Efficient gene editing in Neurospora crassa with CRISPR technology

Abstract

Background: Efficient gene editing is a critical tool for investigating molecular mechanisms of cellular processes and engineering organisms for numerous purposes ranging from biotechnology to medicine. Recently developed RNA-guided CRISPR/Cas9 technology has been used for efficient gene editing in various organisms, but has not been tested in a model filamentous fungus, Neurospora crassa.

Findings: In this report, we demonstrate efficient gene replacement in a model filamentous fungus, Neurospora crassa, with the CRISPR/Cas9 system. We utilize Cas9 endonuclease and single crRNA:tracrRNA chimeric guide RNA (gRNA) to: (1) replace the endogenous promoter of clr-2 with the β-tubulin promoter, and (2) introduce a codon optimized fire fly luciferase under the control of the gsy-1 promoter at the csr-1 locus. CLR-2 is one of the core transcription factors that regulate the expression of cellulases, and GSY-1 regulates the conversion of glucose into glycogen. We show that the β-tubulin promoter driven clr-2 strain shows increased expression of cellulases, and gsy-1-luciferase reporter strain can be easily screened with a bioluminescence assay.

Conclusion: CRISPR/Cas9 system works efficiently in Neurospora crassa, which may be adapted to Neurospora natural isolates and other filamentous fungi. It will be beneficial for the filamentous fungal research community to take advantage of CRISPR/Cas9 tool kits that enable genetic perturbations including gene replacement and insertions.

Keywords: Biofuel; CRISPR/Cas9; Cellulase; Genome editing; Homologous recombination; Neurospora crassa.

Figure 1

System overview of genomic edition in N. crassa using CRISPR/Cas9. a The system consists of two components, a Cas9 protein and a single crRNA:tracrRNA chimeric guide RNA (gRNA), comprising a 20-bp target sequence (red) complementary to the genomic target adjacent to a PAM site of NGG (blue). b Design of the Cas9 and gRNA constructs. The Cas9 protein contained a SV40 nuclear localization signal, and the expression was under the control of the trpC promoter and terminator. The gRNA was expressed under the snoRNA SNR52 promoter and contained a terminator from the 30 region of the yeast SUP4 gene. c Design of gRNA targeted to clr-2 and csr-1 loci.

Figure 2

Evaluation of CRISPR/Cas9 system for transformation of N. crassa. a Diagram of donor plasmids. Blue color regions are the sequence regions that are homologous to genomic DNA. Bar gene cassette which contains the bar gene under the control of trpC promoter and terminator was included in each plasmids for selection. b The number of Ignite-resistant colonies by clr-2 locus targeted transformation with different amount of Cas9 and gRNA plasmids. From left to right: zero, 1, 2.5, and 5 µg each of Cas9 and gRNA plasmid was co-transfected with the donor plasmid (5 µg). **p < 0.01, Tukey’s test. Error bars corresponds to the SEM. c The number of Ignite-resistant colonies by csr-1 locus targeted transformation with zero and 5 µg each of Cas9 and gRNA plasmids. **p < 0.01, student’s t-test. Error bars corresponds to the SEM. d–f Luciferase activity on the plates of gsy-1-luciferase transformants. Luciferase signal (d), under red-light (e), and merged (f) images are shown.

Figure 3

Rate of homologous integration at clr-2 locus in wild type with CRISPR/Cas9 technology versus mus-51 KO with the traditional method. a The number of Ignite-resistant colonies by clr-2 locus targeted transformation in the mus-51 KO and wild type (WT: 74-OR23-1V A) with CRISPR/Cas9. Error bars corresponds to the SEM. b qPCR analysis to assess the number of clr-2 in genomic DNA from the transformants with either WT (right panel) or mus-51 KO backgrounds (left panel). Error bars corresponds to the SEM. c qPCR analysis to assess the number of gh6-2 in genomic DNA from the transformants with either WT (right panel) or mus-51 KO backgrounds (left panel). Error bars corresponds to the SEM. d Schematic overview of the priming sites for PCR analysis to confirm the connection of β-tubulin promoter and clr-2. e PCR assay using β-tubulin-p F and clr-2 R primers. Expected fragment size: 1,343 bp.

Figure 4

Amplified expression of cellulase genes by enhanced expression of clr-2 with β-tubulin promoter. The mRNA expression of clr-2 and cellulase genes are measured by qRT-PCR. The expression of clr-2 (a), cbh-1 (b), gh5-1 (c), and gh6-2 (d) are shown. White and black bars show mRNA expressions in wild type (WT: 74-OR23-1V A) and β-tubulin-clr-2 strains, respectively. Each strain is cultured in the media containing 2% glucose as a sole carbon source. All the expressions were normalized by the expressions in WT. **p < 0.01, student’s t-test. Error bars corresponds to the SEM.