Team:Jiangnan China/Engineering2

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The core of our project is to construct a recombinant strain of Starmerella bombicola to produce the sophorolipid with our expectation. Step by step, we faced a lot of problems and failures. With careful consideration and effective improvement, finally we achieved engineering success.

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Construction of CRISPR/Cas9 gene-editing system

Background:

The only successful and mature case of constructing the Starmerella bombicola was using the homologous recombination method, which performs a low efficiency and costs a long time. But the time left for experiments was less than one year. The first priority was to construct a new gene-editing system in S. bombicola.

Research:

The investigation focused on some successful cases of modifying other kinds of yeasts similar to S. bombicola and more gene-editing methods with high efficiency.[6] Finally, CRISPR/Cas9, famous for its high efficiency and successfully applied in many bacteria and fungi especially yeasts, became our final choice.

Imagine

We were hoping CRISPR/Cas9 system constructed could function in S. bombicola and show high gene-editing efficiency.

Design

The homologous arms were designed for integrating the Cas9 gene into the genome of S. bombicola. The use of the strongest promoter Ptef1 to express Cas9 protein and sgRNA could increase the gene-editing efficiency.[7] As the Cas9 protein was too big to transport into the nucleus, a nuclear localization sequence (NLS) was needed to help the transportation of Cas9 protein.

Bulid

The homologous arms “upSBLE” and “doSBLE” were obtained by inverse PCR,[3] using the vector with the SBLE gene as the template.

The fragment “Cas9-NLS-Tsyn7 was chemically synthesized by the company (Jinweizhi, suzhou, China).

Then the fragment was fused with Ptef1 by fusion PCR.

The fragment “Ptef1-Cas9-NLS-Tsyn7” was linked to the homologous arms by the one-step connection and then the recombinant vector was acquired. After that, the fragment below can be acquired by linearization.

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Fig.4 Cas9 expression cassette

Test

The Cas9 expression cassette was fused to the self-excising hygromycin marker cassette to obtain the recombinant vector. After linearization, it was electroporated into wild-type S. bombicola. The positive transformants were induced by galactose to eject hygromycin resistance gene. Then the recombinant strain with Cas9 gene and hygromycin resistance gene deletion was carried on the verification experiment of single, double and triple gene-editing efficiency.

Single gene-editing efficiency:

We designed the sgRNA that targeted at Pxa1 site. The hygromycin resistance gene[8] was also added to help find the positive transformants. We constructed the Pxa1 editing expression cassette below, and transformed it into the recombinant strain.

Fig.5 Pxa1 editing expression cassette

This result might be related to low conversion efficiency. But the more important reason is that Starmerella bombicola is eukaryote, it is more complex for exogenous gene to express in it.

Fig.6 Gel electrophoresis analysis of positive transformants (1)

The single gene-editing efficiency was 100%.

Double gene-editing efficiency:

Based on Pxa1 editing expression vector, designed the sgRNA that targeted at GFP site. We constructed the Pxa1, GFP editing expression cassette (Fig.7), and transformed it into the recombinant strain.

Fig.7 Pxa1, GFP editing expression cassette

The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies were extracted genomes for PCR, gel electrophoresis analysis and conducted green fluorescence observation. As a result, all of the target fragments were verified to be right in gel electrophoresis analysis and only two positive colonies showed fluorescence.

Fig.8 Gel electrophoresis analysis of positive transformants (2)

Fig.9 Green fluorescence observation in fluorescence

The double gene-editing efficiency was 99%.

Triple gene-editing efficiency:

Based on Pxa1, GFP editing expression cassette, we designed the sgRNA that targeted at Leu site. We constructed the Pxa1, GFP, Leu editing expression cassette (Fig.10), and transformed it into the recombinant strain.

Fig.10 Pxa1, GFP, Leu editing expression cassette

The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies were conducted green fluorescence observation. After that, the positive transformants without fluorescence were dotted on the Complete Medium (CM) and Minimal Medium (MM). Then the colonies grown on the CM but did not grow on the MM were the correct transformants.

Fig.11 Green fluorescence observation in fluorescence

Fig.12 colony observation (Left: CM; Right: MM)

The triple gene-editing efficiency was 30%.

Learn

The CRISPR/Cas9 system had been successfully applied in the gene-editing of S. bombicola, which showed a high gene-editing efficiency. The Cas9 gene was inserted into the genome of S. bombicola and may keep working.

Improve

As the Cas9 gene was inserted into genome of S. bombicola, it may be continually expressed and has some potential safety hazard. By transiently expressing Cas9 protein, the Cas9 gene can be degraded after working in the cytoplasm. This can be a solution to the problem.

Research:

We did some research on the defense mechanism of yeast against heterogeneous genes. We found that it was difficult for heterogeneous genes to express freely in S. bombicola.

Imagine:

The Cas9 protein could be transiently expressed in S. bombicola if it is not inserted into its genome.

Design

The strongest promoter Ptef1 was used to express Cas9 protein and sgRNA and could increase the gene-editing efficiency. As the Cas9 protein was too big to transport into the nucleus, a nuclear localization sequence (NLS) was needed to improve the transportation of Cas9 protein.

Bulid

The fragment “Cas9-NLS-Tsyn7” was chemically synthesized by the company (Jinweizhi, suzhou, China).

Then the fragment was fused with Ptef1 using fusion PCR.

Fig.13 Cas9 expression cassette without homologous arms

Test

Cas9 protein was transiently expressed to edit GFP site to verify if the newly built CRISPR/Cas9 gene-editing system could work in S. bombicola.

Construction of the GFP editing plasmid: Based on Cas9 expression plasmid, we added upGFP and doGFP as homologous arms to recombine with the GFP site. We designed the sgRNA to guide the Cas9 protein to nucleus and specific site. The hygromycin resistance gene was built between the upGFP and the doGFP as the marker gene to determine if the transformation was successful. Then the correct plasmid was linearized.

Fig.13 Cas9 expression cassette without homologous arms

The GFP editing cassette was transformed into the S. bombicola and incubated for 14 hours. Then the transformed yeasts were cultured on solid YPD medium with hygromycin. After incubation, there were a lot of transformants on the plate. Then the green fluorescence intensity of the transformants were determined. But none of them showed green fluorescence.

Learn

Transient expression of Cas9 protein could function in S. bombicola and the CRISPR/Cas9 gene-editing system showed a high gene-editing efficiency.