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Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor
Saturday, 2020/11/21 | 07:02:22
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Phillip A. Cleves, Amanda I. Tinoco, Jacob Bradford, Dimitri Perrin, Line K. Bay, and John R. Pringle PNAS November 17, 2020 117 (46) 28899-28905 SignificanceCoral reefs are biodiversity hotspots of great ecological, economic, and aesthetic importance. Their global decline due to climate change and other stressors has increased the urgency of understanding the molecular bases of corals’ responses to stress. Analyses of coral genomes and gene-expression patterns have identified many genes that may be important in stress resistance, but rigorous testing of their function will require the analysis of appropriate mutants. Here, we used CRISPR technology to show that mutational loss of a putative regulator of gene expression in response to heat stress indeed produced a loss of heat tolerance. Such use of CRISPR to generate mutations in corals should illuminate many aspects of coral biology and, thus, help to guide conservation efforts. AbstractReef-building corals are keystone species that are threatened by anthropogenic stresses including climate change. To investigate corals’ responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating many hypotheses about the roles of particular genes and molecular pathways. However, it has not generally been possible to test these hypotheses rigorously because of the lack of genetic tools for corals or closely related cnidarians. CRISPR technology seems likely to alleviate this problem. Indeed, we show here that microinjection of single-guide RNA/Cas9 ribonucleoprotein complexes into fertilized eggs of the coral Acropora millepora can produce a sufficiently high frequency of mutations to detect a clear phenotype in the injected generation. Based in part on experiments in a sea-anemone model system, we targeted the gene encoding Heat Shock Transcription Factor 1 (HSF1) and obtained larvae in which >90% of the gene copies were mutant. The mutant larvae survived well at 27 °C but died rapidly at 34 °C, a temperature that did not produce detectable mortality over the duration of the experiment in wild-type (WT) larvae or larvae injected with Cas9 alone. We conclude that HSF1 function (presumably its induction of genes in response to heat stress) plays an important protective role in corals. More broadly, we conclude that CRISPR mutagenesis in corals should allow wide-ranging and rigorous tests of gene function in both larval and adult corals.
See https://www.pnas.org/content/117/46/28899
Figure 1: Highly effective disruption of HSF1 by CRISPR/Cas9 in A. millepora larvae. (A) Structure of HSF1 and locations of the targeted sites. Five of the 12 exons are shown; the distance between exons 3 and 9 is ∼2.5 kb. Approximate locations of the sites targeted by sgRNAs 1 and 2 are shown (see SI Appendix, Table S1, for sequences), as are the locations of the primers (arrowheads 1–4) used to amplify genomic DNA for sequencing to test for the presence of induced mutations. (B) Fractions of larvae in which ≥1, ≥70%, or 100% of the PCR clones sequenced (10–16 per animal) showed mutations and overall percentages of mutant sequences observed (see SI Appendix, Table S2 for details). Experiments 1–3 represent the three nights on which spawning occurred and freshly fertilized eggs were injected. For control animals injected with Cas9 only (no sgRNA), data are combined for the five animals injected on each night of spawning. (C) Patterns of the mutations seen in two individual larvae. Black bars, positions of the sgRNA target sites; red nucleotides, Protospacer-adjacent-motif (PAM) sites. Dashes, nucleotides deleted in the mutant alleles; blue letters, inserted/replaced nucleotides. |
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(96).png "Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor")
