Improved base editing and functional screening in Leishmania via co-expression of the AsCas12a ultra variant, a T7 RNA polymerase, and a cytosine base editor
- Department of Cell and Developmental Biology, Biocentre, University of Würzburg, Am Hubland, Germany
- Division of Immunology, Paul-Ehrlich-Institut, Germany
Figures
Figure 1
Optimization of cytosine base editor (CBE) single-guide RNA (sgRNA) expression in L. major.
Schematics of base editing strategies to transfect Leishmania parasites either with a (A) single or (B) dual base editing system. (A) Plasmid pLdCH-hyBE4max (Engstler and Beneke, 2023) contains (from left to right) a L. donovani-derived ribosomal RNA (rRNA) promoter, sgRNA expression cassette, hepatitis delta virus (HDV) ribozyme containing transsplice sequence (TSS), hyBE4max CBE, L. donovani-derived A2 intergenic sequence, and hygromycin-resistance marker. (B) Plasmid pTB007-hyBE4max (Engstler and Beneke, 2023) contains separated through intergenic regions (from left to right) a hyBE4max CBE, T7 RNA polymerase (RNAP), and hygromycin-resistance marker. This construct is integrated into the β-tubulin locus of L. major parasites (LmjF.33:339,096-341,104). The CBE sgRNA expression vector contains a puromycin-resistance marker, T7 RNAP promoter, sgRNA expression cassette, and HDV ribozyme. (C) Different versions of the T7 RNAP promoter for CBE sgRNA expression have been tested. (D) Doubling times of tdTomato-expressing L. major transfected non-clonal populations with the single plasmid system and variants of the dual plasmid system for targeting of tdTomato. Error bars show standard deviations of triplicates. Tar: Cells transfected with a CBE sgRNA expression plasmid that facilitates the introduction of a STOP codon within the tdTomato open-reading frame (ORF); Ctrl: Cells transfected with a CBE sgRNA expression plasmid that causes a codon mutation without amino acid change. Asterisks indicate Student’s t-test: *p>0.05. (E) FACS plot of parasites shown in (D), analyzed 7 days post transfection. (F) FACS plot showing tdTomato-expressing and wildtype L. major parasites.
Figure 2 with 1 supplement
Developing an AsCas12a-mediated integration system.
(A) Schematic of the Cas12a-mediated integration approach. A stable L. mexicana cell line maintains plasmid pTB107 as an episome and is co-transfected with a donor DNA reporter construct and a Cas12a crRNA template DNA. Using a T7 RNA polymerase (RNAP) promoter, the Cas12a crRNA template DNA is in vivo transcribed to facilitate a Cas12a-mediated double-strand break (DSB) at the 18S rRNA SSU locus. Using homology flanks the reporter construct is efficiently integrated at the site of the DSB. Plasmid pTB107 thereby allows for the expression of hyBE4max cytosine base editor (CBE), AsCas12a ultra (Zhang et al., 2021), T7 RNAP, and hygromycin-resistance marker. AsCas12a ultra and T7 RNAP are expressed as a fusion protein but then cleaved through a P2A self-cleaving peptide. The reporter constructs consist of a pPLOT plasmid tagging cassette (Beneke et al., 2017) with additional homology flanks, allowing for the expression of mNeonGreen and a blasticidin-resistance marker. (B) Schematic of the Cas12a crRNA template DNA generation. Two overlapping primers are amplified in a PCR. The forward primer is common and contains an unmodified T7 RNAP promoter and an optimized Cas12a direct repeat variant (DR) (DeWeirdt et al., 2021). The guide reverse primer contains the crRNA-specific spacer sequence aligning to the targeted locus and the Cas12a DR to enable the overlap with the forward primer. (C and E) FACS plot showing L. mexicana pTB107 non-clonal populations following the transfection described in (A). Percentages represent the remaining proportion of mNeonGreen-expressing cells. (C) The homology flank (HF) length used for reporter construct integration has been varied and the additional use of Cas12a crRNA was tested. The transfection of a circular plasmid, containing the reporter construct, was included as a control (episomal). (E) Six different Cas12a crRNAs have been tested using two different integration loci (as described in the main text). (D and F) Efficiency of transfections shown in (C and E) were measured. The number of cells required for the transfection to obtain one transfectant is shown. Error bars show standard deviations of triplicates.
Figure 2—figure supplement 1
Verification of AsCas12a-mediated reporter construct integration.
(A) Locus map of the 18S rRNA SSU locus in L. mexicana pTB107 cells before and after co-transfection of Cas12a crRNA-1 and a reporter construct (as described in Figure 2A, C, and D). The positions of Cas12a crRNA-1 and homology flanks (HFs) used for the integration of the reporter are highlighted. Amplicons of PCRs spanning the integration sites are indicated and amplification results are shown in (B), with DNA sizes highlighted in bp. (C) Sanger sequencing trace plots of amplicons from non-clonal populations shown in (B). The site of integration into the 18S rRNA locus is highlighted with a dotted line. (D) Locus map of the 18S rRNA SSU locus in L. mexicana pTB107 cells expressing tdTomato before and after co-transfection of various Cas12a crRNAs and a reporter construct (as described in Figure 2A, E, and F). The positions of Cas12a crRNA-1, 2, 3, 4, 5, and 6 are highlighted. Indicated HF positions have been used for the integration of the tdTomato expression construct on one allele but individual HFs adjacent to crRNA-1–6 have been used for integration of the reporter construct. Amplicons of PCRs spanning the integration sites are indicated and amplification results are shown in (E) for verification of crRNA-4. (F) Sanger sequencing trace plots of amplicons from non-clonal populations shown in (E).
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Figure 2—figure supplement 1—source data 1
Raw DNA images of Figure 2—figure supplement 1B and E with labels.
- https://cdn.elifesciences.org/articles/97437/elife-97437-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Raw DNA images of Figure 2—figure supplement 1B and E without labels.
- https://cdn.elifesciences.org/articles/97437/elife-97437-fig2-figsupp1-data2-v1.zip
Figure 3
Integration of cytosine base editor (CBE) single-guide RNA (sgRNA) expression cassettes via AsCas12a ultra.
(A) Schematic of Cas12a-mediated integration of the CBE sgRNA expression construct. The pTB107 stable cell line is co-transfected with two constructs: (1) a Cas12a crRNA template DNA and (2) a CBE sgRNA expression construct. The Cas12a crRNA template DNA is in vivo transcribed and consists of an unmodified T7 RNA polymerase (RNAP) promoter (T7wt, light green), a Cas12a direct repeat (DR, red) and a 20 nt Cas12a guide target sequence (SPACER, blue). The CBE sgRNA expression construct is integrated into the 18S rRNA SSU locus, following the Cas12a-mediated double-strand break (DSB). This donor construct contains two homology flanks (HF, yellow), a puromycin-resistance marker (gray), a T7 T-10 GG promoter (green), a guide target sequence (orange), a Cas9 scaffold (dark green), and an HDV (purple). (B) FACS plots show pTB107 parasites that express tdTomato and have been transfected with pTB104 and pTB105 CBE sgRNA expression cassettes, containing a tdTomato-targeting guide. Percentages represent the remaining proportion of non-clonal tdTomato-expressing cells. (C) Following dilutions after transfection, the number of puromycin-resistant transfectants obtained per transfected cell was calculated. Error bars show standard deviations of triplicates. (D) Doubling times for transfected Leishmania parasites shown in (B). PAR: parental cell line; UT: Un-transfected control.
Figure 4 with 4 supplements
Targeting PF16 via Cas12a-delivered cytosine base editor (CBE) single-guide RNA (sgRNA) expression cassettes.
L. major, L. mexicana, L. donovani wildtype, and tdTomato/pTB107 cell lines were transfected with a range of CBE sgRNA expression constructs in order to functionally mutate PF16. Different strategies for construct delivery and sgRNA expression were tested, including the integration of constructs into the neomycin-resistance marker (using Cas12a sgRNA-6) and the 18S rRNA SSU locus (Cas12a sgRNA-4), each using 60 nt homology flanks. For comparison to our previous system (Engstler and Beneke, 2023), we also expressed CBE sgRNAs from an episome and transfected pLdCH-hyBE4max into L. major. (A) Violin plot of pooled replicates from motility tracked non-clonal populations. The mean velocity of tracked cells was plotted 14 days post transfection for L. major, and 6 and 16 days post transfection for L. mexicana and L. donovani. The total percentage of tracked cells showing a velocity of less than 1 µm/s is highlighted. Each population was analyzed using a Cramér-von Mises test to detect any shift in the population distribution toward lower speed. Percentages are marked with an asterisk when that shift was significant (*p>0.05). (B) Corresponding Sanger sequencing trace plots. Blue shading: 20 nt guide target sequence. Red dotted lines: hyBE4max editing window.
Figure 4—figure supplement 1
Oxford Nanopore Technology (ONT) and Illumina sequencing of non-clonal transfectants.
L. mexicana pTB107 cells expressing tdTomato were co-transfected with Cas12a crRNA-4 template DNA constructs and a cytosine base editor (CBE) single-guide RNA (sgRNA) expression cassette containing a PF16 targeting guide sequence. Isolated DNA from non-clonal mutant populations were subjected to ONT and Illumina sequencing 16 days post transfection. (A) Illumina sequencing reads were mapped against the L. mexicana MHOMGT2001U1103 genome annotation. The edited consensus sequence is shown alongside with the reference sequence and calculated editing rates (based on 35× genome coverage). (B and C) ONT reads were mapped against two customized L. mexicana MHOMGT2001U1103 genome annotations and viewed in the IGV genome browser (Robinson et al., 2011), with uniquely mapped reads in gray and reads with multiple alignments in white. Reads mapped against the PGKB 5’ and 3’UTR are over-represented as these UTRs are also present in the pTB107 plasmid. (B) ONT reads were mapped to a customized genome in which we assumed that the 18S rRNA SSU locus had one integrated copy of the tdTomato expression cassette on one allele and one integrated copy of the CBE sgRNA expression construct on the other allele. Schematics show the annotation of genetic elements assumed to be on each allele. The position of one allele in respect to the position on the other allele is indicated with red arrows. The position of the Cas12a crRNA-4 to integrate the CBE sgRNA expression cassette is additionally highlighted. Black arrows indicate unique ONT reads that cover the entire customized locus. Reads are trimmed as the genome annotation is incomplete (NNN gap highlighted). (C) ONT reads were mapped to a customized genome in which we assumed that both cassettes were integrated on the same allele and could be detected on a single ONT read. (D) Illumina read coverage was normalized and chromosome coverage was compared to coverage of individual genetic features on these chromosomes.
Figure 4—figure supplement 2
Blast analysis of Oxford Nanopore Technology (ONT) sequencing.
(A) Schematic of tdTomato and cytosine base editor (CBE) single-guide RNA (sgRNA) expression cassette integration into the 18S rRNA locus. (B) Overview of the standard nucleotide Blast search process, where ONT fastq reads were queried against the coding sequences of the tdTomato reporter gene and the sgRNA expression cassette. (C and D) Alignment of matched ONT reads against the 18S rRNA locus, confirming that contigs correspond to the integration site and adjacent genomic sequences. The IDs for each matching contig are provided, and the data are available at the European Nucleotide Archive (ENA) (Accession number: PRJEB83088).
Figure 4—figure supplement 3
Sequencing of clones isolated from an AsCas12a-mediated library transfection.
L. mexicana, L. major, and L. donovani tdTomato/pTB107 cell lines were co-transfected with a library containing a mixture of 15 guides and Cas12a crRNA-4 targeting the 18S rRNA SSU locus. Library clones from library transfection were isolated and their cytosine base editor (CBE) single-guide RNA (sgRNA) amplified for Sanger sequencing. (A) Sanger sequencing trace plots of the library before and 6 days post transfection. The T7 T10 GG promoter, sgRNA target sequence, and the beginning of the sgRNA scaffold sequence are highlighted. (B) Sanger sequencing trace plots of guide cassettes sequenced from clones. Highlighted in red are clones that maintain more than one sgRNA.
Figure 4—figure supplement 4
Novel scoring for cytosine base editor (CBE) single-guide RNA (sgRNA) sorting.
Schematic of new CBE scoring matrix. An sgRNA receives the highest score, when the ratio of available cytosines to resulting STOP codons is 1:1. Additionally, the final score depends on the number of possible STOP codons and position of cytosines within the editing window (activities indicated above: high, mid, and low).
Figure 5
Small-scale loss-of-function screen.
(A) Cas12a-mediated integration of the cytosine base editor (CBE) single-guide RNA (sgRNA) expression construct library involves co-transfecting a cell line expressing tdTomato and containing either pTB107 or pTB106 with two constructs: (1) a Cas12a crRNA template DNA and (2) a CBE sgRNA expression construct library. Integration is facilitated by Cas12a-mediated double-strand breaks (DSBs), as described in Figure 3. (B) The library, transfected in three independent replicates, contains 15 non-targeting sgRNAs and 24 sgRNAs targeting essential or fitness-associated genes. Plasmid library DNA and genomic DNA from transfected cells were extracted before transfection (0 hr) and after several subcultures (168, 216, and 288 hr post-transfection). (C) Samples underwent Illumina amplicon sequencing for downstream analysis. (D and E) For each sgRNA, the ratio of normalized counts between 0 hr and 168, 216, or 288 hr was calculated and plotted against raw read counts. Averages from triplicates are displayed with error bars indicating the standard deviation. A 0.99 confidence ellipse, calculated from non-targeting controls, highlights significant sgRNA depletion when data points fall outside the ellipse. CBEs used in the screen differ in the RAD51 single-stranded DNA-binding domain within the hyBE4max enzyme, derived from either L. major (pTB106: L.m. RAD51-BD) or Homo sapiens (pTB107: H.s. RAD51-BD). (E) For the comparison, data from Engstler and Beneke, 2023 (LeishBASEedit v1) were re-analyzed and plotted alongside the newly generated data from this study (LeishBASEedit v2).
Figure 6
PF16 gene replacement by using CRISPR/AsCas12a ultra.
(A) Map of the PF16 locus before and after targeting with Cas12a 5’ and 3’crRNA positions indicated. The PF16-ORF is replaced with a pTBlast and pTPuro cassette. Primers used for verification of the ORF replacement are highlighted with PCR amplicon length. (B) Visualization of PCR amplicons, showing expected products for pTPuro and pTBlast in the ΔPF16 lane and the amplicon of the PF16-ORF in the PARENT lane (L. mexicana tdTomato/pTB107 cell line). (C and D) Sanger sequencing trace plots of amplicons in (B), showing sequencing of the upstream region in (C) and downstream region in (D). Black arrows indicate Cas12a double-strand breaks (DSBs). (E) Violin plot of pooled replicates from motility tracked non-clonal populations, including the ΔPF16 and PARENT cell line. The total percentage of tracked cells showing a velocity of less than 1 µm/s is highlighted. Each population was analyzed using a Cramér-von Mises test to detect any shift in the population distribution toward lower speed. Percentages are marked with an asterisk when that shift was significant (*p>0.05). ORF, open-reading frame.
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Figure 6—source data 1
Raw DNA images of Figure 6B with labels.
- https://cdn.elifesciences.org/articles/97437/elife-97437-fig6-data1-v1.zip
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Figure 6—source data 2
Raw DNA images of Figure 6B without labels.
- https://cdn.elifesciences.org/articles/97437/elife-97437-fig6-data2-v1.zip
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Leishmania mexicana) | L. mexicana wildtype | Eva Gluenz laboratory | WHO strain MNYC/BZ/62/M379 | Used TriTrypDB (release 59, Aslett et al., 2010) reference annotation: L. mexicana MHOMGT2001U1103 |
| Cell line (Leishmania major) | L. major wildtype | Eva Gluenz laboratory | Strain Friedlin | Used TriTrypDB (release 59, Aslett et al., 2010) reference annotation: L. major Friedlin |
| Cell line (Leishmania donovani) | L. donovani wildtype | Joachim Clos laboratory (Decuypere et al., 2005) | Strain BPK190 | Used TriTrypDB (release 59, Aslett et al., 2010) reference annotation: L. donovani BPK282A1 |
| Recombinant DNA reagent | pTB007-hyBE4max | Engstler and Beneke, 2023 | Contains CBE hyBE4max and T7 RNAP | |
| Recombinant DNA reagent | pLdCH-hyBE4max | Engstler and Beneke, 2023 | Contains CBE hyBE4max and CBE sgRNA | |
| Recombinant DNA reagent | pLdCH-hyBE4max-LmajDBD | Engstler and Beneke, 2023 | Contains Leishmania-optimized CBE hyBE4max and CBE sgRNA | |
| Recombinant DNA reagent | pTB107 | This study | Contains CBE hyBE4max, AsCas12a ultra, and T7 RNAP (available in versions with either a hygromycin or phleomycin-resistance marker) | |
| Recombinant DNA reagent | pTB106 | This study | Contains Leishmania-optimized CBE hyBE4max, AsCas12a ultra, and T7 RNAP (available in versions with either a hygromycin or phleomycin-resistance marker) | |
| Recombinant DNA reagent | pTB102 | This study | Contains T7 RNAP promoter-driven CBE sgRNA expression cassette without homology flanks | |
| Recombinant DNA reagent | pTB104 | This study | Contains T7 RNAP promoter-driven CBE sgRNA expression cassette with homology flanks for neomycin-resistance gene | |
| Recombinant DNA reagent | pTB105 | This study | Contains T7 RNAP promoter-driven CBE sgRNA expression cassette with homology flanks for 18S rRNA SSU locus | |
| Software | TriTrypDB (release 59) | Aslett et al., 2010 | https://tritrypdb.org/tritrypdb/app | |
| Software | LeishBASEedit | This study | http://www.leishbaseedit.net/ | See description under ‘Automated CBE guide design using LeishBASEedit’ |
| Gene (L. donovani BPK282A1, L. major Friedlin, L. mexicana MHOMGT2001U1103) | PF16 | TriTrypDB (release 59) (Aslett et al., 2010) | LdBPK_201450.1, LmjF.20.1400, LmxM.20.1400 |
Additional files
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Supplementary file 1
Primers used in this study.
- https://cdn.elifesciences.org/articles/97437/elife-97437-supp1-v1.xlsx
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Supplementary file 2
Plasmid maps.
GenBank files of pTB102, pTB104, pTB105, pTB106, and pTB107.
- https://cdn.elifesciences.org/articles/97437/elife-97437-supp2-v1.zip
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Supplementary file 3
A protocol for AsCas12a-mediated transfection of cytosine base editor (CBE) single-guide RNA (sgRNA) expression cassettes.
Step-by-step protocol includes guidelines for PCR amplification of donor DNA and Cas12a crRNA template DNA, as well as preparations for transfection.
- https://cdn.elifesciences.org/articles/97437/elife-97437-supp3-v1.pdf
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Supplementary file 4
Analysis of small-scale loss-of-function screen.
The analysis file contains raw and normalized amplicon sequencing counts for each single-guide RNA (sgRNA) subjected to the screen, along with the ratio analysis shown in Figure 5.
- https://cdn.elifesciences.org/articles/97437/elife-97437-supp4-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/97437/elife-97437-mdarchecklist1-v1.pdf
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Source data 1
Raw DNA images of Figure 2—figure supplement 1B and E.
Details are described in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/97437/elife-97437-data1-v1.zip
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Source data 2
Raw DNA images of Figure 6B.
Details are described in Figure 6.
- https://cdn.elifesciences.org/articles/97437/elife-97437-data2-v1.zip
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Source code 1
Novel scoring for cytosine base editor (CBE) single-guide RNA (sgRNA) sorting.
Source code for CBE sgRNA scoring described above. The updated data sets can be accessed at https://www.leishbaseedit.net/. The source code for the guide scoring has been uploaded to https://github.com/ElisabethMeiser/Collaboration_Beneke_Meiser, copy archived at Meiser, 2024.
- https://cdn.elifesciences.org/articles/97437/elife-97437-code1-v1.zip
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Improved base editing and functional screening in Leishmania via co-expression of the AsCas12a ultra variant, a T7 RNA polymerase, and a cytosine base editor
eLife 13:RP97437.
https://doi.org/10.7554/eLife.97437.3
