CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

Table 1 Numbers of colonies regenerating from protoplasts of F. venenatum transformed with AMA1 ‘CRISPR’ vectors

Vector	CRISPR constituents	Experiment 1				Experiment 2
		A	B	C	Total	A	B	C	Total
pFCFvCas9	Fv Cas9	0	1	0	1	0	1	1	2
pFC332	A.niger Cas9	11	0	1	12	14	6	3	23
pFCFvCas9::PolII-PK3/14	Fv Cas9 PFv5SrRNA-sgRNA	0	0	0	0	1	0	0	1
pFC332::5S-PK3/14	A.niger Cas9 PFv5SrRNA-sgRNA	0	1	0	1	2	6	3	11
pFC332::PolII-PK3/14	A.niger Cas9 PgdpA/ribozyme-sgRNA	10	11	4	25	16	5	9	30

CRISPR constituents were Cas9 codon optimised for Fusarium venenatum (Fv) or Aspergillus niger and sgRNAs targeting the PKS12 gene were transcribed using PolIII promoter PFv5SrRNA or PolII promoter PgdpA (PgdpA/ribozyme). Numbers given are for colonies counted on selection plates 10 days after transformation of approximately 1 × 10⁸ protoplasts with 5 µg vector DNA. Three replicates (A-C) were performed for each vector transformation and the experiment was repeated (Experiments 1 and 2).

ISSN: 2054-3085