A Nickase Cas9 Gene-Drive System Promotes Super-Mendelian inheritance in Drosophila

CRISPR-based gene-drives have been proposed for managing insect populations, including disease-transmitting mosquitoes, due to their ability to bias their inheritance toward super-Mendelian rates (\u3e50%). Current technologies use a Cas9 that introduces DNA double-strand breaks into the opposing wild-type allele to replace it with a copy of the gene-drive allele via DNA homology-directed repair. However, the use of different Cas9 versions is unexplored, and alternative approaches could increase the available toolkit for gene-drive designs. Here, we report a gene-drive that relies on Cas9 nickases that generate staggered paired nicks in DNA to propagate the engineered gene-drive cassette. We show that generating 5\u27 overhangs in the system yields efficient allelic conversion. The nickase gene-drive arrangement produces large, stereotyped deletions that are advantageous to eliminate viable animals carrying small mutations when targeting essential genes. Our nickase approach should expand the repertoire for gene-drive arrangements aimed at applications in mosquitoes and beyond

Efficient allelic-drive in Drosophila.

Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus

Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test a CRISPR-based homing gene drive for Culex quinquefasciatus, and show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes

BIOSAFETY. Safeguarding gene drive experiments in the laboratory.

Multiple stringent confinement strategies should be used whenever possibleThis is the author accepted manuscript. The final version is available from AAAS via http://dx.doi.org/10.1126/science.aac793

The mutagenic chain reaction : from Evo-Devo to active genetics

While there has been a great deal of research trying to understand the link between DNA information the regulatory level in the final morphological outcome of an organism, a deep understanding of this relationship remains elusive. Here I attempted a new integrated approach to problem by : 1) comparing DNA sequences taken from different animals that have been diversified during evolution to generate altered function. 2) exploring a novel approach to dissecting cis-regulatory modules (CRMs) using in locus Cas9 genome editing; and 3) developing the mutagenic chain reaction, a novel active genetic element that can be use to easily perform cis-regulatory analysis in organisms with no existing genetic tool

The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations

An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype, whereas individuals homozygous for two copies of the allele will display a mutant phenotype. We have developed a method called the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome-editing system for generating autocatalytic mutations, to produce homozygous loss-of-function mutations. In Drosophila, we found that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome, thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms

Gene editing technologies and applications for insects

Initially discovered in bacteria, CRISPR-based genome editing endonucleases have proven remarkably amenable for adaptation to insects. To date, these endonucleases have been utilized in a plethora of both model and non-model insects including diverse flies, bees, beetles, butterflies, moths, and grasshoppers, to name a few, thereby revolutionizing functional genomics of insects. In addition to basic genome editing, they have also been invaluable for advanced genome engineering and synthetic biology applications. Here we explore the recent genome editing advancements in insects for generating site-specific genomic mutations, insertions, deletions, as well as more advanced applications such as Homology Assisted Genome Knock-in (HACK), potential to utilize DNA base editing, generating predictable reciprocal chromosomal translocations, and development gene drives to control the fate of wild populations