Generation and validation of genome editing tools for mouse CatSper β & Ɣ using CRISPR/Cas9 and split-EGFP systems

Mammalian sperms swim through convoluted female reproductive system and interact with the eggs in the fallopian tube. Sperm cells have the capacity to fertilize eggs but they cannot achieve this until they go through capatitation acrosome reaction. During capacitation, plasma membrane, permeability and motility patterns and flagellar activity change and Ca2+ is increased that causes hyperactivation, an asymmetric whip-like movement of the flagellum. This entry of Ca2+ is crucial for sperm hyperactivation and Ca2+ ions during this reaction flows through a flagellar specific ion channel called as Cation channel of sperm (CatSper). CatSper channel is a Ca2+ selective ion channel and maintains the Ca2+ entry into sperm cells. CatSper channel is comprised of CatSpers1–4, CatSper β (beta), CatSper γ (gamma) and CatSper δ (delta) and the loss of these genes in mice causes infertility. CatSper channel is localized at the principal piece of the sperm flagellum CatSper 1–4 mutations show no CatSper current detection, indicating all α subunits are required for the functional CatSper channel complex. It is shown that CatSper δ null mutants are infertile and they lose hyperactivated motility and Ca2+ current in the spermatozoa. The loss of CatSper δ also reduces CatSper1 detection, showing that CatSper δ auxiliary subunit is crucial and responsible for the formation of the CatSper channel assembly. Recombinant DNA technology provides the power to modify DNA molecules at a precise and targeted fashion within a cell, offering the ability to understand the gene functions, develop innovative therapeutic drugs, and biotechnological products. With the new developments in genomic engineering, in theory, it is now possible to directly edit or regulate the functions of DNA sequences in their endogenous conditions for any organism. Sequence specific DNA nucleases can be programmed to edit any endogenous genomic sequences with high precision and efficacy at a predetermined locus in a broad range of organisms and cell types, thus effectively promoting genome editing. They can either nick single strand DNA or cleave both strands of DNA either within or nearby the targeted sequence in a non-specific manner since they have sequence specific DNA binding domains bound to a nuclease. These nucleases have sequence-specific nuclase domains that can cut DNA strands within or out of the targeted locus by nicking one strand or cutting the double strands on DNA molecules. Cells activate of of two repair mechanisms when the DNA is damaged. They can activate either non-holomogous end joining (NHEJ) or homologous recombination (HR). NHEJ can introduce indels, thus causing frame-shift mutations or knock genes out of function. In HDR repair, cells can repair the damage in by recombining donor DNA as homologous to the targeted site with explicit genomic modifications. Recent developments in genome editing has lead to a revolutionizing Clustered, Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system as it allows gene manipulations in many cell lines and organisms including human. CRISPR is an immune system for bacteria and archaea. These organisms detect and eliminate foreign DNA and viral phages with its CRISPR accociated gene product, Cas9 and add small fragments into their CRISPR cluster. Bu using this ability of Cas9, CRISPR/Cas9 gene editing system is developed. Cas9 has a recognition and nuclease site. Both domains of the nuclease cuts different DNA strands. Cas) recognizes ~20 bp sequence with NGG at the beginning of the sequence. By using this 20 nucleotide sequence with NGG and guide RNA with Cas9, almost any sequence beginning with NGG can be targeted and edited. In our study, we used pCAG-EGxxFP and pX330 plasmids containing CatSper ion channel auxiliary subunits CatSper β ve CatSper γ gene fragments and co-transfected them together in HEK293 cells to express EGFP gene. We cloned CatSper β ve CatSper γ gene fragments individually into pX330 to obtain pX330-β ve pX330-γ constructs. pX330 contains Cas9 gene and single guide RNA. We also used pCAG-EGxxFP plasmid which has CMV promoter and EGFP genes. This plasmid has 482 bp homology sequence with 5` and 3` EGFP fragments and placed under ubiquitous CAG promoter. The vector is designed that we can insert out targed sequence into EGFP gene. We cloned CatSper β ve CatSper γ gene fragments into pCAGxxFP individually to obtain pCAG-EG-β-FP and pCAG-EG-γ-FP constructs. Our aim is to co-transfect pX330-β with pCAG-EG-β-FP and pX330-γ with pCAG-EG-γ-FP into HEK293 cells to check whether guide RNAs containing CatSper β ve CatSper γ fragments can bind to targeted gene fragments in pCAG-EG-β-FP and pCAG-EG-γ-FP constructs, thus cleaving the targeted sequences with the expressed Cas9 enzyme. If Cas9 cleaves the DNA fragments, pCAGxxFP plasmid will reconstitute the EGFP and give green fluoresence. Therefore, we transfected pX330-β, pCAG-EG-β-FP, pX330-γ and pCAG-EG-γ-FP constructs individually as negative controls into HEK293 cells. And we also co-transfected pX330-β with pCAG-EG-β-FP and pX330-γ with pCAG-EG-γ-FP into HEK293 cells and incubated them at 37 °C for 24 hours. When we checked HEK293 cells under confocal microscope, we observed green flourescence in co-transfected constructs but not in individually transfected constructs. This indicates that single guide RNAs recognized CatSper β and CatSper γ gene fragments and Cas9 enzyme cleaved the targeted sequences. With this study, we demonstared that our designed CRISPR/Cas9 constructs are working in HEK293 cell lines.