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Fig. 5 | Microbial Cell Factories

Fig. 5

From: Current genetic strategies to investigate gene functions in Trichoderma reesei

Fig. 5

Schematic diagram of the CRISPR/Cas9 system developed inT. reesei. (a) Two plasmids expressing NLS-Cas9 fusion protein and sgRNA were constructed, respectively, and were delivered together to cells. (b) The NLS-Cas9 protein and sgRNA were synthesized and assembled in vitro, and then they were transformed together into cells. (c) A plasmid expressing NLS-Cas9 fusion protein was constructed and delivered to cells. The sgRNA was prepared by in vitro transcription and transformed into host cells harboring the NLS-Cas9 expression cassette. The Cas9-sgRNA complex unwinds the double-stranded DNA (dsDNA) and the sgRNA binds to one of the DNA strands. Upon binding, the Cas9 nuclease cleaves both DNA strands upstream of the PAM sequence to form DSB, which is repaired either by the HR pathway or the NHEJ pathway

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