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Fig. 2 | Fungal Biology and Biotechnology

Fig. 2

From: Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes

Fig. 2

a CRISPR/Cas9 facilitated deletion of A. niger gaaX gene based on in vitro assembled RNP complex and donor DNA containing pyrG selection marker. Targeting efficiency was analysed by using colony PCR ensuring the absence of gaaX ORF (primers 1, oPEEL-294/295) and the presence of donor DNA cassette at the correct genomic locus (primers 2, oPEEL-279/292 and 3, oPEEL-293/334). Targeting efficiency and the colony forming units (CFU) per 105 protoplasts and 1 µg donor DNA b with 1500 bp flanking sequence in the donor DNA and different concentrations of RNP complex and c with 90 nm RNP complex and varying lengths of flanking sequence in the donor DNA. 3–12 colonies from each transformation depending on colony number on plates were analyzed

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