Research ArticleMICROBIOLOGY

RELATe enables genome-scale engineering in fungal genomics

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Science Advances  18 Sep 2020:
Vol. 6, no. 38, eabb8783
DOI: 10.1126/sciadv.abb8783
  • Fig. 1 Workflow of sgRNA library design and functional screen.

    (1) C. neoformans genomic DNA was digested with restriction enzyme cocktail, Hpa II, Scr FI, and Bfa I; (2) the sgRNA library was cloned into T-DNA vector pDHt-SK-NEO-CnU6; (3) the sgRNA library was introduced into A. tumefaciens; (4) the sgRNA library was delivered into C. neoformans H99CAS9; (5) transcytosis assay in HBMEC; (6) intravenous inoculation in mice; (7) intranasal inoculation in mice; (8) the recovered sgRNA library was amplified by polymerase chain reaction (PCR) and submitted for deep sequencing.

  • Fig. 2 Agrobacterium-based delivery of sgRNA produces efficient deletion of target gene.

    (A) T-DNA expression vector for sgRNA. LB, left border of T-DNA; RB, right border of T-DNA; NEO, drug G418 resistance gene; U6, C. neoformans U6 promoter. (B) Melanization assay. U6, transformants containing only C. neoformans U6 promoter; LAC1 sgRNA1 and sgRNA2, transformants containing sgRNAs expressed under U6 promoter. Dark brown colony, melanization; white colony, no melanization. Photo credit: Z. Li, Johns Hopkins University. (C) Indel mutation in the targeting site of LAC1. Sequence targeted by LAC1 sgRNA2, highlighted in orange; PAM (protospacer adjacent motif) site, highlighted in blue; red dashes, deleted bases; red bases, insertion. Photo credit: Z. Li, Johns Hopkins University.

  • Fig. 3 sgRNA library targeting C. neoformans genomic sequences.

    (A) Length distribution of the protospacers (region between U6 promoter and sgRNA guide body). (B) Distribution of the protospacers within C. neoformans. (C) Cumulative frequency of sgRNAs. YPD4, initial sgRNA library; YPD22, sgRNA library cultured for additional 18 days; L, sgRNA library recovered from lung after intranasal infection; B, sgRNA recovered from brain after intranasal infection; Biv, sgRNA recovered from brain after intravenous infection; T, sgRNA library recovered from transcytosis assay. (D) Cumulative frequency of genes.

  • Fig. 4 Functional screen reveals cryptococcal factors contributing to penetration of the blood-brain barrier.

    (A and B) Venn diagram on distribution of the depleted sgRNAs. Transcytosis, sgRNAs depleted in penetration of the HBMEC monolayer in vitro; Brain IV, sgRNAs depleted in brain invasion after intravenous infection; Brain IN, sgRNAs depleted in disseminating from lung to brain after intranasal infection; Brain IV & IN & Transcytosis, overlap of depleted sgRNAs between Transcytosis, Brain IV, and Brain IN; YPD, sgRNAs depleted in in vitro growth; Lung IN, sgRNAs depleted in lung survival. Threshold of depletion defined as normalized log2 fold change ≤ −1. (C) Heat map showing the enrichment change of the depleted sgRNAs. Each column corresponds to a sample, and rows represent the 144 sgRNAs that were depleted during transcytosis and intravenous and intranasal infection (normalized log2 fold change ≤ −1). Sequencing read counts of the sgRNA were normalized and log2-transformed and are displayed as colors ranging from blue to red as shown in the key. The sgRNA order was determined by hierarchical clustering, and their corresponding gene IDs are denoted at the right. (D) Function categories of the 142 depleted genes. The number of genes in each functional category is shown on the x axis.

  • Fig. 5 Sfp1 and Wdr1 promote C. neoformans penetration of the blood-brain barrier in vitro and in vivo.

    (A and B) Transcytosis assay using HBMEC monolayer. HBMEC monolayer was incubated with 1 × 106 C. neoformans for 4 hours. Transcytosis frequency (%) was determined [(total CFUs recovered from the lower chamber/total number of cryptococcal cells added to the upper chamber) × 100]. Relative transcytosis (%) was determined [(transcytosis frequency of mutant or complemented strain/transcytosis frequency of wild-type strain) × 100]. (C and D) C. neoformans penetration into brain. CFUs from the brains were determined 24 hours after intravenous infection with 1 × 105 cells. Wild type (Wt) (n = 3); Mutants sfp1∆ and wdr1∆ (n = 3). Complemented strains sfp1::SFP1 and wdr1::WDR1 (n = 4). P value is determined by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., no significant difference. Data shown are means ± SEM. (E and F) Growth test of mutants sfp1∆ and wdr1∆ on YPD agar for 3 days at 37°C. Photo credit: Z. Li, Johns Hopkins University.

Supplementary Materials

  • Supplementary Materials

    RELATe enables genome-scale engineering in fungal genomics

    Zhongming Li and Kwang Sik Kim

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