Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

Abstract

Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR‐Cas9 has been applied successfully to these parasites providing a robust tool to study non‐essential gene functions. Here, we have developed a versatile inducible system combining Di‐Cre recombinase and CRISPR‐Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre‐recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di‐Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed “universal” template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR‐based (molecular cloning‐free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.

CLK1/CLK2-driven signalling at the Leishmania kinetochore is captured by spatially referenced proximity phosphoproteomics

“…Attempts to delete KKT24 or KKT26 using CRISPR- Cas9 in L. mexicana promastigotes were unsuccessful, indicating these kinetochore proteins are essential to parasite survival. We, therefore, opted for an inducible gene deletion strategy, using Cas9-directed insertion to introduce loxP sites flanking the endogenous genes 26 . The addition of rapamycin induces dimerisation of DiCre recombinase, which then excises the loxP-flanked gene (Fig.…”

Protein kinase signalling at the Leishmania kinetochore captured by XL-BioID

“…Primers used to generate sgRNA expression cassettes and repair cassettes were designed using http://www.leishgedit.net 15 or manually for the LoxP lines 25 . Cassettes were generated by PCR, precipitated with glycogen, resuspended in 10μl water for each transfection and heat sterilised for 5mins at 95°C.…”

“…Attempts to knockout KKT24 or KKT26 by Cas9 in L. mexicana promastigotes were unsuccessful, indicating these kinetochore proteins are essential to parasite survival. We therefore opted for an inducible gene deletion strategy, using Cas9-directed insertion to introduce loxP sites flanking the endogenous genes 25 . Addition of rapamycin induces dimerisation of DiCre recombinase which then excises the loxP flanked gene (Fig.…”

Cell Cycle, Telomeres, and Telomerase in Leishmania spp.: What Do We Know So Far?

“…Our very little knowledge about telomerases in Leishmania spp. reflects the challenges involved in the genome manipulation of these parasites [114,115]. However, the recent advent of genome editing using CRISPR-Cas9 and its variations should help resolve some of these functional questions that are still lingering, as has been done for other genes and in other species [116][117][118].…”