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Research Article
Shiroh Iwanaga
Osaka University
Corresponding Author
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The current CRISPR/Cas9 system for Plasmodium falciparum suffers from technical problems caused by plasmid constructs, such as delays in establishing transgenic parasites during drug selection and unexpected integration of circular donor DNA by single-crossover recombination. Although these problems can be solved by using linear donor templates, such an approach requires highly efficient introduction of DNA and rapid completion of recombination because linear DNA is easily lost from the parasites during multiplication. Here, we overcame these problems by developing a highly efficient DNA transfer method and Cas9-expressing parasites. Using our new CRISPR/Cas9 system, transgenic parasites were established in two weeks without any unexpected recombination or off-target mutations. Furthermore, with our system, two genes on different chromosomes were successfully modified in one transfection. Because of its high efficiency and robustness, our new CRISPR/Cas9 system will become a standard technique for genetic engineering of P. falciparum and dramatically advance studies of this parasite.
Keywords
CRISPR/Cas9, Plasmodium falciparum, Cas9-expressing parasites, donor template, parasite
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No competing interests reported.
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CURRENT STATUS: Under Review
Version 1
Posted 12 Apr, 2021
No community comments so far
Editorial decision: Major revision
On 20 May, 2021
Reviews received
Received 16 May, 2021
Reviews received
Received 06 May, 2021
Reviewers agreed
On 17 Apr, 2021
Reviewers agreed
On 13 Apr, 2021
Reviewers invited
Invitations sent on 13 Apr, 2021
Editor assigned
On 13 Apr, 2021
Editor invited
On 12 Apr, 2021
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On 09 Apr, 2021
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On 01 Apr, 2021
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This preprint is under consideration at Scientific Reports. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Shiroh Iwanaga
Osaka University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 12 Apr, 2021
No community comments so far
Editorial decision: Major revision
On 20 May, 2021
Reviews received
Received 16 May, 2021
Reviews received
Received 06 May, 2021
Reviewers agreed
On 17 Apr, 2021
Reviewers agreed
On 13 Apr, 2021
Reviewers invited
Invitations sent on 13 Apr, 2021
Editor assigned
On 13 Apr, 2021
Editor invited
On 12 Apr, 2021
Submission checks complete
On 09 Apr, 2021
First submitted
On 01 Apr, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The current CRISPR/Cas9 system for Plasmodium falciparum suffers from technical problems caused by plasmid constructs, such as delays in establishing transgenic parasites during drug selection and unexpected integration of circular donor DNA by single-crossover recombination. Although these problems can be solved by using linear donor templates, such an approach requires highly efficient introduction of DNA and rapid completion of recombination because linear DNA is easily lost from the parasites during multiplication. Here, we overcame these problems by developing a highly efficient DNA transfer method and Cas9-expressing parasites. Using our new CRISPR/Cas9 system, transgenic parasites were established in two weeks without any unexpected recombination or off-target mutations. Furthermore, with our system, two genes on different chromosomes were successfully modified in one transfection. Because of its high efficiency and robustness, our new CRISPR/Cas9 system will become a standard technique for genetic engineering of P. falciparum and dramatically advance studies of this parasite.
Figure 1
Figure 2
Figure 3
Figure 4
No competing interests reported.
This is a list of supplementary files associated with this preprint. Click to download.
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