Multiple Applications of a Transient CRISPR-Cas9 Coupled with Electroporation (TRACE) System in the Cryptococcus neoformans Species Complex

Abstract

Cryptococcus neoformans is a fungal pathogen that claims hundreds of thousands of lives annually. Targeted genetic manipulation through biolistic transformation in C. neoformans drove the investigation of this clinically important pathogen at the molecular level. Although costly and inefficient, biolistic transformation remains the major method for editing the Cryptococcus genome as foreign DNAs introduced by other methods such as electroporation are predominantly not integrated into the genome. Although the majority of DNAs introduced by biolistic transformation are stably inherited, the transformation efficiency and the homologous integration rate (∼1-10%) are low. Here, we developed a Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation (TRACE) system for targeted genetic manipulations in the C. neoformans species complex. This method took advantages of efficient genome integration due to double-strand breaks created at specific sites by the transient CRISPR-Cas9 system and the high transformation efficiency of electroporation. We demonstrated that TRACE can efficiently generate precise single-gene deletion mutants using the ADE2 locus as an example. This system can also effectively delete multiple genes in a single transformation, as evident by the successful generation of quadruple mfα1Δ2Δ3Δ4Δ mutants. In addition to generating gene deletion mutants, we complemented the ade2Δ mutant by integrating a wild-type ADE2 allele at the "safe haven" region (SH2) via homologous recombination using TRACE. Interestingly, introduced DNAs can be inserted at a designated genetic site without any homologous sequences, opening up numerous other applications. We expect that TRACE, an efficient, versatile, and cost-effective gene editing approach, will greatly accelerate research in this field.

Keywords: CRISPR-Cas9; Cryptococcus neoformans; biolistic transformation; double-strand break; ectopic integration; electroporation; gene complementation; gene disruption; gene family.

Figure 1

Construction of the Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation (TRACE) system. A diagram of the three elements of the TRACE system generated via PCR (left panel). Arrows represent the position and the direction of the primers. A diagram for the working concept of the TRACE system (right panel). When the three elements are all expressed in Cryptococcus cells, single-guide RNA (sgRNA) will guide Cas9 to the specific site that matches the target sequence and then Cas9 will generate a double-strand break (DSB). The deletion construct will serve as the template during DSB repair and eventually replace the gene of interest with the drug-resistant marker by homologous recombination. NAT: nourseothricin resistance cassette

Figure 2

TRACE [Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation] can dramatically increase the gene disruption rate. The TRACE system was used to generate ADE2 deletion mutants in both serotype A (H99 and cku80Δ H99) and serotype D (JEC21 and XL280) strains. Electroporation with no Cas9/sgRNA (single-guide RNA) was done in parallel. The deletion construct was maintained at the same concentration. Red colonies indicate mutants with disrupted ADE2.

Figure 3

Deletion of ADE2 (1 kb arms) using TRACE [Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation] showed transient CAS9 expression and high efficiency of homologous integration in both serotype A and D strain backgrounds. (A) A diagram for the diagnostic RFLP for correct homologous replacement of the ADE2 gene (i). Arrows indicate the positions of the primers for PCR. For candidates in the H99 background, PCR product was digested by the restriction enzyme NotI. A diagram of Cas9 with arrows pointing to the positions of the primers used to amplify the Cas9 coding sequence (ii). (B) RFLP analysis of randomly selected ade2Δ candidates in the H99 background. Candidates that showed correct bands (2.3 and 1.8 kb) after NotI digestion were marked with stars. Wild-type (WT) (H99) showed one single 4.2-kb band because it lacks a NotI cutting site. Genomic DNA of an ade2Δ strain from our previous study (YP27) was used as a positive control (PC). M, marker. (C) No Cas9 coding sequence (4.4 kb) could be detected in any of the selected ade2Δ candidates (H99) or the WT strain. Plasmid pDD162 was used as a PC. (D) In the PCR analysis of ade2Δ candidates in the XL280 background, candidates that showed the correct band (4.4 kb) were marked with stars. WT showed one single 3.6-kb band. NAT: nourseothricin resistance cassette

Figure 4

Homologous arms of 500 bp, but not 50 bp, are sufficient for efficient homologous replacement. (A) The ade2Δ candidates (H99) generated with the deletion construct carrying 500-bp homologous arms were screened for homologous replacement by RFLP analysis. Candidates with correct 2.3- and 1.8-kb bands after NotI digestion were marked with stars. Wild-type (WT) (H99) showed one single 4.2-kb band. Genomic DNA of a previously confirmed ade2Δ mutant was used as a positive control (PC). M, marker. (B) A diagram for the insertion rather than replacement of the ADE2 locus with the construct carrying 50-bp arms. Positions of the primers used for the PCR screen in (C) are indicated with arrows. (C) PCR screen of transformants generated with the deletion construct with 50-bp arms. Candidates with insertion should yield a band of 2.1 kb while WT should yield a band of ∼200 bp. The negative control (NC) had no DNA template. NAT: nourseothricin resistance cassette

Figure 5

Transformation efficiency and gene disruption rate are dependent on the dose of the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 elements. Different doses of Cas9 and single-guide RNA (sgRNA) were used to transform the same batch of H99 cells using TRACE (Transient CRISPR-Cas9 coupled with Electroporation).

Figure 6

Low concentration of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 elements are sufficient for correct gene deletion and can reduce the chance of single-guide RNA (sgRNA) integration. (A) Transformation of serotype A and serotype D strains with TRACE (Transient CRISPR-Cas9 coupled with Electroporation) (100 ng sgRNA and 170 ng Cas9) or without TRACE to disrupt the ADE2 gene. (B) After five consecutive passages on nonselective YPD medium, red and white colonies generated by TRACE were replicated onto nonselective and selective medium. Nonstable transformants showed spotty growth on selective medium. (C) White colonies generated from TRACE and normal electroporation were tested for stability after five passages. Black arrows indicated stable candidates. (D) The ade2Δ candidates generated by TRACE with the low dose of sgRNA and Cas9 were screened for homologous replacement by RFLP. Candidates that yielded correct bands of 2.3 and 1.8 kb after NotI digestion were marked with stars. Wild-type (WT) (H99) showed one single 4.2-kb band. Genomic DNA of one ade2Δ strain served as the positive control (PC). M, marker. (E and F) The ade2Δ candidates generated by TRACE with the low dose of sgRNA and Cas9 were screened for the presence of sgRNA (E) and CAS9 (F). The negative control (NC) had no DNA template.

Figure 7

TRACE [Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation] can generate a quadruple mfα1Δ2Δ3Δ4Δ mutant in H99 with a single transformation. (A) A diagram of the mating type locus of H99. The mating type locus contains four pheromone genes, MFα1–4. Three pheromone genes—MFα2, MFα3, and MFα4—are identical in DNA sequence. (B) A diagram of the two deletion constructs carrying NAT and NEO drug-resistance markers (NAT: nourseothricin resistance cassette; NEO: Neomycin resistance cassette). The constructs were designed to delete MFα1–2 or MFα3–4 by homologous recombination. Asterisks indicate the target sequences of single-guide RNAs (sgRNAs). (C) Sequence alignment of the four pheromone genes. The coding sequences are underlined. Red and blue sequences are the target sequences of sgRNAs. The corresponding PAM (protospacer-adjacent motif) sequences are highlighted in yellow. (D) A diagram of the cellular process of bisexual mating in C. neoformans. Cell–cell recognition and cell fusion is controlled by the pheromone pathway activated by Mat2. (E) α isolates of wild-type (WT) H99, mat2Δ, mfα1Δ2Δ3Δ4Δ (three randomly selected transformants), and mfα1Δ2Δ were cocultured with the mating type a partner JEC20. Successful mating leads to filamentation that will confer a white and fluffy appearance at the colony edge. Upper panel shows the images of the whole colonies and the lower panel shows the images of the edge of each colony.

Figure 8

ADE2 complementation in the H99 safe haven (SH2) region by TRACE [Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation]. (A) Complementation of ade2Δ by TRACE with the complementation construct carrying the homologous arms (left) or the complementation construct without (W/O) any homologous arms (right). (B and C) A diagram of the complementation construct with the homologous arms (B) or without arms (C) (NEO: neomycin resistance cassette). Positions of the primers used for further PCR analysis in (D and E) are indicated with arrows. (D and E) PCR screening of randomly selected white colonies from transformation with the complementation construct with the homologous arms (D) or without the homologous arms (E) for the integration events at the SH2 region. Correct integration into the SH2 region should yield a band of 7.4 kb. Wild-type (WT) should yield a band of 2 kb.