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Abstract Despite the revolutionary impacts of CRISPR-Cas gene editing systems, the effective and widespread use of CRISPR technologies in emerging model organisms still faces significant challenges. These include the inefficiency in generating heritable mutations at the organismal level, limited knowledge about the genomic consequences of gene editing, and an inadequate understanding of the inheritance patterns of CRISPR-Cas-induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for CRISPR editing in the microcrustaceanDaphnia pulex; 2) assessing the editing efficiency of Cas9 and Cas12a nucleases, examining mutation inheritance patterns, and analyzing the local and global mutation spectrum in thescarletmutants; and 3) investigating the transcriptomes ofscarletmutants to understand the pleiotropic effects ofscarletunderlying their swimming behavior changes. Our reengineered CRISPR microinjection method results in efficient biallelic editing with both nucleases. While indels are dominant in Cas-induced mutations, a few on-site large deletions (>1kb) are observed, most likely caused by microhomology-mediated end joining repair. Knock-in of a stop codon cassette to thescarletlocus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progenies. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes (e.g., NMDA1, ABAT, CNTNAP2) involved in human neurodegenerative diseases. This study expands our understanding of the dynamics of gene editing in the tractable model organismDaphniaand highlights its promising potential as a neurological disease model. 

Abstract Theories predict that directional selection during adaptation to a novel habitat results in elevated meiotic recombination rate. Yet the lack of population-level recombination rate data leaves this hypothesis untested in natural populations. Here, we examine the population-level recombination rate variation in two incipient ecological species, the microcrustacean Daphnia pulex (an ephemeral-pond species) and Daphnia pulicaria (a permanent-lake species). The divergence of D. pulicaria from D. pulex involved habitat shifts from pond to lake habitats as well as strong local adaptation due to directional selection. Using a novel single-sperm genotyping approach, we estimated the male-specific recombination rate of two linkage groups in multiple populations of each species in common garden experiments and identified a significantly elevated recombination rate in D. pulicaria. Most importantly, population genetic analyses show that the divergence in recombination rate between these two species is most likely due to divergent selection in distinct ecological habitats rather than neutral evolution. 

Mutation rate in the nuclear genome differs between sexes, with males contributing more mutations than females to their offspring. The male-biased mutation rates in the nuclear genome is most likely to be driven by a higher number of cell divisions in spermatogenesis than in oogenesis, generating more opportunities for DNA replication errors. However, it remains unknown whether male-biased mutation rates are present in mitochondrial DNA (mtDNA). Although mtDNA is maternally inherited and male mtDNA mutation typically does not contribute to genetic variation in offspring, male mtDNA mutations are critical for male reproductive health. In this study, we measured male mtDNA mutation rate using publicly available whole-genome sequences of single sperm of the freshwater microcrustacean Daphnia pulex . Using a stringent mutation detection pipeline, we found that the male mtDNA mutation rate is 3.32 × 10 −6 per site per generation. All the detected mutations are heteroplasmic base substitutions, with 57% of mutations converting G/C to A/T nucleotides. Consistent with the male-biased mutation in the nuclear genome, the male mtDNA mutation rate in D. pulex is approximately 20 times higher than the female rate per generation. We propose that the elevated mutation rate per generation in male mtDNA is consistent with an increased number of cell divisions during male gametogenesis.