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.
Jaeyeon, L., Kyeongjun, K., Hyeonmin, R., Hyeonsik, Y., Kerstin, D., Mid-Eum, P., Joong-Tak, Y., Bon-Kyoung, K., Ji-Hye, Y., Ho-Seok, L., Eun Yu, K.
Advertisement
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 17
- Comments 0