Heterologous and endogenous U6 snRNA promoters enable CRISPR/Cas9 mediated genome editing in Aspergillus niger

Table 1 Comparison on CRISPR/Cas9 system for A. niger

Cas9 expression		sgRNA expression		Targeted gene	Donor DNA (SM/homology arm size, bp)	Gene editing efficiency	Notes	References
Promoter	Terminator	Promoter	Terminator	Targeted gene	Donor DNA (SM/homology arm size, bp)	Gene editing efficiency	Notes	References
Ptef1	Ttef1	PgpdA	TtrpC	albA	–	Some	Requiring to add HH and HDV for processing sgRNA	[10]
PcoxA	Ttef1	PmbfA	TtrpC	pyrG	–	Obtaining 25 colonies on MM with 5′-FOA	Requiring to add self-cleaving ribozymes for processing sgRNA	[11]
PcoxA	Ttef1	PmbfA	TtrpC	MttA	pyrG/690 and 834^b	100% (7/7)		[11]
Ptef1	Ttef1	In vitro synthesis		1090836	pyrG/1500	28% (11/40)	3% (1/30)^a	[12]
				1117792		100% (8/8)	43% (13/30)^a
				1141260		100% (8/8)	0% (0/30)^a
				1121140		38% (3/8)	2% (1/60)^a
				1146483		88% (7/8)
				1170646		63% (5/8)
PglaA	TglaA	PhU6	Ploy(T)₆	albA	–	15% (4/27)	Without any selection pressure for targeted gene editing	This study
		PyU6			–	20% (1/5)
		PanU6			–	23% (3/13)
		PanU6		albA	hph/40	36% (5/14)	79% (11/14)^c

^aThe gene deletion efficiency without CRISPR/Cas9 system
^bThis donor DNA for gene integration was flanked by a 5′ flanking sequence of 690 bp homologous to the promoter region of the pyrG gene, while the 3′ flanking sequence was a mutated and truncated pyrG CDS of 834 bp (pyrG^{m2, trunc})
^cAfter co-transformation of donor DNA MHi-albA-hph, sgRNA3.1 and pCas9, the outgrown albino colonies with albA disruption reached up to 79% (11/14). In some albino colonies, some unexpected base pair errors were mediated by NHEJ at the 5′-junction or 3′-junction of DBSs. Therefore, the precise gene integration was only 36% (5/14)

ISSN: 2054-3085