Dodging silver bullets: good CRISPR gene-drive design is critical for eradicating exotic vertebrates.

Self-replicating gene drives that can spread deleterious alleles through animal populations have been promoted as a much needed but controversial 'silver bullet' for controlling invasive alien species. Homing-based drives comprise an endonuclease and a guide RNA (gRNA) that are replicated during meiosis via homologous recombination. However, their efficacy for controlling wild populations is threatened by inherent polymorphic resistance and the creation of resistance alleles via non-homologous end-joining (NHEJ)-mediated DNA repair. We used stochastic individual-based models to identify realistic gene-drive strategies capable of eradicating vertebrate pest populations (mice, rats and rabbits) on islands. One popular strategy, a sex-reversing drive that converts heterozygous females into sterile males, failed to spread and required the ongoing deployment of gene-drive carriers to achieve eradication. Under alternative strategies, multiplexed gRNAs could overcome inherent polymorphic resistance and were required for eradication success even when the probability of NHEJ was low. Strategies causing homozygotic embryonic non-viability or homozygotic female sterility produced high probabilities of eradication and were robust to NHEJ-mediated deletion of the DNA sequence between multiplexed endonuclease recognition sites. The latter two strategies also purged the gene drive when eradication failed, therefore posing lower long-term risk should animals escape beyond target islands. Multiplexing gRNAs will be necessary if this technology is to be useful for insular extirpation attempts; however, precise knowledge of homing rates will be required to design low-risk gene drives with high probabilities of eradication success.

Mapping combinatorial expression of non-clustered protocadherins in the developing brain identifies novel PCDH19-mediated cell adhesion properties.

Non-clustered protocadherins (ncPcdhs) are adhesive molecules with spatio-temporally regulated overlapping expression in the developing nervous system. Although their unique role in neurogenesis has been widely studied, their combinatorial role in brain physiology and pathology is poorly understood. Using probabilistic cell typing by in situ sequencing, we demonstrate combinatorial inter- and intra-familial expression of ncPcdhs in the developing mouse cortex and hippocampus, at single-cell resolution. We discovered the combinatorial expression of Protocadherin-19 (Pcdh19), a protein involved in PCDH19-clustering epilepsy, with Pcdh1, Pcdh9 or Cadherin 13 (Cdh13) in excitatory neurons. Using aggregation assays, we demonstrate a code-specific adhesion function of PCDH19; mosaic PCDH19 absence in PCDH19+9 and PCDH19 + CDH13, but not in PCDH19+1 codes, alters cell-cell interaction. Interestingly, we found that PCDH19 as a dominant protein in two heterophilic adhesion codes could promote trans-interaction between them. In addition, we discovered increased CDH13-mediated cell adhesion in the presence of PCDH19, suggesting a potential role of PCDH19 as an adhesion mediator of CDH13. Finally, we demonstrated novel cis-interactions between PCDH19 and PCDH1, PCDH9 and CDH13. These observations suggest that there is a unique combinatorial code with a cell- and region-specific characteristic where a single molecule defines the heterophilic cell-cell adhesion properties of each code.

Generation of Gene Drive Mice for Invasive Pest Population Suppression.

Gene drives are genetic elements that are transmitted to greater than 50% of offspring and have potential for population modification or suppression. While gene drives are known to occur naturally, the recent emergence of CRISPR-Cas9 genome-editing technology has enabled generation of synthetic gene drives in a range of organisms including mosquitos, flies, and yeast. For example, studies in Anopheles mosquitos have demonstrated >95% transmission of CRISPR-engineered gene drive constructs, providing a possible strategy for malaria control. Recently published studies have also indicated that it may be possible to develop gene drive technology in invasive rodents such as mice. Here, we discuss the prospects for gene drive development in mice, including synthetic "homing drive" and X-shredder strategies as well as modifications of the naturally occurring t haplotype. We also provide detailed protocols for generation of gene drive mice through incorporation of plasmid-based transgenes in a targeted and non-targeted manner. Importantly, these protocols can be used for generating transgenic mice for any project that requires insertion of kilobase-scale transgenes such as knock-in of fluorescent reporters, gene swaps, overexpression/ectopic expression studies, and conditional "floxed" alleles.

Progress Toward Zygotic and Germline Gene Drives in Mice.

CRISPR-based synthetic gene drives have the potential to deliver a more effective and humane method of invasive vertebrate pest control than current strategies. Relatively efficient CRISPR gene drive systems have been developed in insects and yeast but not in mammals. Here, we investigated the efficiency of CRISPR-Cas9-based gene drives in Mus musculus by constructing "split drive" systems where gRNA expression occurs on a separate chromosome to Cas9, which is under the control of either a zygotic (CAG) or germline (Vasa) promoter. While both systems generated double-strand breaks at their intended target site in vivo, no homology-directed repair between chromosomes ("homing") was detectable. Our data indicate that robust and specific Cas9 expression during meiosis is a critical requirement for the generation of efficient CRISPR-based synthetic gene drives in rodents.

Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression.

CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs. Through nucleofection of mouse embryonic stem cells, we demonstrate highly efficient cleavage of the target loci using the dual-guide plasmids, which are available as Cas9-nuclease or Cas9-nickase expression constructs, with or without selection markers. These vectors are a valuable addition to the CRISPR/Cas9 toolbox and will be made available to all interested researchers via the Addgene plasmid repository.