Rotifers (Brachionus plicatilis) have many features that make them a suitable model organism for research on the genetics of aging, such as parthenogenic reproduction and a life span of only two weeks. The B. plicatilis annotated genome was published in 2019, which allows researchers to make precise genetic modifications. This field is relatively new, and among a small handful of publications, there is no standardized technique for inserting nucleic acids into B. plicatilis. Microinjection is a form of mechanical delivery of biological materials into living cells and is a vital practice in genetic editing research. During this project, we developed a protocol for the microinjection of B. plicatilis eggs. Here, I work with these microinjection techniques to genetically modify B. plicatilis using CRISPR-Cas12a to knock out the insulin-like growth factor receptor 1 (IGFR1) gene.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is often delivered to cells via microinjection. The protocol for the genetic modification of rotifer amictic eggs with lipophilic vesicles containing CRISPR-Cas12a complexed with a gRNA targeting the B. plicatilis IGFR1 locus is currently being developed. I hypothesize that this will knock out the IGFR1 gene and that this will extend the rotifer lifespan, similar to what is seen in C. elegans. gRNAs have been designed and tested in vitro with CRISPR-Cas12a and a PCR product of rotifer DNA. We have successfully developed gRNA capable of efficient cutting at the desired location. The injection will also contain a repair template containing GFP (green fluorescent protein). This GFP will also provide a visual indicator that the IGFR1 gene has been knocked out, and the repair template has been knocked in via homology-driven repair.

This genetic modification has many potential applications, such as enhancing diagnostic and research techniques regarding both larval fish, as well as CRISPR and genetic editing techniques.

Christopher Burtner

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