human cells, but not in photosynthetic eukaryotes. Here we tested TIGR–Tas in the green alga Chlamydomonas reinhardtii. Recombinant TaTas and ParTas assembled with in vitro-transcribed tigRNAs cleaved target DNA in a concentration-dependent manner, and electroporated TaTas protein was detected in C. reinhardtii cells. Targeted deep sequencing of the endogenous MAA7 locus recovered indel-containing reads in ParTas-treated cells that were absent from wild-type controls, including a 1-bp deletion located at the position previously reported for ParTas-associated cleavage. Co-delivery of a double-stranded non-homologous oligonucleotide (dsNHO) with a 24-bp duplex and 8-nt 3′ overhangs, designed to match the staggered ends generated by TasR-family nucleases, increased the recovery of indel-containing reads for both proteins. The frequency of these events was very low, and the present data do not establish TIGR–Tas as an efficient genome-editing system in this host. They nonetheless provide initial evidence that TIGR–Tas can act on the nuclear genome of a photosynthetic eukaryote, and indicate that dsNHO-assisted recovery of sequence alterations is not restricted to conventional CRISPR– Cas nucleases.