The latest Paper of the Month from Parasitology is ‘New and emerging uses of CRISPR/Cas9 to genetically manipulate apicomplexan parasites’ by Manlio Di Cristina and Vern B. Carruthers.

So what’s with all the hype about CRISPR/Cas9 leading a genetic revolution? Simply put, these molecular tools – or rather scissors in their most common form – allow researchers to make genetic changes faster and more precisely than ever before. Borrowed from a bacterial defense system against viruses, CRISPR/Cas9 genome editing involves two basic entities, namely an enzyme that cuts nucleotides called Cas9 and a guide RNA that binds to Cas9 and directs it to a complementary sequence in the genome. Depending on the particular type of Cas9, CRISPR/Cas9 can be used to localize a complementary sequence in the cell nucleus, block or activate expression of specific genes, or cut one or both strands of the DNA double helix. Cutting DNA at a specific site in the genome is especially useful because it allows researchers to precisely disrupt a gene, insert a tag such as a fluorescent protein to localize the encoded protein within the cell, or even create specific mutations to define how amino acid sequences confer protein function.

Manlio Di Cristina and I wrote the review on recent advances using CRISPR/Cas9 tools in apicomplexan parasites because this topic is evolving so rapidly that we felt the community could benefit from consolidation of the latest findings. Apicomplexan parasites include many pathogens of humans and animals, most notably Plasmodium (the agents of malaria), Toxoplasma (a brain parasite affecting babies and immune deficient patients), and Cryptosporidium (a rather unpleasant gastrointestinal bug). Implementing CRISPR/Cas9 tools into these systems has greatly accelerated the understanding of how such parasites cause disease, what makes them susceptible or resistant to treatment, and which genes are most important to their survival and transmission.

As an example of how we used CRISPR/Cas9 in our recent work, we were interested in understanding how Toxoplasma manages to persist indefinitely as a slow growing stage within neurons of an infected individual. To survive indefinitely the parasite probably needs to renew its cellular components, and we reasoned that it might use proteases in its endolysosomal system to degrade such components. We were particularly interested in a digestive protease termed CPL, but we were hampered by the difficulties of deleting genes in Toxoplasma strains that efficiently convert to the persistent stage. CRISPR/Cas9 enabled us to precisely introduce cuts at either end of the CPL gene to kick it out of the genome. Whereas previously it would have taken several tries and up to one year to create such a gene deletion mutant, CRISPR/Cas9 tools allowed us to make a CPL mutant strain in roughly two months. We also used CRISPR/Cas9 to precisely reintroduce the CPL coding sequence into the mutant strain. A series of experiments with mutant and restored strains showed that CPL digestion is crucial for parasite persistence in neurons during infection. This discovery has opened a door toward developing new therapeutic compounds that block CPL function and potentially diminish persistent infection.

Notable samplings of other recent CRISPR/Cas9-enabled work include identifying malaria transcription factors required for parasite development in mosquitoes, defining the importance of every Toxoplasma gene to parasite fitness, and establishing a contributor to Cryptosporidium resistance to antifolate treatment.

Don’t expect the hype to fade anytime soon. New and clever innovations of CRISPR/Cas9 tools are enabling gene silencing via epigenetic manipulations or targeting RNA instead of DNA, along with revealing how DNA is organized within the nucleus. Also, the commercial availability of recombinant Cas9 together with custom synthesized guide RNA opens the prospect of using premade complexes of Cas9 and guide RNA for genetic manipulation without the time consuming task of creating plasmids to express these components. After reading this post, I hope you agree there are ample reasons for apicomplexan sleuths to exclaim “Viva la (CRISPR) revolution!!”

 

Read the full article ‘New and emerging uses of CRISPR/Cas9 to genetically manipulate apicomplexan parasites in full for free until 5th May, 2018.

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