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Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum.

Wagner JC, Platt RJ, Goldfless SJ, Zhang F, Niles JC - Nat. Methods (2014)

Bottom Line: Malaria is a major cause of global morbidity and mortality, and new strategies for treating and preventing this disease are needed.Here we show that the Streptococcus pyogenes Cas9 DNA endonuclease and single guide RNAs (sgRNAs) produced using T7 RNA polymerase (T7 RNAP) efficiently edit the Plasmodium falciparum genome.Targeting the genes encoding native knob-associated histidine-rich protein (kahrp) and erythrocyte binding antigen 175 (eba-175), we achieved high (≥ 50-100%) gene disruption frequencies within the usual time frame for generating transgenic parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

ABSTRACT
Malaria is a major cause of global morbidity and mortality, and new strategies for treating and preventing this disease are needed. Here we show that the Streptococcus pyogenes Cas9 DNA endonuclease and single guide RNAs (sgRNAs) produced using T7 RNA polymerase (T7 RNAP) efficiently edit the Plasmodium falciparum genome. Targeting the genes encoding native knob-associated histidine-rich protein (kahrp) and erythrocyte binding antigen 175 (eba-175), we achieved high (≥ 50-100%) gene disruption frequencies within the usual time frame for generating transgenic parasites.

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Related in: MedlinePlus

Assessing the fate of kahrp-sgRNA-T-induced cleavage of the kahrp locus in the absence of a homologous donor plasmid. (a) Schematic of the validated pT7 RNAP and pCas9-kahrp sgRNA-T plasmids used in this experiment. (b) Deep sequencing analysis of the region targeted for cleavage by kahrp-sgRNA-T. A representative set (1 of 3 independent experiments) of twenty bacterial clones analyzed by Sanger sequencing is illustrated.
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Figure 3: Assessing the fate of kahrp-sgRNA-T-induced cleavage of the kahrp locus in the absence of a homologous donor plasmid. (a) Schematic of the validated pT7 RNAP and pCas9-kahrp sgRNA-T plasmids used in this experiment. (b) Deep sequencing analysis of the region targeted for cleavage by kahrp-sgRNA-T. A representative set (1 of 3 independent experiments) of twenty bacterial clones analyzed by Sanger sequencing is illustrated.

Mentions: The potential for unintended gene disruptions due to induced off-target strand breaks and repair by the error-prone non-homologous end joining (NHEJ) mechanism has been described in human cells 17–20. This has led to exploration of strategies to mitigate these undesirable outcomes 21–23. Therefore, we sought preliminary insight into how frequent such off-target events could be in P. falciparum. Notably, bioinformatics analyses have revealed that P. falciparum lacks canonical NHEJ components24, 25. Additionally, a recent study examining chromosomal double strand breaks induced by the meganuclease I-Sce1 showed that these are very efficiently and exclusively repaired by homologous donor sequence when present. In the absence of a suitable donor, however, an NHEJ-like repair process of unknown mechanism that resulted in elimination of the I-Sce1 target site was observed 26. To understand how an off-target Cas9 and sgRNA-T-induced chromosomal double strand break might be processed in P. falciparum, we simulated such an event by expressing the kahrp-sgRNA-T used earlier to mediate efficient editing of the kahrp locus, but in the absence of a suitable homologous donor plasmid [pCas9-kahrp sgRNA-T and pT7 RNAP]. Control parasites expressing pUC19-sgRNA-T [pCas9-pUC19 sgRNA-T and pT7 RNAP] were transfected in parallel. In two independent experiments, we observed no gross defects in relative growth between the two parasite lines generated, as both reached working parasitemias at similar times post-transfection, and qualitatively expanded comparably thereafter. To determine whether NHEJ-like events had occurred within the kahrp region targeted for cleavage, we isolated total genomic DNA from the kahrp- and pUC19- sgRNA-T transfected lines for deep sequencing. With greater than 3x106 reads each from the kahrp- and pUC19- sgRNA-T experiments, we were unable to detect indels above the limit of detection for the MiSeq sequencing method. The MiSeq error rate is about 0.001 events per 100 bp27, which leads to an expected background of ~2,000 total indels at our read length (70 bp) and depth (~3x106). We observed ~1,500 total indels in both test and control samples, which is indistinguishable from the expected background level for the sequencing method (Fig 3b). These data suggest that NHEJ-like events occurring at Cas9-kahrp-sgRNA-T-induced cleavage sites are likely to be infrequent. Presently, we do not understand why cleavage induced by Cas9-sgRNA versus I-Sce1 meganuclease in the absence of a suitable donor sequence produces different repair outcomes. However, discrepancies in how double strand breaks induced by various nuclease platforms, including zinc finger nucleases, TALENs and I-Sce1, are repaired have been described 28. Understanding the basis for these differences in repair is an active research area given the implications for improving genome-editing efficiency. With respect to applications in P. falciparum, our findings taken together with previous genome editing studies using zinc finger nucleases5 and I-Sce126 suggest that homologous repair events should be strongly favored during CRISPR/Cas9-mediated genome editing over potentially deleterious off-target NHEJ-like outcomes.


Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum.

Wagner JC, Platt RJ, Goldfless SJ, Zhang F, Niles JC - Nat. Methods (2014)

Assessing the fate of kahrp-sgRNA-T-induced cleavage of the kahrp locus in the absence of a homologous donor plasmid. (a) Schematic of the validated pT7 RNAP and pCas9-kahrp sgRNA-T plasmids used in this experiment. (b) Deep sequencing analysis of the region targeted for cleavage by kahrp-sgRNA-T. A representative set (1 of 3 independent experiments) of twenty bacterial clones analyzed by Sanger sequencing is illustrated.
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Related In: Results  -  Collection

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Figure 3: Assessing the fate of kahrp-sgRNA-T-induced cleavage of the kahrp locus in the absence of a homologous donor plasmid. (a) Schematic of the validated pT7 RNAP and pCas9-kahrp sgRNA-T plasmids used in this experiment. (b) Deep sequencing analysis of the region targeted for cleavage by kahrp-sgRNA-T. A representative set (1 of 3 independent experiments) of twenty bacterial clones analyzed by Sanger sequencing is illustrated.
Mentions: The potential for unintended gene disruptions due to induced off-target strand breaks and repair by the error-prone non-homologous end joining (NHEJ) mechanism has been described in human cells 17–20. This has led to exploration of strategies to mitigate these undesirable outcomes 21–23. Therefore, we sought preliminary insight into how frequent such off-target events could be in P. falciparum. Notably, bioinformatics analyses have revealed that P. falciparum lacks canonical NHEJ components24, 25. Additionally, a recent study examining chromosomal double strand breaks induced by the meganuclease I-Sce1 showed that these are very efficiently and exclusively repaired by homologous donor sequence when present. In the absence of a suitable donor, however, an NHEJ-like repair process of unknown mechanism that resulted in elimination of the I-Sce1 target site was observed 26. To understand how an off-target Cas9 and sgRNA-T-induced chromosomal double strand break might be processed in P. falciparum, we simulated such an event by expressing the kahrp-sgRNA-T used earlier to mediate efficient editing of the kahrp locus, but in the absence of a suitable homologous donor plasmid [pCas9-kahrp sgRNA-T and pT7 RNAP]. Control parasites expressing pUC19-sgRNA-T [pCas9-pUC19 sgRNA-T and pT7 RNAP] were transfected in parallel. In two independent experiments, we observed no gross defects in relative growth between the two parasite lines generated, as both reached working parasitemias at similar times post-transfection, and qualitatively expanded comparably thereafter. To determine whether NHEJ-like events had occurred within the kahrp region targeted for cleavage, we isolated total genomic DNA from the kahrp- and pUC19- sgRNA-T transfected lines for deep sequencing. With greater than 3x106 reads each from the kahrp- and pUC19- sgRNA-T experiments, we were unable to detect indels above the limit of detection for the MiSeq sequencing method. The MiSeq error rate is about 0.001 events per 100 bp27, which leads to an expected background of ~2,000 total indels at our read length (70 bp) and depth (~3x106). We observed ~1,500 total indels in both test and control samples, which is indistinguishable from the expected background level for the sequencing method (Fig 3b). These data suggest that NHEJ-like events occurring at Cas9-kahrp-sgRNA-T-induced cleavage sites are likely to be infrequent. Presently, we do not understand why cleavage induced by Cas9-sgRNA versus I-Sce1 meganuclease in the absence of a suitable donor sequence produces different repair outcomes. However, discrepancies in how double strand breaks induced by various nuclease platforms, including zinc finger nucleases, TALENs and I-Sce1, are repaired have been described 28. Understanding the basis for these differences in repair is an active research area given the implications for improving genome-editing efficiency. With respect to applications in P. falciparum, our findings taken together with previous genome editing studies using zinc finger nucleases5 and I-Sce126 suggest that homologous repair events should be strongly favored during CRISPR/Cas9-mediated genome editing over potentially deleterious off-target NHEJ-like outcomes.

Bottom Line: Malaria is a major cause of global morbidity and mortality, and new strategies for treating and preventing this disease are needed.Here we show that the Streptococcus pyogenes Cas9 DNA endonuclease and single guide RNAs (sgRNAs) produced using T7 RNA polymerase (T7 RNAP) efficiently edit the Plasmodium falciparum genome.Targeting the genes encoding native knob-associated histidine-rich protein (kahrp) and erythrocyte binding antigen 175 (eba-175), we achieved high (≥ 50-100%) gene disruption frequencies within the usual time frame for generating transgenic parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

ABSTRACT
Malaria is a major cause of global morbidity and mortality, and new strategies for treating and preventing this disease are needed. Here we show that the Streptococcus pyogenes Cas9 DNA endonuclease and single guide RNAs (sgRNAs) produced using T7 RNA polymerase (T7 RNAP) efficiently edit the Plasmodium falciparum genome. Targeting the genes encoding native knob-associated histidine-rich protein (kahrp) and erythrocyte binding antigen 175 (eba-175), we achieved high (≥ 50-100%) gene disruption frequencies within the usual time frame for generating transgenic parasites.

Show MeSH
Related in: MedlinePlus