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Over the course of millions of years, modifications in mammalian genomes have produced an incredible variety of phenotypes, from the antlers of deer, to the breathing capacity of whales, to the dexterous fingers of a human. This remarkable diversity is perhaps nowhere more evident than in the limb of the mammal, which, despite its underlying structural similarities, exhibits specialized morphologies that look strikingly different in different species. Thus the hands typing this abstract are structurally similar but morphologically and functionally divergent from the fins of a manatee, the wings of a bat, or the legs of a horse. Comparing the genetic and regulatory underpinnings of this divergence (and indeed, of the similarities between species) allows us to simultaneously reconstruct genomic evolution and delve into the developmental mechanisms that produce limbs. The lesser Egyptian jerboa, Jaculus jaculus, is genomically similar to the common laboratory mouse, Mus musculus, but has highly divergent derived characteristics in the hindlimb. The hindlimb of the jerboa features a striking elongation of metatarsals and tibia, the loss of two digits, and the fusing of metatarsals into one central bone. By engineering ‘jerboanized’ mouse models, where regions of the mouse genome are replaced with their jerboa homologues to produce animals with jerboa-esque phenotypes, we can elucidate the mechanisms of divergence and development in the jerboa in a well-established and highly manipulable model system. Here, I present work on two genetic engineering techniques that promise to transform interspecies comparisons in rodent systems. First, I introduce a large interspecies conversion that simultaneously deletes a ~26kb region of the mouse genome and replaces it with ~31kb of homologous sequence from the jerboa. Second, I present the first demonstration of active genetics, a technique that makes use of the CRISPR Cas9 system to convert genotypes, in a rodent model. Active genetics in the rodent may be used to increase the rate of inheritance of an allele, which has profound implications the future of genetic engineering in both laboratory and applied settings. Together these tools may be used to develop complex mouse models of jerboanized phenotypes to probe the specific genomic changes responsible for the evolution of the development of the jerboa morphology.

Pdf of the poster (HAG_TAGC2020 Poster.pdf) and Video/Audio walk through (HAG_TAGC2020_Poster_Vid.mov)Title:Super-Mendelian inheritance mediated by CRISPR–Cas9 in the female mouse germline.Abstract:A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems that have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR–Cas9 and endogenous homology- directed repair mechanisms to convert heterozygous genotypes to homozygosity. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR–Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology- directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR–Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.

Pdf of the poster (HAG_TAGC2020 Poster.pdf) and Video/Audio walk through (HAG_TAGC2020_Poster_Vid.mov)Title:Super-Mendelian inheritance mediated by CRISPR–Cas9 in the female mouse germline.Abstract:A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems that have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR–Cas9 and endogenous homology- directed repair mechanisms to convert heterozygous genotypes to homozygosity. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR–Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology- directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR–Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.

This is the dataset for all genotyping for the paper "Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline." Abstract:A gene drive biases the transmission of a particular allele of a gene such that it is inherited at a greater frequency than by Mendelian segregation. Recently, highly efficient gene drive systems were developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR/Cas9 and endogenous homology directed repair mechanisms to convert heterozygous genotypes to homozygosity. If implemented in laboratory rodents, similarly powerful systems would enable the rapid assembly of genotypes that involve multiple genes (e.g., to model multigenic human diseases). Such complex genetic models are currently precluded by time, cost, and a requirement for a large number of animals to obtain a few individuals of the desired genotype. However, the efficiency of a CRISPR/Cas9 gene drive system in mammals has not yet been determined. Here, we utilize an active genetic “CopyCat” element embedded in the mouse Tyrosinase gene to detect genotype conversions after Cas9 activity in the embryo and in the germline. Although Cas9 efficiently induces double strand DNA breaks in the early embryo and is therefore highly mutagenic, these breaks are not resolved by homology directed repair. However, when Cas9expression is limited to the developing female germline, resulting double strand breaks are resolved by homology directed repair that copies the CopyCat allele from the donor to the receiver chromosome and leads to its super-Mendelian inheritance. These results demonstrate feasibility of the CRISPR/Cas9 gene drive mechanism in mice, which has potential to transform the use of rodent models in basic and biomedical research and also contributes valuable data to the ongoing discussion of possible approaches to suppress invasive and disease-harboring wild rodent populations., Please use the Word.docx file titled "Instructions" for instructions on how to navigate this dataset., {"references":["https://www.biorxiv.org/content/early/2018/07/04/362558"]}

This is the dataset for all genotyping for the paper "Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline." Abstract:A gene drive biases the transmission of a particular allele of a gene such that it is inherited at a greater frequency than by Mendelian segregation. Recently, highly efficient gene drive systems were developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR/Cas9 and endogenous homology directed repair mechanisms to convert heterozygous genotypes to homozygosity. If implemented in laboratory rodents, similarly powerful systems would enable the rapid assembly of genotypes that involve multiple genes (e.g., to model multigenic human diseases). Such complex genetic models are currently precluded by time, cost, and a requirement for a large number of animals to obtain a few individuals of the desired genotype. However, the efficiency of a CRISPR/Cas9 gene drive system in mammals has not yet been determined. Here, we utilize an active genetic “CopyCat” element embedded in the mouseTyrosinasegene to detect genotype conversions after Cas9 activity in the embryo and in the germline. Although Cas9 efficiently induces double strand DNA breaks in the early embryo and is therefore highly mutagenic, these breaks are not resolved by homology directed repair. However, whenCas9expression is limited to the developing female germline, resulting double strand breaks are resolved by homology directed repair that copies the CopyCat allele from the donor to the receiver chromosome and leads to its super-Mendelian inheritance. These results demonstrate feasibility of the CRISPR/Cas9 gene drive mechanism in mice, which has potential to transform the use of rodent models in basic and biomedical research and also contributes valuable data to the ongoing discussion of possible approaches to suppress invasive and disease-harboring wild rodent populations.