| abstract |
A method of producing a mutant host cell with altered expression of a gene of interest (GOI) by a temperature-inducible CRISPR/Cas system, which comprises: na) at a first working temperature, introducing into a host cell a CRISPR/Cas system employing ni) an inactive pre-guide RNA (pre-gRNA) which is a ribozyme-extended guide RNA comprising a guide RNA (gRNA) that recognizes a predetermined target site at the GOI, terminally extended by at least one self-processing Hammerhead (HH) ribozyme characterized by the presence of a thermosensitive hairpin and a cleavage site adjacent to the gRNA; and nii) a RNA-guided Cas endonuclease which recognizes the target site upon hybridizing with the gRNA; n nb) increasing the first working temperature to a second working temperature, thereby changing the structure of the thermosensitive hairpin and activating the pre-gRNA by self-catalyzed cleavage at the cleavage site to obtain the gRNA, which induces binding of the endonuclease to the target site, thereby altering expression of the GOI. |