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Simplified and effective RNA interference and CRISPR-Cas9 systems for Cryptococcus neoformans
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  • Qiying Chen,
  • Yifan Wu,
  • Xinyuan Peng,
  • Min Peng,
  • Chen Zhang,
  • Yifan He,
  • Qing tao Kong,
  • Hong Sang
Qiying Chen
Southern Medical University
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Yifan Wu
Nanjing Medical University
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Xinyuan Peng
Nanjing University
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Min Peng
Nanjing University
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Chen Zhang
Nanjing University
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Yifan He
Nanjing Medical University
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Qing tao Kong
Nanjing Jinling Hospital
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Hong Sang
Southern Medical University

Corresponding Author:[email protected]

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Abstract

The 3,4-dihydroxyphenylalanine (DOPA) melanin is one of important virulence factors for Cryptococcus neoformans, which may trigger immune responses in the host. It is worth exploring the genetic function of C. neoformans, by which we may derive more antifungal strategies. Therefore, we established two systems that were constructed quickly and easily for the knock-down/knock-out of LAC1 gene: RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. The RNAi system used pSilencer 4.1-CMV neo plasmid and short hairpin RNA to realize the effective transcriptional suppression. The CRISPR-Cas9 system used the PNK003 vectors to obtain a stable albino mutant strain. The results of phenotype, qRT-PCR, Transmission Electron Microscope (TEM) and spectrophotometry were used to assess the ability of melanin production. As a result, the RNAi system displayed attenuation of transcriptional suppression when the transformants continuously passed on new plates. However, the transcriptional suppression of long loop in short hairpin RNA were more powerful and lasted longer. The CRISPR-Cas9 system constructed an albino strain completely without the ability to produce melanin. Considering the weakening of transcriptional suppression, we recommend using a long loop for the RNAi system and 1st or 2nd passage of knockdown strains for the subsequent studies. Besides, the different capacities of melanin production might be useful for exploring the linear relation between melanin and immunoreaction of the host. In addition, we recommend applying the PNK003 vectors to other serotypes of C. neoformans for quick screening of possible trait-regulating genes because of its easy construction and valid knockout effect.
28 Feb 2023Submitted to Journal of Basic Microbiology
15 Mar 2023Submission Checks Completed
15 Mar 2023Assigned to Editor
15 Mar 2023Review(s) Completed, Editorial Evaluation Pending
20 Mar 2023Reviewer(s) Assigned
01 Apr 2023Editorial Decision: Revise Major
05 Apr 20231st Revision Received
05 Apr 2023Submission Checks Completed
05 Apr 2023Assigned to Editor
05 Apr 2023Review(s) Completed, Editorial Evaluation Pending
11 Apr 2023Reviewer(s) Assigned
21 Apr 2023Editorial Decision: Revise Major
30 Apr 20232nd Revision Received
01 May 2023Submission Checks Completed
01 May 2023Assigned to Editor
01 May 2023Review(s) Completed, Editorial Evaluation Pending
01 May 2023Reviewer(s) Assigned
24 May 2023Editorial Decision: Revise Minor
25 May 20233rd Revision Received
25 May 2023Assigned to Editor
25 May 2023Submission Checks Completed
25 May 2023Review(s) Completed, Editorial Evaluation Pending
28 May 2023Editorial Decision: Revise Minor
29 May 20234th Revision Received
01 Jun 2023Assigned to Editor
01 Jun 2023Submission Checks Completed
01 Jun 2023Review(s) Completed, Editorial Evaluation Pending
02 Jun 2023Editorial Decision: Accept