Table 1 Characteristics and efficiency of the CRISPR-Cas9 mediated NHEJ and HDR experiments in the tested M. circinelloides strains.

Host strain	Target	Elements used for transformation^a	Repair mechanism	Selection method	Transformation frequency (per 10⁵ protoplasts)	Targeting efficiency^b
M. circinelloides CBS277.49	carB	Cas9 (50 µM), gRNA (50 µM)	—	white phenotype	0	—
M. circinelloides CBS277.49	carB	Cas9 (100 µM), gRNA (100 µM)	NHEJ	white phenotype	2 × 10⁴	100%
M. circinelloides CBS277.49	carB	Cas9 (200 µM), gRNA (200 µM)	NHEJ	white phenotype	1.5 × 10⁴	100%
M. circinelloides MS12	carB	Cas9 (50 µM), gRNA (50 µM)	—	white phenotype	0	—
M. circinelloides MS12	carB	Cas9 (100 µM), gRNA (100 µM)	NHEJ	white phenotype	1.25 × 10⁴	100%
M. circinelloides MS12	carB	Cas9 (200 µM), gRNA (200 µM)	NHEJ	white phenotype	1.2 × 10⁴	100%
M. circinelloides MS12	carB	Cas9 (100 µM), gRNA (100 µM), template DNA (5 µg)	HDR	white phenotype, complementation of uracil auxotrophy	2	100%
M. circinelloides MS12	carB	template DNA (5, 10 or 15 µg)	—	white phenotype, complementation of uracil auxotrophy	0	0
M. circinelloides MS12	carB	Cas9 (100 µM), gRNA (100 µM)	—	white phenotype, complementation of uracil auxotrophy	0	0
M. circinelloides MS12	hmgR2	Cas9 (100 µM), gRNA (100 µM), template DNA (5 µg)	HDR	complementation of uracil auxotrophy	1	100%
M. circinelloides MS12	hmgR2	template DNA (5, 10 or 15 µg)	—	complementation of uracil auxotrophy	0	0
M. circinelloides MS12	hmgR2	Cas9 (100 µM), gRNA (100 µM)	—	complementation of uracil auxotrophy	0	0

^aApplied concentrations of the elements used are presented in parentheses. ^bTargeting efficiency means the percentage of the isolated transformants, in which the mutation occurred in the targeted site.

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