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.