Upgrading of efficient and scalable CRISPR–Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila

Table 1 Summary of the genomic editing in M. thermophila using the CRISPR–Cas system

Host strain^a	Target locus	Elements in co-transformation	No. of analyzed transformants	No. of HR transformants^b	HR efficiency (%)^c	Each gene disruption efficiency (%)
WT	cre-1	Donor-cre1-neo	20	3	15	0
WT	cre-1	Cas12a +donor-cre1-neo	20	3	15	0
WT	cre-1	crRNA +donor-cre1-neo	20	2	10	0
WT	cre-1	Cas12a + crRNA + donor-cre1-neo	20	18	90	60
WT	cre-1, res-1, gh1-1	Donor DNA of cre-1, res-1 and gh1-1	23	0	0	0 0 0
WT	cre-1, res-1, gh1-1	Cas12a + pooled three sets of crRNA + donor DNA of cre-1, res-1 and gh1-1	20	8	40	35 25 25
WT	cre-1, res-1, gh1-1	Cas12a + array1 + donor DNA of cre-1, res-1 and gh1-1	22	7	32	41 32 27
WT	cre-1, res-1, gh1-1	Cas9 + three sets of sgRNA + donor DNA of cre-1, res-1 and gh1-1	23	9	39	39 35 39
M3	neo,alp-1, rca-1, hcr-1	Donor DNA of neo, alp-1, rca-1 and hcr1	23	0	0	0 0 0 0
M3	neo,alp-1, rca-1, hcr -1	Cas12a + array2 + donor DNA of neo, alp-1, rca-1 and hcr-1	23	5	22	26 30 13 22
M3	neo, alp-1, rca-1, hcr-1	Cas9 + four sets of sgRNA + donor DNA of neo, alp-1, rca-1 and hcr-1	23	5	22	35 30 13 30
M7	bar, ap-3, prk-6	Donor DNA of bar, ap-3 and prk-6	22	0	0	0 0 0
M7	bar, ap-3, prk-6	Cas12a + array3 + donor DNA of bar, ap-3 and prk-6	22	9	41	32 27 23
M7	Bar, ap-3, prk-6	Cas9 + three sets of sgRNA + donor DNA of bar, ap-3 and prk-6	21	8	38	33 29 19

^aWT, wild-type strain; M3, triple-mutant Δcre1Δres1Δgh1-1; M7, septuple mutant ∆cre1Δres1Δgh1-1ΔneoΔalp1Δrca1::xyr1Δhcr1
^bHR, homologous recombination
^cHR efficiency, HR frequency of gene replacement (including both homokaryons and heterokaryon)