CRISPR Plasmids: Parasites

Eukaryotic parasites continue to cause substantial death and disease in the world. Parasites such as Plasmodium (malaria), Toxoplasma (toxoplasmosis), Trypanosoma (African sleeping sickness), Cryptosporidium (cryptosporidiosis), Leishmania (leishmaniasis), and others have been the focus of biomedical research for decades. Recent developments with CRISPR-Cas9 gene editing have propelled both reverse and forward genetics of these parasites. Moreover, CRISPR-dCas9 gene regulation has also been established for some of these parasites. Together, these tools have enabled precise genetic analyses to pinpoint genes linked to parasite virulence, transmission, and development. This collection brings together some of the plasmids that have been used in these efforts.

The content for this page was generated with the help of Scott Lindner.

Cut

Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often lead to frameshifts, creating loss of function alleles.

To introduce specific genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs at a much lower efficiency than NHEJ.

Libraries

Toxoplasma CRISPR Knockout Pooled Library - Toxoplasma gondii CRISPR genome-wide knockout gRNA pooled library designed with 10 guides against 8,158 predicted protein-coding genes.