DOI: 10.1101/481416Nov 29, 2018Paper

Ribozyme-Mediated, Multiplex CRISPR Gene Editing and CRISPRi in Plasmodium yoelii

BioRxiv : the Preprint Server for Biology
Michael P Walker, Scott E Lindner

Abstract

Functional characterization of genes in Plasmodium parasites often relies on genetic manipulations to disrupt or modify a gene-of-interest. However, these approaches are limited by the time required to generate transgenic parasites for P. falciparum and the availability of a single drug selectable marker for P. yoelii. In both cases, there remains a risk of disrupting native gene regulatory elements with the introduction of exogenous sequences. To address these limitations, we have developed CRISPR-RGR, a SpCas9-based gene editing system for Plasmodium that utilizes a Ribozyme-Guide-Ribozyme (RGR) sgRNA expression strategy. Using this system with P. yoelii, we demonstrate that both gene disruptions and coding sequence insertions are efficiently generated, producing marker-free and scar-free parasites with homology arms as short as 80-100bp. Moreover, we find that the common practice of using one sgRNA can produce both unintended plasmid integration and the desired locus replacement editing events, while the use of two sgRNAs results in only locus replacement editing. Lastly, we show that CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding dCas9 to targets in the gene control region of a gene-of-interest, resulti...Continue Reading

Related Concepts

Biological Markers
Gene Expression
Genes
Parasites
Plasmids
Plasmodium
Plasmodium falciparum
Plasmodium yoelii
Rodent
Catalytic RNA

Related Feeds

CRISPR for Genome Editing

Genome editing technologies enable the editing of genes to create or correct mutations. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). Here is the latest research on the use of CRISPR-Cas system in gene editing.

BioRxiv & MedRxiv Preprints

BioRxiv and MedRxiv are the preprint servers for biology and health sciences respectively, operated by Cold Spring Harbor Laboratory. Here are the latest preprint articles (which are not peer-reviewed) from BioRxiv and MedRxiv.

CRISPR (general)

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). CRISPR-Cas system enables the editing of genes to create or correct mutations. Discover the latest research on CRISPR here.

CRISPR Genome Editing & Therapy (Preprints)

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on the application of this system for gene editing and therapy in human diseases.

CRISPR Ribonucleases Deactivation

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on mechanisms that underlie deactivation of CRISPR ribonucleases. Here is the latest research.

CRISPR for Genome Editing (Preprints)

Genome editing technologies enable the editing of genes to create or correct mutations. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). Here are the latest preprints on the use of CRISPR-Cas system in gene editing.