Optimisation of CBE 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, single-guide RNA (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 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, single-guide RNA (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 parasites transfected 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 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), analysed 7 days post transfection. (F) FACS plot showing tdTomato-expressing and wildtype L. major parasites.

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 RNAP promoter, the Cas12a crRNA template DNA is in vivo transcribed to facilitate a Cas12a-mediated 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 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 consists 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 reaction. The forward primer is common and contains an unmodified T7 RNAP promoter and an optimised 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 parasites 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.

Integration of CBE 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 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 DSB. This donor construct contains two homology flanks (HF, yellow), a puromycin-resistance marker (grey), 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 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.

Targeting PF16 via Cas12a-delivered CBE 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 analysed using a Cramér-von Mises Test to detect any shift in the population distribution towards lower speed. Percentages are marked with an asterisks 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.

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 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 analysed using a Cramér-von Mises Test to detect any shift in the population distribution towards lower speed. Percentages are marked with an asterisks when that shift was significant (*p > 0.05).

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 HFs used for the integration of the reporter are highlighted. Amplicons of PCR reactions 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 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 to 6 have been used for integration of the reporter construct. Amplicons of PCR reactions 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 shown in (E).

ONT and Illumina sequencing of transfectants.

L. mexicana pTB107 cells expressing tdTomato were co-transfected with Cas12a crRNA-4 template DNA constructs and a CBE sgRNA expression cassette containing a PF16 targeting guide sequence. Isolated DNA from parasites 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 grey 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.

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 mixtures of 15 guides and Cas12a crRNA-4 targeting the 18S rRNA SSU locus. Library clones from library transfection were isolated and their CBE 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.

Novel scoring for CBE sgRNA sorting.

Schematic of new CBE scoring matrix. A 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).