U6
promoters have been used for single guide RNA (sgRNA) transcription in the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) genome editing system. However, no availableU6promoters have been identified inAspergillus niger,which is an important industrial platform for organic acid and protein production. Two CRISPR/Cas9 systems established inA. nigerhave recourse to the RNA polymerase II promoter or in vitro transcription for sgRNA synthesis, but these approaches generally increase cloning efforts and genetic manipulation. The validation of functional RNA polymerase II promoters is therefore an urgent need forA. niger.
Here, we developed a novel CRISPR/Cas9 system inA. nigerfor sgRNA expression, based on one endogenousU6promoter and two heterologousU6promoters. The three testedU6promoters enabled sgRNA transcription and the disruption of the polyketide synthasealbAgene inA. niger. Furthermore, this system enabled highly efficient gene insertion at the targeted genome loci inA. nigerusing donor DNAs with homologous arms as short as 40-bp.
This study demonstrated that both heterologous and endogenousU6promoters were functional for sgRNA expression inA. niger. Based on this result, a novel and simple CRISPR/Cas9 toolbox was established inA. niger,that will benefit future gene functional analysis and genome editing.