CRISPR Helps Identify Key Heat Tolerance Gene in Corals
Climate change has caused enormous shifts and changes in marine ecology, driving research into understanding the maintenance and improvement of marine life to mitigate risks. In a new study published in Proceedings of the National Academy of Sciences (PNAS) on 9th November, a team of researchers from Stanford University, Australian Institute of Marine Science, and Queensland University of Technology demonstrated that CRISPR-mediated mutation of a heat shock transcription factor could result in reduced heat tolerance in corals, suggesting that corals have a heat protection mechanism.
Verifying hypotheses of the impact of climate change on corals is challenging due to the lack of experimental model systems and genetic tools to manipulate their genome. In this novel study, the authors used CRISPR to edit a gene in fertilized eggs of coral to understand its function.
HSF1 Transcription Factor
HSF1- heat shock transcription factor 1 is an important gene for heat tolerance in many organisms. The team sought to understand the effect of disrupting this gene in the coral, Acropora millepora. The ability to withstand heat can have important implications for coral reef recovery and restoration in the face of increasing temperatures in the oceans. It helps to understand how corals deal with climate change and develop coral management techniques such as “selective breeding and movement of corals among reefs,” according to Dr. Line Bay, Principal Scientist at the Australian Institute of Marine Science.
In the past decades, thermal stress has caused the decline of corals across the globe. Hence, to analyze the mechanisms of heat-induced coral bleaching and death and possible protective pathways, the authors zeroed in on HSF1, a gene linked to HSP70 and HSP90, molecular chaperones that regulate heat stress in many eukaryotes. The study entailed optimizing sgRNA design and volume of injection to achieve a high frequency of gene editing.
CRISPR Mediated Mutation of HSF1
The authors used two sgRNAs, one to target exon 3 and another against exon 9. In three consecutive experiments, they injected sgRNA, coupled with fluorescent markers, into multiple batches of freshly fertilized zygotes. After 12h of injection, positive zygotes were assessed based on the fluorescent marker and analyzed for survival and growth. Genotyping by amplicon sequencing was performed for surviving larvae at 6 days post-fertilization. More than 80% of HSF1 gene sequences were mutated in all injected animals.
To understand the impact of HSF1 mutation, uninjected and sgRNA/Cas9-injected larvae were subjected to heat stress at 34°C. HSF1 mutation resulted in over 60% larvae death in 48h, suggesting that HSF1 and its downstream effectors confer protection against heat stress in corals.
“We developed an improved CRISPR-Cas9 method that allowed us to test gene function in coral for the first time. As a proof-of-concept, we used CRISPR-Cas9 genome editing to understand the function of a key gene that influences the ability of coral to survive heat.” lead author Dr. Philip Cleves said. This could pave the way for a broad spectrum of coral genetic studies that can evaluate the functions of many other genes in various pathways. The authors recommend careful selection of genes that CRISPR can study due to the unique biology of corals. Redundant genes, pathways that differ in larval, juvenile, and adult corals, method of sgRNA/Cas9 delivery, and genotype-to-phenotype correlation are important points to consider when designing a CRISPR-based genetic study of corals. However, this study provides an experimental framework to design research plans to understand the molecular, cellular, and developmental biology of corals.
By Sahana Shankar, Ph.D. Candidate
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