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Lentiviral mediated CRISPR/Cas9
- 1. Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system Remi L. Gratacap , Tim Regan, Carola E. Dehler, Samuel A. M. Martin, Pierre Boudinot, Bertrand Collet and Ross D. Houston Chew Chee Kei, Lock Tian Jenq, Soo Xin Lu, Yiap Fu Wei
- 2. INTRODUCTION One of the fastest growing sector in food production One of the fastest growing sector in food production Little is known about the functional genes involved in disease resistance Infectious diseases heavily affects the market and ecosystem Salmonids are one of the most important species in aquaculture AQUACULTURE CRISPR/CAS9 ● Genome editing technique, able to identify genes involved in disease resistance ● CAS9 is an enzyme which cuts specific sites of DNA ● Able to facilitate gene knockout, activation, inactivation, deletion, insertion and modification of genome ● Highly accurate Problem statement: Identify the effectiveness of lentivirus-mediated CRISPR/Cas9 in salmonid cell line as a disease modelling system for disease resistance
- 3. INTRODUCTION Lentivirus-mediated CRISPR/CAS9 Lentivirus can be used for the transduction of the salmonid fish by: 1. Creating a guide RNA (gRNA) library of the gene of interest or non-translated regions 2. Insert the gRNA to the viral vector 3. Virus inserts itself to host’s genome to express CAS9 Why lentivirus? more advantageous than electroporation or transient plasmid transfection because: ● Efficiently integrates into the host genome ● Enables stable cell lines ● Allows integration of antibiotic resistance markers and fluorescent reporters Genes involved in disease resistance of salmonid fish using lentivirus-mediated CRISPR/CAS9 RIG-I Triggers innate interferon immune response when it detects dsRNA (Yoneyama et al., 2004). EGFP Protein which emits green fluorescence
- 4. LENTIVIRAL-TRANSDUCTION CRISPR/CAS9 Guide RNA will bind to the target sequence Cas9 enzyme comes in and bind to guide RNA Cas9 enzyme will cut the both strands of DNA Mutation will occur when cut is repaired Lentivirus is an efficient vector in delivering the CRISPR/Cas9 and flexible to allow combination of design elements to improve CRISPR/Cas9 - Specification - Polycistronic system
- 6. RESULT 1 Optimisation of lentiviral transduction 3 variables were tested: 1. incubation temperature 2. spinfection 3. duration of incubation To determine the transduction efficiency. Optimised protocol for efficient transduction of CHSE cells -neat lentivirus supernatant -2hr spinfection at 100xg -incubation for 24 h at 22 °C Used for downstream experiments
- 7. RESULT 2 47% edited at the desired locus pKLV2 plasmid (containing the human U6 promoter) - Functional - effective at transcribing gRNA in the CHSE cell line Lentivirus delivery strategy (either neat, or diluted together with antibiotic selection for enrichment) leads to very high genome editing efficiency. enriched to 60% using puromycin selection cells cells
- 8. CHALLENGES (LIMITATIONS) ➔ Lentivirus tends to integrate at the site of active transcription ➔ There are possibilities of genome off target How does it work? Induce immune response, inactivate vector, inhibiting transduction, attack transduced cell How does it work? - High off target: induced mutation at site - Present study: 2 top off target regions were sequenced and show no editing WAYS TO RESOLVE: Use of CHSE-EC cell line - constitutively expressed Cas9 - construct targeting EGFP or RIG-I Use of electroporation - Editing efficiency using electroporation of the gRNA was improved to 90% editing of EGFP - successful in edit the genome of mammalian immune cells WAYS TO RESOLVE: - Be extra careful when editing genome with Cas9 - Minimise possibilities of genome off target
- 9. CURRENT DEVELOPMENT Microinjection of CRISPR/Cas9 Other potential method (TALEN) ❏ Zygote microinjection ❏ Knockout myostatin (MSTN) gene in channel catfish genome ❏ Enhance growth and increase productivity (Radev et al. 2015) ❏ Zebrafish line with mutation of COL6A1 gene ❏ Model of collagen VI-related diseases ❏ Drug discovery assay and research on this disease (Valero et al. 2018)
- 10. CONCLUSION ❏ Lentivirus as the delivery system for CRISPR/CAS9 improve the efficiency of transduction of the RIG I & EGFP gene into the CHSE salmonid fish cell line ❏ CRISPR/CAS9 has been proven for it’s efficiency among all the others method even though it still have some limitations ❏ More studies involved in host-pathogen interactions in fish will be conducted in future
- 11. REFERENCES Gratacap, R., Regan, T., Dehler, C., Martin, S., Boudinot, P., Collet, B. and Houston, R., 2020. Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system. BMC Biotechnology, 20(1) Liu, C, Zhang, L, Liu, H & Cheng, K 2017, "Delivery Strategies of the CRISPR-Cas9 Gene-Editing System for Therapeutic Applications", J Control Release. viewed 29 September 2020, <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723556/>. National Library of Medicine (US), 2020. Genetics Home Reference. What Are Genome Editing And CRISPR- Cas9?. [online] Available at: <https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting> [Viewed 17 September 2020]. Radev, Z, Hermel, J, Elipot, Y, Bretaud, S, Arnould, S, Duchateau, P, Ruggiero, F, Joly, J & Sohm, F 2015, "A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish", PLOS ONE, vol. 10, no. 7, p. E0133986. Valero, Y, Saraiva‐Fraga, M, Costas, B & Guardiola, F 2018, "Antimicrobial peptides from fish: beyond the fight against pathogens", Reviews in Aquaculture, vol. 12, no. 1, pp. 224-253. What is CRISPR-Cas9? 2020 Yourgenomeorg. viewed 29 September 2020, <https://www.yourgenome.org/facts/what-is-crispr-cas9>.
Editor's Notes
- Aquaculture is one of the fastest growing sector in food production, salmonid fishes are one of the most important in aquaculture, infectious diseases is one of the biggest challenges in aquaculture as it affects the market and ecosystem Crispr/cas9 Problem statement
- Genes involved in disease resistance of salmonid fish: Both genes were targeted by the grna and were used to prove they were edited with high efficiency
- A piece of RNA (guide RNA) consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold will binds to DNA and makes sure that the Cas9 enzyme cuts at the right point in the genome. Cas9 will bind to the guide RNA and acting like a molecular scissor cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed. The Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut across both strands of the DNA. At this stage the cell recognises that the DNA is damaged and tries to repair it, and mutation might occur. https://www.yourgenome.org/facts/what-is-crispr-cas9 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723556/
- Lentiviral transduction was optimised for use in salmonid cell lines. This is to improve CRISPR/Cas9 delivery and genome editing efficiency. DIAGRAM: An optimised protocol for efficient transduction of CHSE cells using neat lentivirus supernatant on suspended cells, together with a spinfection step (2 h at 100 xg) and incubation for 24 h at 22 °C Representative image of the CHSE cells 8 days post transduct with p-LentiGFP Incubation temperature, spinfection and duration of incubation affects the transduction efficiency.
- DIAGRAM: A-d: efficient editing of EGFP in CHSE-EC Chinook salmon cell line by lentivirus using the optimised protocol. A: recorded flow cytometry results Plasmid cloned (human U6 promoter, EGFP-gRNA, CHSE-EC cells) using optimised protocol drive expression of second-generation gRNA scaffold and puromycin resistance gene. Editing efficiency was determined by loss of fluorescence for EGFP.
- The first challenge in this research is the lentivirus tend to integrate at the site of the active transcription which will induced a immune response. When immune response is induced it will inactive the vector , inhibiting transduction and attack transduced cell. Ways to resolve it is by using the CHSE-EC cell that expresses the Cas9, in combination with a lentivirus packaging a gRNA construct targeting EGFP or RIG-I, the genome of the salmonid cells was edited at very high efficiency. Second , the use of electroporation also improve the editing efficiency of the gRNA to about 90% , where indicated in the research, electroporation is successful in editing genome of mammalian immune cells. The second challenge is the possibilities of genome off target as high off target will induced mutation at the site which is not preferable when doing genome editing. As from the present study, the 2 top off target regions were sequenced and show no editing. Ways to resolve it, be extra careful when editing the genome with Cas9 to minimize the isibilities of genome off target .
- Microinjection of CRISPR/Cas9: MSTN gene is important because of its role in regulation of skeletal muscle growth in all vertebrate the overall mutation rates and survival rate were higher than those obtained in Atlantic salmon, Common carp and Nile tilapia https://www.nature.com/articles/s41598-017-07223-7 Other potential method (TALEN): Collagen VI protein is involved in cell-cell attachment and interact with collagen IV of the basement membrane, thus playing an important role in muscle maintenance Deficiency of the gene disrupt the protein structure *However, CRISPR is a more useful and flexible tool as compare to TALEN and ZFNs due to higher efficiency