Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology.

Related papers:

title: An Inherited Male-Producing Factor in Aedes aegypti
abstract: An inherited factor causes a predominance of males in certain strains and in progeny of single pairs of Aedes aegypti L. This factor appears to be transmitted only by males and is not due to differential mortality, at least in postgametic stages. Mass release of male-producing males might be used in control operations.

title: Transporting the marker gene re (red eye) into a laboratory cage population of Aedes aegypti (Diptera: Culicidae), using meiotic drive at the MD locus.
abstract: An attempt has been made to use the meiotic drive gene MD to transport a marker re (red eye) into a laboratory population of the mosquito Aedes aegypti . The experiment produced an increase in re frequency, but also indicated that this gene has unexpectedly high fitness in the laboratory. The need for field estimates of fitness is indicated.

title: Gene drive systems for insect disease vectors
abstract: The elegant mechanisms by which naturally occurring selfish genetic elements, such as transposable elements, meiotic drive genes, homing endonuclease genes and Wolbachia, spread at the expense of their hosts provide some of the most fascinating and remarkable subjects in evolutionary genetics. These elements also have enormous untapped potential to be used in the control of some of the world's most devastating diseases. Effective gene drive systems for spreading genes that can block the transmission of insect-borne pathogens are much needed. Here we explore the potential of natural gene drive systems and discuss the artificial constructs that could be envisaged for this purpose.

title: Site-specific selfish genes as tools for the control and genetic engineering of natural populations
abstract: Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.

title: Homing endonuclease mediated gene targeting in Anopheles gambiae cells and embryos
abstract: Homing endonuclease genes (HEGs) are ‘selfish’ genetic elements that combine the capability to selectively disrupt specific gene sequences with the ability to rapidly spread from a few individuals to an entire population through homologous recombination repair events. Because of these properties, HEGs are regarded as promising candidates to transfer genetic modifications from engineered laboratory mosquitoes to wild-type populations including Anopheles gambiae the vector of human malaria. Here we show that I-SceI and I-PpoI homing endonucleases cleave their recognition sites with high efficiency in A. gambiae cells and embryos and we demonstrate HEG-induced homologous and non-homologous repair events in a variety of functional assays. We also propose a gene drive system for mosquitoes that is based on our finding that I-PpoI cuts genomic rDNA located on the X chromosome in A. gambiae, which could be used to selectively incapacitate X-carrying spermatozoa thereby imposing a severe male-biased sex ratio.

title: Targeting the X Chromosome during Spermatogenesis Induces Y Chromosome Transmission Ratio Distortion and Early Dominant Embryo Lethality in Anopheles gambiae
abstract: We have exploited the high selectivity of the homing endonuclease I-PpoI for the X-linked Anopheles gambiae 28S ribosomal genes to selectively target X chromosome carrying spermatozoa. Our data demonstrated that in heterozygous males, the expression of I-PpoI in the testes induced a strong bias toward Y chromosome–carrying spermatozoa. Notably, these male mosquitoes also induced complete early dominant embryo lethality in crosses with wild-type females. Morphological and molecular data indicated that all spermatozoa, irrespectively of the inheritance of the transgene, carried a substantial amount of I-PpoI protein that could attack the maternally inherited chromosome X of the embryo. Besides the obvious implications for implementing vector control measures, our data demonstrated the feasibility of generating synthetic sex distorters and revealed the intriguing possibility of manipulating maternally inherited genes using wild-type sperm cells carrying engineered endonucleases.

title: A synthetic homing endonuclease-based gene drive system in the human malaria mosquito
abstract: Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity. The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof of principle, the possibility of targeting the mosquito’s ability to serve as a disease vector. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations. We have suggested previously that homing endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose. Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions and the homing endonuclease gene I-SceI, can substantially increase its transmission to the progeny in transgenic mosquitoes of the human malaria vector Anopheles gambiae. We show that the I-SceI element is able to invade receptive mosquito cage populations rapidly, validating mathematical models for the transmission dynamics of HEGs. Molecular analyses confirm that expression of I-SceI in the male germline induces high rates of site-specific chromosomal cleavage and gene conversion, which results in the gain of the I-SceI gene, and underlies the observed genetic drive. These findings demonstrate a new mechanism by which genetic control measures can be implemented. Our results also show in principle how sequence-specific genetic drive elements like HEGs could be used to take the step from the genetic engineering of individuals to the genetic engineering of populations.

title: A synthetic sex ratio distortion system for the control of the human malaria mosquito
abstract: It has been theorized that inducing extreme reproductive sex ratios could be a method to suppress or eliminate pest populations. Limited knowledge about the genetic makeup and mode of action of naturally occurring sex distorters and the prevalence of co-evolving suppressors has hampered their use for control. Here we generate a synthetic sex distortion system by exploiting the specificity of the homing endonuclease I-PpoI, which is able to selectively cleave ribosomal gene sequences of the malaria vector Anopheles gambiae that are located exclusively on the mosquito’s X chromosome. We combine structure-based protein engineering and molecular genetics to restrict the activity of the potentially toxic endonuclease to spermatogenesis. Shredding of the paternal X chromosome prevents it from being transmitted to the next generation, resulting in fully fertile mosquito strains that produce >95% male offspring. We demonstrate that distorter male mosquitoes can efficiently suppress caged wild-type mosquito populations, providing the foundation for a new class of genetic vector control strategies.

title: The ecology of genetically modified mosquitoes.
abstract: Ecological and population biology issues constitute serious challenges to the application of genetically modified mosquitos (GMM) for disease control.

title: Genome-block expression-assisted association studies discover malaria resistance genes in Anopheles gambiae
abstract: We developed an approach to fine-map large genomic regions that play important roles for malaria parasite resistance in the vector mosquito, Anopheles gambiae. Direct-association studies in wild mosquitoes from malaria-endemic areas in Africa identified three mutations in three genes that are associated significantly with Plasmodium falciparum infection. We confirmed the involvement of these genes in parasite infection by silencing them. The discovered genes will help us understand interactions between the mosquitoes and parasites. The identified single-nucleotide polymorphisms could be used to develop a malaria risk-assessment tool and may help eliminate parasite-susceptible mosquitoes. The malaria parasite-resistance island (PRI) of the African mosquito vector, Anopheles gambiae, was mapped to five genomic regions containing 80 genes, using coexpression patterns of genomic blocks. High-throughput sequencing identified 347 nonsynonymous single-nucleotide polymorphisms within these genes in mosquitoes from malaria-endemic areas in Kenya. Direct association studies between nonsynonymous single-nucleotide polymorphisms and Plasmodium falciparum infection identified three naturally occurring genetic variations in each of three genes (An. gambiae adenosine deaminase, fibrinogen-related protein 30, and fibrinogen-related protein 1) that were associated significantly with parasite infection. A role for these genes in the resistance phenotype was confirmed by RNA interference knockdown assays. Silencing fibrinogen-related protein 30 increased parasite infection significantly, whereas ablation of fibrinogen-related protein 1 transcripts resulted in mosquitoes nearly free of parasites. The discovered genes and single-nucleotide polymorphisms are anticipated to be useful in the development of tools for malaria control in endemic areas in Africa.

title: Reprogramming homing endonuclease specificity through computational design and directed evolution
abstract: Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally.

title: Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
abstract: Specific gene-targeting nucleases are critical for genetic engineering and therapy. Meganucleases have been shown to harbor many properties, including small monomeric folds and high cleavage specificities, that are appropriate for this purpose. However, they are rarely used for genome modification because their DNA specificity requires an extensive network of up to 50 amino acids, making redesign difficult. Because extensively reprogramming molecular recognition across such a complex interface is a fundamental biotechnology challenge, meganucleases are used here as a model system for the development of an efficient approach to alter their specificity. The nucleases produced by this method, when tethered to transcription activator-like effector domains, resulted in highly active DNA targeting reagents with notable recognition properties. LAGLIDADG homing endonucleases (meganucleases) are sequence-specific DNA cleavage enzymes used for genome engineering. Recently, meganucleases fused to transcription activator-like effectors have been demonstrated to efficiently introduce targeted genome modifications. However, retargeting meganucleases to genomic sequences of interest remains challenging because it usually requires extensive alteration of a large number of amino acid residues that are situated in and near the DNA interface. Here we describe an effective strategy to extensively redesign such an extensive biomolecular interface. Well-characterized meganucleases are computationally screened to identify the best candidate enzyme to target a genomic region; that protein is then redesigned using iterative rounds of in vitro selections within compartmentalized aqueous droplets, which enable screening of extremely large numbers of protein variants at each step. The utility of this approach is illustrated by engineering three different meganucleases to cleave three human genomic sites (found in two exons and one flanking intron in two clinically relevant genes) and a fourth endonuclease that discriminates between single-nucleotide polymorphism variants of one of those targets. Fusion with transcription activator-like effector DNA binding domains significantly enhances targeted modification induced by meganucleases engineered in this study. Simultaneous expression of two such fusion endonucleases results in efficient excision of a defined genomic region.

title: Multiplex Genome Engineering Using CRISPR/Cas Systems
abstract: Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells.

title: RNA-programmed genome editing in human cells
abstract: Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.

title: Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease
abstract: We employ the CRISPR-Cas system of Streptococcus pyogenes as programmable RNA-guided endonucleases (RGENs) to cleave DNA in a targeted manner for genome editing in human cells. We show that complexes of the Cas9 protein and artificial chimeric RNAs efficiently cleave two genomic sites and induce indels with frequencies of up to 33%.

title: CRISPR-assisted editing of bacterial genomes
abstract: Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.

title: One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering
abstract: Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

title: Heritable genome editing in C. elegans via a CRISPR-Cas9 system
abstract: We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.

title: Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.

title: Efficient In Vivo Genome Editing Using RNA-Guided Nucleases
abstract: In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator–like effector nucleases.

title: Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice
abstract: The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5′ coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

title: Heritable gene targeting in the mouse and rat using a CRISPR-Cas system.
abstract: CRISPR-Cas systems have been developed as an efficient gene editing technology in cells and model organisms. Here we use a CRISPR-Cas system to induce genomic DNA fragment deletion in mice by coinjecting two single-guide RNAs (sgRNAs) targeting the Uhrf2 locus with Cas9 mRNA. Furthermore, we report the generation of a Mc3R and Mc4R double-gene knockout rat by means of a single microinjection. High germline-transmission efficiency was observed in both mice and rats. The clustered, regularly interspaced, short palindromic repeats (CRISPR)associated protein (Cas) system has evolved in bacteria and archaea as an RNA-based adaptive immune system against viral and plasmid invasion1. Based on gene conservation and locus organization, three major types of CRISPR systems have been identified2,3. In the type II systems, the complex of a CRISPR RNA (crRNA) annealed to a trans-activating crRNA (tracrRNA) is sufficient to guide the Cas9 endonuclease to a specific genomic sequence to generate double-strand breaks in target DNA4. Previous studies established a strategy for multiplex genome engineering with the Cas9 RNA-guided endonuclease in mammalian cells5,6. Recently, efficient genome editing by the CRISPR-Cas system has been shown in multiple organisms, including zebrafish, mice and bacteria7–9. Several groups have demonstrated that compared with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR-Cas–mediated gene targeting has similar or greater efficiency in cells and zebrafish5–7,10. Although it has been demonstrated that multiple genes can be disrupted in individual mouse embryos using CRISPR-Cas–mediated systems9, germline transmission of Cas9-mediated mutations in animals has not yet been reported. In addition, whether long, specific, genomic DNA target fragments

title: Efficient nonmeiotic allele introgression in livestock using custom endonucleases
abstract: Selective breeding has long been practiced to enrich for desirable DNA variation that influences livestock traits. We demonstrate that genetic variants can be directly introgressed into livestock genomes using a modified transcription activator-like effector nuclease system. The transient exposure of livestock cells to sequence-targeted editors stimulates homology-directed repair to levels that eliminate the need for transgene-dependent selection. Use of oligonucleotide template enables efficient single nucleotide changes to the genome and permits the transmission of both natural and novel DNA sequence variants into naïve livestock breeds and species. Gene editing offers a powerful method for accelerating the genetic improvement of livestock for food and also for developing swine as a resource for regenerative medicine and models of human disease. We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10–50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.

title: Biosafety and risk assessment in the use of genetically modified mosquitoes for disease control
abstract: The development and implementation of the release of genetically modified mosquitoes (GMM) for interrupting pathogen transmission represent a major challenge, despite the fact that several achievements have been made about Anopheles and Aedes mosquitoes. There are major biotechnology challenges remaining about the improvement of the stability of a gene construct and its expression for a robust and complete interruption of pathogen transmission and the devise of safe means of spreading foreign antipathogen genes through mosquito populations in the wild. The implementation obstacles to overcome include proper risk assessment and management, conduct of studies to ensure safety for humans and the environment, devise of appropriate control strategies based on sound gene-driving systems, address properly ethical, legal and social implications of the release of GMM and public concerns. Although the development of GMM as disease-control tool is technically feasible, for proper implementation no field release must be undertaken until clear scientific proof of safety for humans and the environment and efficacy is provided and ELSI concerns and public acceptance are properly addressed

title: Guidance for contained field trials of vector mosquitoes engineered to contain a gene drive system: recommendations of a scientific working group.
abstract: THE FOLLOWING RECOMMENDATIONS represent the response of a group of involved scientists to the need for guidance to aid researchers, government authorities, and community leaders as they consider the design and implementation of field trials to assess the safety and efficacy of genetic strategies for reducing the transmission of diseases by mosquito vectors. Guidance is provided for contained (caged) field trials of genetically-engineered (GE) vector mosquitoes that are fertile and contain novel genetic constructs designed to spread through natural mosquito populations (“gene drive systems”). An effort is made to raise the practical issues that must be considered in advance of such testing, provide generalized recommendations based on currently available information, and identify “points to consider” regarding additional information that may be required in order to make informed decisions on a case-by-case basis. This guidance is intended to clarify the pathway for further assessment of the potential utility of such GE mosquitoes as a tool for the improvement of public health in disease-endemic regions. 2.

title: Release of genetically engineered insects: a framework to identify potential ecological effects
abstract: Genetically engineered (GE) insects have the potential to radically change pest management worldwide. With recent approvals of GE insect releases, there is a need for a synthesized framework to evaluate their potential ecological and evolutionary effects. The effects may occur in two phases: a transitory phase when the focal population changes in density, and a steady state phase when it reaches a new, constant density. We review potential effects of a rapid change in insect density related to population outbreaks, biological control, invasive species, and other GE organisms to identify a comprehensive list of potential ecological and evolutionary effects of GE insect releases. We apply this framework to the Anopheles gambiae mosquito – a malaria vector being engineered to suppress the wild mosquito population – to identify effects that may occur during the transitory and steady state phases after release. Our methodology reveals many potential effects in each phase, perhaps most notably those dealing with immunity in the transitory phase, and with pathogen and vector evolution in the steady state phase. Importantly, this framework identifies knowledge gaps in mosquito ecology. Identifying effects in the transitory and steady state phases allows more rigorous identification of the potential ecological effects of GE insect release.

title: Homing endonuclease genes: the rise and fall and rise again of a selfish element.
abstract: Homing endonuclease genes (HEGs) are selfish genetic elements that spread by first cleaving chromosomes that do not contain them and then getting copied across to the broken chromosome as a byproduct of the repair process. The success of this strategy will depend on the opportunities for homing--in other words, the frequency with which HEG(+) and HEG(-) chromosomes come into contact--which varies widely among host taxa. HEGs are also unusual in that the selection pressure for endonuclease function disappears if they become fixed in a population, which makes them susceptible to degeneration and imposes a need for regular horizontal transmission between species. HEGs will be selected to reduce the harm done to the host organism, and this is expected to influence the evolution of their sequence specificity and maturase functions. HEGs may also be domesticated by their hosts, and are currently being put to human uses.

title: The population genetics of Drosophila transposable elements.
abstract: The theoretical and empirical studies reviewed here yield the conclusion that Drosophila TEs are maintained in populations as a result of transpositional increase in copy number, and that their spread is checked by one or more opposing forces. In other words, the concept that TEs are essentially intragenomic parasites is supported

title: Maternal-effect selfish genes in flour beetles.
abstract: A previously unknown class of dominant, maternal-effect lethal M factors was found to be widespread in natural populations of the flour beetle, Tribolium castaneum, collected on several continents. Such factors are integrated into the host chromosomes at variable locations and show the remarkable property of self-selection by maternal-effect lethality to all hatchlings that do not inherit a copy of the factor itself. Offspring are rescued by either paternally or maternally inherited copies. The M-bearing chromosome is thereby perpetuated at the expense of its non-M homolog. M factors that map to different regions of the genome do not rescue one another's maternal-effect lethality. Factors expressing these properties are predicted to spread in a population, even in the absence of any additional selective advantage. Similar factors also occur in the related species T. confusum.

title: A synthetic maternal-effect selfish genetic element drives population replacement in Drosophila.
abstract: One proposed strategy for controlling the transmission of insect-borne pathogens uses a drive mechanism to ensure the rapid spread of transgenes conferring disease refractoriness throughout wild populations. Here, we report the creation of maternal-effect selfish genetic elements in Drosophila that drive population replacement and are resistant to recombination-mediated dissociation of drive and disease refractoriness functions. These selfish elements use microRNA-mediated silencing of a maternally expressed gene essential for embryogenesis, which is coupled with early zygotic expression of a rescuing transgene.

title: A cytogenetic analysis of meiotic drive in the mosquito, Aedes aegypti (L.)
abstract: Meiotic drive in Aedes aegypti (L.) is shown by a Giemsa C-banding technique to be associated with preferential isochromatid breakage of the X chromosome during male meiosis. These breaks remain open at least until anaphase-I and, since the range of cells affected is proportional to the sensitivity of the X chromosome to the Distorter gene, it is argued that they are directly related to the decreased number of spermatozoa found in distorting males. This reduction is considered to be attributable to the degeneration of more X- than Y-bearing spermatids but it is probable that some non-functional X-bearing spermatozoa are also produced. Chromosome breakage is almost completely confined to four sites, two adjacent to the centromere, one just proximal to the intercalary band and another about the centre of the unbanded arm. Although the first three of these lie within a region in which crossing-over does not take place, fragmentation occurs more frequently in a chiasmate arm than in one devoid of chromatid exchange.

title: Sex-Ratio Distortion Caused by Meiotic Drive in Mosquitoes
abstract: Meiotic-drive genes have been described in two species of mosquito, Aedes aegypti and Culex quinquefasciatus. In both species, a Y (M)-linked gene causes a change in sex ratio in favor of males. More is known about the Distorter gene (D) in A. aegypti, but thc gene in C. quinquefasciatus appears to be similar. D is located on the right arm of the sex chromosome, is marked with the intercalary Giemsa C-band when present, and is linked closely to the sex locus or region. D undergoes meiotic drive only when in coupling with M, the male-determining gene, which does not recombine with the centromere. Sensitivity to the MD haplotype is controlled at m, the female-determining gene. Therefore, m is considered to be the responder. The m haplotype is polymorphic for sensitivity to MD (mr1, mr2, ms1-ms6). Sensitivity is influenced, in some strains, by a second sex-linked gene t (tolerance to Distorter). Another sex-linked gene, A, enhances the effect of D. Meiotic drive is associated with breaks on the sex chromosomes, First seen at diplotene, mainly at four discrete positions, 90% of these breaks occur on the X (m) chromosome, They usually appear on a chiasmic arm of the bivalent, with the result that most acentric fragments remain attached to the unbroken homologue by a chiasma. Examination of developing spermatozoa in Distorter males reveals extreme sperm depletion, a high degree of abnormality (e.g., multiple axonemes), and an increased DNA content up to the 4C level. Surprisingly, however, the effective fertility of these males is hardly reduced. Evidently, spermatozoa are produced in nondistorter males in vast excess. The Distorter gene has been found in populations from Africa, America, Australia, and Sri Lanka. Resistance to it is much more widespread. The presence of natural resistance argues against the use of D for population control, but its effect has been shown to be enhanced in the presence of certain translocations and the A gene, causing it to become effective in field cage trials, even in the presence of some resistance. There is also experimental support for using D to bring about changes in populations ("population replacement").

title: Pest Control by Genetic Manipulation of Sex Ratio
abstract: We model the release of insects carrying an allele at multiple loci that shifts sex ratios in favor of males. We model two approaches to sex ratio alteration. In the first (denoted SD), meiotic segregation (or sperm fertility) is distorted in favor of gametes carrying the male-determining genetic element (e.g., Y-chromosome). It is assumed that any male carrying at least one copy of the SD allele produces only genotypically male offspring. In the second approach (denoted PM), the inserted allele alters sex ratio by causing genetically female individuals to become phenotypically male. It is assumed that any insect carrying at least one copy of the PM allele is phenotypically male. Both approaches reduce future population growth by reducing the number of phenotypic females. The models allow variation in the number of loci used in the release, the size of the release, and the negative fitness effect caused by insertion of each sex ratio altering allele. We show that such releases may be at least 2 orders of magnitude more effective than sterile male releases (SIT) in terms of numbers of surviving insects. For example, a single SD release with two released insects for every wild insect and a 5% fitness cost per inserted allele could reduce the target population to 1/1000th of the no-release population size, whereas a similar-sized SIT release would only reduce the population to one-fifth of its original size. We also compare these two sex ratio alteration approaches to a female-killing (FK) system and the sterile male technique when there are repeated releases over a number of generations. In these comparisons, the SD approach is the most efficient with equivalent pest suppression achieved by release of ≈1 SD, 1.5–20 PM, 2–70 FK, and 16–3,000 SIT insects, depending on conditions. We also calculate the optimal number of SD and PM allele insertions to be used under various conditions, assuming that there is an additional genetic load incurred for each allelic insertion.

title: The Population Genetics of Using Homing Endonuclease Genes in Vector and Pest Management
abstract: Homing endonuclease genes (HEGs) encode proteins that in the heterozygous state cause double-strand breaks in the homologous chromosome at the precise position opposite the HEG. If the double-strand break is repaired using the homologous chromosome, the HEG becomes homozygous, and this represents a powerful genetic drive mechanism that might be used as a tool in managing vector or pest populations. HEGs may be used to decrease population fitness to drive down population densities (possibly causing local extinction) or, in disease vectors, to knock out a gene required for pathogen transmission. The relative advantages of HEGs that target viability or fecundity, that are active in one sex or both, and whose target is expressed before or after homing are explored. The conditions under which escape mutants arise are also analyzed. A different strategy is to place HEGs on the Y chromosome that cause one or more breaks on the X chromosome and so disrupt sex ratio. This strategy can cause severe sex-ratio biases with efficiencies that depend on the details of sperm competition and zygote mortality. This strategy is probably less susceptible to escape mutants, especially when multiple X shredders are used.

title: INTRODUCING DESIRABLE TRANSGENES INTO INSECT POPULATIONS USING Y-LINKED MEIOTIC DRIVE—A THEORETICAL ASSESSMENT
abstract: The use of genetic drive mechanisms to replace native mosquito genotypes with individuals bearing antipathogen transgenes is a potential strategy for repressing insect transmission of human diseases such as malaria and dengue. Antipathogen transgenes have been developed and tested, but efficient gene drive mechanisms are lacking. Here we theoretically assess the feasibility of introducing antipathogen genes into wild Aedes aegypti populations by using a naturally occurring meiotic drive system. We consider the release of males having both a Y-linked meiotic drive gene and an X-linked drive-insensitive response allele to which an antipathogen gene is linked. We use mathematical models and computer simulations to determine how the post-introduction dynamics of the antipathogen gene are affected by specific genetic characteristics of the system. The results show that when the natural population is uniformly sensitive to the meiotic drive gene, the antipathogen gene may be driven close to fixation if the fitness costs of the drive gene, the insensitive response allele, and the antipathogen gene are low. However, when the natural population has a small proportion of an X-linked insensitive response allele or an autosomal gene that strongly reduces the effect of the drive gene, the antipathogen gene does not spread if it has an associated fitness cost. Our modeling results provide a theoretical foundation for further experimental tests.

title: Evolutionary maintenance of selfish homing endonuclease genes in the absence of horizontal transfer
abstract: Homing endonuclease genes are “selfish” mobile genetic elements whose endonuclease promotes the spread of its own gene by creating a break at a specific target site and using the host machinery to repair the break by copying and inserting the gene at this site. Horizontal transfer across the boundary of a species or population within which mating takes place has been thought to be necessary for their evolutionary persistence. This is based on the assumption that they will become fixed in a host population, where opportunities of homing will disappear, and become susceptible to degeneration. To test this hypothesis, we modeled behavior of a homing endonuclease gene that moves during meiosis through double-strand break repair. We mathematically explored conditions for persistence of the homing endonuclease gene and elucidated their parameter dependence as phase diagrams. We found that, if the cost of the pseudogene is lower than that of the homing endonuclease gene, the 2 forms can persist in a population through autonomous periodic oscillation. If the cost of the pseudogene is higher, 2 types of dynamics appear that enable evolutionary persistence: bistability dependent on initial frequency or fixation irrespective of initial frequency. The prediction of long persistence in the absence of horizontal transfer was confirmed by stochastic simulations in finite populations. The average time to extinction of the endonuclease gene was found to be thousands of meiotic generations or more based on realistic parameter values. These results provide a solid theoretical basis for an understanding of these and other extremely selfish elements.

title: Requirements for effective malaria control with homing endonuclease genes
abstract: Malaria continues to impose a substantial burden on human health. We have previously proposed that biological approaches to control the mosquito vector of disease could be developed using homing endonuclease genes (HEGs), a class of selfish or parasitic gene that exists naturally in many microbes. Recent lab studies have demonstrated that HEGs can function in mosquitoes. We constructed and analyzed a model of mosquito population genetics and malaria epidemiology to determine how well HEGs need to function in order to have a significant effect on the burden of disease. Our model, combined with currently available data, indicates that populations of Anopheles gambiae could be eliminated by releasing 2–3 HEGs targeting female fertility genes, or a driving-Y chromosome that is transmitted to 75–96% of progeny. Combinations of fertility-targeting HEGs and Y drive may also be effective. It is possible to eliminate the disease without eliminating the vector, but the parameter space producing this outcome appears to be small. HEGs causing a quantitative reduction in adult survival can be more effective than those targeting female fertility, but the selection coefficients that need to be imposed are still large, unless many HEGs are to be released. Simulations show that HEG-based strategies can be effective over socially relevant time frames. Important limiting assumptions of the models are that there is only a single vector species, and we model a homogeneous population, not a landscape. Nevertheless, we conclude that HEG-based approaches could have a transformational effect on malaria control efforts.

title: Interplay of population genetics and dynamics in the genetic control of mosquitoes
abstract: Some proposed genetics-based vector control methods aim to suppress or eliminate a mosquito population in a similar manner to the sterile insect technique. One approach under development in Anopheles mosquitoes uses homing endonuclease genes (HEGs)—selfish genetic elements (inherited at greater than Mendelian rate) that can spread rapidly through a population even if they reduce fitness. HEGs have potential to drive introduced traits through a population without large-scale sustained releases. The population genetics of HEG-based systems has been established using discrete-time mathematical models. However, several ecologically important aspects remain unexplored. We formulate a new continuous-time (overlapping generations) combined population dynamic and genetic model and apply it to a HEG that targets and knocks out a gene that is important for survival. We explore the effects of density dependence ranging from undercompensating to overcompensating larval competition, occurring before or after HEG fitness effects, and consider differences in competitive effect between genotypes (wild-type, heterozygotes and HEG homozygotes). We show that population outcomes—elimination, suppression or loss of the HEG—depend crucially on the interaction between these ecological aspects and genetics, and explain how the HEG fitness properties, the homing rate (drive) and the insect's life-history parameters influence those outcomes.

title: Insect Population Control by Homing Endonuclease-Based Gene Drive: An Evaluation in Drosophila melanogaster
abstract: Insects play a major role as vectors of human disease as well as causing significant agricultural losses. Harnessing the activity of customized homing endonuclease genes (HEGs) has been proposed as a method for spreading deleterious mutations through populations with a view to controlling disease vectors. Here, we demonstrate the feasibility of this method in Drosophila melanogaster, utilizing the well-characterized HEG, I-SceI. In particular, we show that high rates of homing can be achieved within spermatogonia and in the female germline. We show that homed constructs continue to exhibit HEG activity in the subsequent generation and that the ectopic homing events required for initiating the strategy occur at an acceptable rate. We conclude that the requirements for successful deployment of a HEG-based gene drive strategy can be satisfied in a model dipteran and that there is a reasonable prospect of the method working in other dipterans. In characterizing the system we measured repair outcomes at the spermatogonial, spermatocyte, and spermatid stages of spermatogenesis. We show that homologous recombination is restricted to spermatogonia and that it immediately ceases when they become primary spermatocytes, indicating that the choice of DNA repair pathway in the Drosophila testis can switch abruptly during differentiation.

title: Targeting the X Chromosome during Spermatogenesis Induces Y Chromosome Transmission Ratio Distortion and Early Dominant Embryo Lethality in Anopheles gambiae
abstract: We have exploited the high selectivity of the homing endonuclease I-PpoI for the X-linked Anopheles gambiae 28S ribosomal genes to selectively target X chromosome carrying spermatozoa. Our data demonstrated that in heterozygous males, the expression of I-PpoI in the testes induced a strong bias toward Y chromosome–carrying spermatozoa. Notably, these male mosquitoes also induced complete early dominant embryo lethality in crosses with wild-type females. Morphological and molecular data indicated that all spermatozoa, irrespectively of the inheritance of the transgene, carried a substantial amount of I-PpoI protein that could attack the maternally inherited chromosome X of the embryo. Besides the obvious implications for implementing vector control measures, our data demonstrated the feasibility of generating synthetic sex distorters and revealed the intriguing possibility of manipulating maternally inherited genes using wild-type sperm cells carrying engineered endonucleases.

title: Highly efficient endogenous human gene correction using designed zinc-finger nucleases
abstract: Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency (SCID) mutation in the IL2Rγ gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.

title: Targeting DNA Double-Strand Breaks with TAL Effector Nucleases
abstract: Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

title: Genome-scale engineering for systems and synthetic biology
abstract: Genome‐modification technologies enable the rational engineering and perturbation of biological systems. Historically, these methods have been limited to gene insertions or mutations at random or at a few pre‐defined locations across the genome. The handful of methods capable of targeted gene editing suffered from low efficiencies, significant labor costs, or both. Recent advances have dramatically expanded our ability to engineer cells in a directed and combinatorial manner. Here, we review current technologies and methodologies for genome‐scale engineering, discuss the prospects for extending efficient genome modification to new hosts, and explore the implications of continued advances toward the development of flexibly programmable chasses, novel biochemistries, and safer organismal and ecological engineering.

title: CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III
abstract: CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.

title: A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
abstract: Ditching Invading DNA Bacteria and archaea protect themselves from invasive foreign nucleic acids through an RNA-mediated adaptive immune system called CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated (Cas). Jinek et al. (p. 816, published online 28 June; see the Perspective by Brouns) found that for the type II CRISPR/Cas system, the CRISPR RNA (crRNA) as well as the trans-activating crRNA—which is known to be involved in the pre-crRNA processing—were both required to direct the Cas9 endonuclease to cleave the invading target DNA. Furthermore, engineered RNA molecules were able to program the Cas9 endonuclease to cleave specific DNA sequences to generate double-stranded DNA breaks. A prokaryotic RNA–directed targeting system can be designed to cleave any DNA sequence. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

title: Improving CRISPR-Cas nuclease specificity using truncated guide RNAs
abstract: Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools. CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target. Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.

title: Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems.
abstract: Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems

title: CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering
abstract: Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

title: DNA targeting specificity of RNA-guided Cas9 nucleases
abstract: The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
abstract: The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 1012 potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

title: CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
abstract: The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

title: Optimising Homing Endonuclease Gene Drive Performance in a Semi-Refractory Species: The Drosophila melanogaster Experience
abstract: Homing endonuclease gene (HEG) drive is a promising insect population control technique that employs meganucleases to impair the fitness of pest populations. Our previous studies showed that HEG drive was more difficult to achieve in Drosophila melanogaster than Anopheles gambiae and we therefore investigated ways of improving homing performance in Drosophila. We show that homing in Drosophila responds to increased expression of HEGs specifically during the spermatogonia stage and this could be achieved through improved construct design. We found that 3′-UTR choice was important to maximise expression levels, with HEG activity increasing as we employed Hsp70, SV40, vasa and βTub56D derived UTRs. We also searched for spermatogonium-specific promoters and found that the Rcd-1r promoter was able to drive specific expression at this stage. Since Rcd-1 is a regulator of differentiation in other species, it suggests that Rcd-1r may serve a similar role during spermatogonial differentiation in Drosophila. Contrary to expectations, a fragment containing the entire region between the TBPH gene and the bgcn translational start drove strong HEG expression only during late spermatogenesis rather than in the germline stem cells and spermatogonia as expected. We also observed that the fraction of targets undergoing homing was temperature-sensitive, falling nearly four-fold when the temperature was lowered to 18°C. Taken together, this study demonstrates how a few simple measures can lead to substantial improvements in the HEG-based gene drive strategy and reinforce the idea that the HEG approach may be widely applicable to a variety of insect control programs.

title: Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells
abstract: Improving Whole-Genome Screens Improved methods are needed for the knockout of individual genes in genome-scale functional screens. Wang et al. (p. 80, published online 12 December) and Shalem et al. (p. 84, published online 12 December) used the bacterial CRISPR/Cas9 system to power-screen protocols that avoid several of the pitfalls associated with small interfering RNA (siRNA) screens. Genome editing by these methods completely disrupts target genes, thus avoiding weak signals that can occur when transcript abundance is partially decreased by siRNA. Furthermore, gene targeting by the CRISPR system is more precise and appears to produce substantially fewer off-target effects than existing methods. Genome-editing technology allows improved positive or negative selection screens. The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.

title: Genetic Screens in Human Cells Using the CRISPR-Cas9 System
abstract: Improving Whole-Genome Screens Improved methods are needed for the knockout of individual genes in genome-scale functional screens. Wang et al. (p. 80, published online 12 December) and Shalem et al. (p. 84, published online 12 December) used the bacterial CRISPR/Cas9 system to power-screen protocols that avoid several of the pitfalls associated with small interfering RNA (siRNA) screens. Genome editing by these methods completely disrupts target genes, thus avoiding weak signals that can occur when transcript abundance is partially decreased by siRNA. Furthermore, gene targeting by the CRISPR system is more precise and appears to produce substantially fewer off-target effects than existing methods. Genome-editing technology allows improved positive or negative selection screens. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

title: High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells
abstract: Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell–based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.

title: Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification
abstract: Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ∼15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases', recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least fourfold higher than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the versatility of this approach for highly specific genome-wide editing.

title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
abstract: Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5′ end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.

title: Highly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila
abstract: We report a simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila. The system comprises two transgenic strains: one expressing Cas9 protein from the germline-specific nanos promoter and the other ubiquitously expressing a custom guide RNA (gRNA) that targets a unique site in the genome. The two strains are crossed to form an active Cas9–gRNA complex specifically in germ cells, which cleaves and mutates the target site. We demonstrate rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described. Founder animals stably expressing Cas9–gRNA transmitted germline mutations to an average of 60% of their progeny, a dramatic improvement in efficiency over the previous methods based on transient Cas9 expression. Simultaneous cleavage of two sites by co-expression of two gRNAs efficiently induced internal deletion with frequencies of 4.3–23%. Our method is readily scalable to high-throughput gene targeting, thereby accelerating comprehensive functional annotation of the Drosophila genome.

title: A System for the Continuous Directed Evolution of Biomolecules
abstract: Laboratory evolution has generated many biomolecules with desired properties, but a single round of mutation, gene expression, screening or selection, and replication typically requires days or longer with frequent human intervention. Because evolutionary success is dependent on the total number of rounds performed, a means of performing laboratory evolution continuously and rapidly could dramatically enhance its effectiveness. Although researchers have accelerated individual steps in the evolutionary cycle, the only previous example of continuous directed evolution was the landmark study of Wright and Joyce, who continuously evolved RNA ligase ribozymes with an in vitro replication cycle that unfortunately cannot be easily adapted to other biomolecules. Here we describe a system that enables the continuous directed evolution of gene-encoded molecules that can be linked to protein production in Escherichia coli. During phage-assisted continuous evolution (PACE), evolving genes are transferred from host cell to host cell through a modified bacteriophage life cycle in a manner that is dependent on the activity of interest. Dozens of rounds of evolution can occur in a single day of PACE without human intervention. Using PACE, we evolved T7 RNA polymerase (RNAP) variants that recognize a distinct promoter, initiate transcripts with ATP instead of GTP, and initiate transcripts with CTP. In one example, PACE executed 200 rounds of protein evolution over the course of 8 days. Starting from undetectable activity levels in two of these cases, enzymes with each of the three target activities emerged in less than 1 week of PACE. In all three cases, PACE-evolved polymerase activities exceeded or were comparable to that of the wild-type T7 RNAP on its wild-type promoter, representing improvements of up to several hundred-fold. By greatly accelerating laboratory evolution, PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions about molecular evolution.

title: Cas9 as a versatile tool for engineering biology
abstract: RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have dramatically transformed our ability to edit the genomes of diverse organisms. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic.

title: Growing dictyate oocytes, but not early preimplantation embryos, of the mouse display high levels of DNA homologous recombination by single-strand annealing and lack DNA nonhomologous end joining.
abstract: We have investigated the ability of growing dictyate oocytes and early preimplantation embryos of the mouse to process extrachromosomal DNA molecules with free ends by intranuclearly microinjecting DNA fragments containing a region of homology of various extent at either the 5' or 3' terminus. Homologous recombination of these fragments by single-strand annealing (SSA), but not other DNA recombination/joining mechanisms, resulted in the formation of a full-length hsp-lacZ-pA fusion gene that was transcriptionally activated by heat shock in growing oocytes and spontaneously at the early two-cell stage in the embryos, making it possible to quantitatively evaluate SSA activities of these cells by the beta-galactosidase produced. SSA activities of oocytes and embryos were similar in their general properties and in the activity levels observed with saturating amounts of DNA. However, embryo SSA was almost one order of magnitude less effective than that of oocytes. Oocyte and embryo 5' --> 3' exonuclease (a key function of the SSA pathway) and DNA nonhomologous end joining (NHEJ) activities were also investigated using an asymmetric PCR assay. Results showed that NHEJ is lacking in oocytes and is very prominent in the embryos, where it competes with SSA for the injected DNA.

title: Differential Usage of Alternative Pathways of Double-Strand Break Repair in Drosophila
abstract: Double-strand DNA breaks can be repaired by any of several alternative mechanisms that differ greatly in the nature of the final repaired products. We used a reporter construct, designated “Repair reporter 3” (Rr3), to measure the relative usage of these pathways in Drosophila germ cells. The method works by creating a double-strand break at a specific location such that expression of the red fluorescent protein, DsRed, in the next generation can be used to infer the frequency at which each pathway was used. A key feature of this approach is that most data come from phenotypic scoring, thus allowing large sample sizes and considerable precision in measurements. Specifically, we measured the proportion of breaks repaired by (1) conversion repair, (2) nonhomologous end joining (NHEJ), or (3) single-strand annealing (SSA). For conversion repair, the frequency of mitotic crossing over in the germ line indicates the relative prevalence of repair by double Holliday junction (DHJ) formation vs. the synthesis-dependent strand annealing (SDSA) pathway. We used this method to show that breaks occurring early in germ-line development were much more frequently repaired via single-strand annealing and much less likely to be repaired by end joining compared with identical breaks occurring later in development. Conversion repair was relatively rare when breaks were made either very early or very late in development, but was much more frequent in between. Significantly, the changes in relative usage occurred in a compensatory fashion, such that an increase in one pathway was accompanied by decreases in others. This negative correlation is interpreted to mean that the pathways for double-strand break repair compete with each other to handle a given breakage event.

title: Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells.
abstract: DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G2 and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G1/G0 (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G1/G0 cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G1/G0 and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected.

title: Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks
abstract: The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DNA repair pathways in resolving nuclease-induced DNA breaks into genome editing outcomes, we previously developed a fluorescent-based reporter system, designated the Traffic Light Reporter, which provides a readout of gene targeting and gene disruption downstream of a targeted DNA double-strand break. Here we describe two related but novel reporters that extend this technology: one that allows monitoring of the transcriptional activity at the reporter locus, and thus can be applied to interrogate break resolution at active and repressed loci; and a second that reads out single-strand annealing in addition to gene targeting and gene disruption. Application of these reporters to assess repair pathway usage in several common gene editing contexts confirms the importance that chromatin status and initiation of end resection have on the resolution of nuclease-induced breaks.

title: Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system
abstract: The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.

title: DNA interrogation by the CRISPR RNA-guided endonuclease Cas9
abstract: The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA–DNA base-pairing to target foreign DNA in bacteria. Cas9–guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9–RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9–RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9–RNA. Competition assays provide evidence that DNA strand separation and RNA–DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

title: Probing the Limits of Genetic Recoding in Essential Genes
abstract: Changing the Code Easily and efficiently expanding the genetic code could provide tools to genome engineers with broad applications in medicine, energy, agriculture, and environmental safety. Lajoie et al. (p. 357) replaced all known UAG stop codons with synonymous UAA stop codons in Escherichia coli MG1655, as well as release factor 1 (RF1; terminates translation at UAG), thereby eliminating natural UAG translation function without impairing fitness. This made it possible to reassign UAG as a dedicated codon to genetically encode nonstandard amino acids while avoiding deleterious incorporation at native UAG positions. The engineered E. coli incorporated nonstandard amino acids into its proteins and showed enhanced resistance to bacteriophage T7. In a second paper, Lajoie et al. (p. 361) demonstrated the recoding of 13 codons in 42 highly expressed essential genes in E. coli. Codon usage was malleable, but synonymous codons occasionally were nonequivalent in unpredictable ways. Thirteen codons could be removed from all essential ribosomal protein-coding genes across 80 Escherichia coli strains. Engineering radically altered genetic codes will allow for genomically recoded organisms that have expanded chemical capabilities and are isolated from nature. We have previously reassigned the translation function of the UAG stop codon; however, reassigning sense codons poses a greater challenge because such codons are more prevalent, and their usage regulates gene expression in ways that are difficult to predict. To assess the feasibility of radically altering the genetic code, we selected a panel of 42 highly expressed essential genes for modification. Across 80 Escherichia coli strains, we removed all instances of 13 rare codons from these genes and attempted to shuffle all remaining codons. Our results suggest that the genome-wide removal of 13 codons is feasible; however, several genome design constraints were apparent, underscoring the importance of a strategy that rapidly prototypes and tests many designs in small pieces.

title: Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing
abstract: The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas acquired immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but it can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences, and we identify an unexpectedly versatile Cas9 protein from Neisseria meningitidis. We provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins.

title: Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
abstract: The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

title: Beyond insecticides: new thinking on an ancient problem
abstract: Vector-borne disease is one of the greatest contributors to human mortality and morbidity throughout the tropics. Mosquito-transmitted diseases such as malaria, dengue, yellow fever and filariasis are the main contributors to this burden. Although insecticides have historically been used to try to control vector populations, over the past 15 years, substantial progress has been made in developing alternative vector control strategies ranging from biocontrol methods through to genetic modification of wild insect populations. Here, we review recent advances concerning these strategies and consider the potential impediments to their deployment, including the challenges of obtaining regulatory approval and community acceptance.

title: Methodologies and special considerations for environmental risk analysis of genetically modified aquatic biocontrol organisms
abstract: Genetic biocontrol of invasive aquatic species proposes to introduce, for control purposes, a genetically modified (GM) version of an invasive fish species to a targeted aquatic environment. Safe deployment and long term use of such technologies will depend on identifying and managing possible unintended effects to the natural environment. Environmental risk analysis (ERA) is a method for identifying the likelihood and consequences of unintended impacts, and for developing risk management strategies. For the unique situation of genetically modified biocontrol organisms (GMBOs), we review the latest thinking in ERA methodologies for GM fish and explore how terminology and assumptions from ERAs of traditional, non-modified biocontrol organisms and GM fish will need to be recast in ERAs of GMBOs. We also outline some special considerations that an ERA of a GMBOs will have to contend with: non-intuitive potential hazards; uncertainty introduced by extrapolating from domestic systems to natural ecosystems; redundancy in risk management options; and challenges of stakeholder engagement related to new technologies.

title: CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering
abstract: Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

title: Targeted enzyme prodrug therapies.
abstract: The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

title: Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite
abstract: Malaria is estimated to cause 0.7 to 2.7 million deaths per year, but the actual figures could be substantially higher owing to under-reporting and difficulties in diagnosis. If no new control measures are developed, the malaria death toll is projected to double in the next 20 years. Efforts to control the disease are hampered by drug resistance in the Plasmodium parasites, insecticide resistance in mosquitoes, and the lack of an effective vaccine. Because mosquitoes are obligatory vectors for malaria transmission, the spread of malaria could be curtailed by rendering them incapable of transmitting parasites. Many of the tools required for the genetic manipulation of mosquito competence for malaria transmission have been developed. Foreign genes can now be introduced into the germ line of both culicine and anopheline mosquitoes, and these transgenes can be expressed in a tissue-specific manner. Here we report on the use of such tools to generate transgenic mosquitoes that express antiparasitic genes in their midgut epithelium, thus rendering them inefficient vectors for the disease. These findings have significant implications for the development of new strategies for malaria control.

title: Engineered Anopheles Immunity to Plasmodium Infection
abstract: A causative agent of human malaria, Plasmodium falciparum, is transmitted by Anopheles mosquitoes. The malaria parasite is under intensive attack from the mosquito's innate immune system during its sporogonic development. We have used genetic engineering to create immune-enhanced Anopheles stephensi mosquitoes through blood meal-inducible expression of a transgene encoding the IMD pathway-controlled NF-kB Rel2 transcription factor in the midgut and fat-body tissue. Transgenic mosquitoes showed greater resistance to Plasmodium and microbial infection as a result of timely concerted tissue-specific immune attacks involving multiple effectors. The relatively weak impact of this genetic modification on mosquito fitness under laboratory conditions encourages further investigation of this approach for malaria control.

title: Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development
abstract: Anopheles stephensi mosquitoes expressing m1C3, m4B7, or m2A10 single-chain antibodies (scFvs) have significantly lower levels of infection compared to controls when challenged with Plasmodium falciparum, a human malaria pathogen. These scFvs are derived from antibodies specific to a parasite chitinase, the 25 kDa protein and the circumsporozoite protein, respectively. Transgenes comprising m2A10 in combination with either m1C3 or m4B7 were inserted into previously-characterized mosquito chromosomal “docking” sites using site-specific recombination. Transgene expression was evaluated at four different genomic locations and a docking site that permitted tissue- and sex-specific expression was researched further. Fitness studies of docking site and dual scFv transgene strains detected only one significant fitness cost: adult docking-site males displayed a late-onset reduction in survival. The m4B7/m2A10 mosquitoes challenged with P. falciparum had few or no sporozoites, the parasite stage infective to humans, in three of four experiments. No sporozoites were detected in m1C3/m2A10 mosquitoes in challenge experiments when both genes were induced at developmentally relevant times. These studies support the conclusion that expression of a single copy of a dual scFv transgene can completely inhibit parasite development without imposing a fitness cost on the mosquito.

title: Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti.
abstract: Mosquitoes (Aedes aegypti) were genetically modified to exhibit impaired vector competence for dengue type 2 viruses (DENV-2). We exploited the natural antiviral RNA interference (RNAi) pathway in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA genome. The A. aegypti carboxypeptidase A promoter was used to express the IR RNA in midgut epithelial cells after ingestion of a bloodmeal. The promoter and effector gene were inserted into the genome of a white-eye Puerto Rico Rexville D (Higgs' white eye) strain by using the nonautonomous mariner MosI transformation system. A transgenic family, Carb77, expressed IR RNA in the midgut after a bloodmeal. Carb77 mosquitoes ingesting an artificial bloodmeal containing DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. DENV-2 titration of individual mosquitoes showed that most Carb77 mosquitoes poorly supported virus replication. Transmission in vitro of virus from the Carb77 line was significantly diminished when compared to control mosquitoes. The presence of DENV-2-derived siRNAs in RNA extracts from midguts of Carb77 and the loss of the resistance phenotype when the RNAi pathway was interrupted proved that DENV-2 resistance was caused by a RNAi response. Engineering of transgenic A. aegypti that show a high level of resistance against DENV-2 provides a powerful tool for developing population replacement strategies to control transmission of dengue viruses.

title: Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize
abstract: Crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) kill pest insects and reduce the use of conventional insecticides. However, the evolution of Bt resistance can diminishes these benefits. The western corn rootworm is a serious pest of maize and is managed with Bt maize. Beginning in 2009, western corn rootworm with resistance to maize producing the Bt toxin Cry3Bb1 imposed severe injury to Cry3Bb1 maize in Iowa. We show that cross-resistance exists between Cry3Bb1 maize and mCry3A maize and is associated with severe injury to Bt maize in farmers’ fields. These results illustrate that Bt crops producing less than a high dose of toxin against target pests may select for resistance rapidly; consequently, current approaches for managing Bt resistance should be reexamined. The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin.

title: Gene amplification confers glyphosate resistance in Amaranthus palmeri
abstract: The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F2 populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology.

title: Rapid vacuolar sequestration: the horseweed glyphosate resistance mechanism.
abstract: Glyphosate-resistant (GR) weed species are now found with increasing frequency and threaten the critically important glyphosate weed-management system [corrected].



The reported (31)P NMR experiments on glyphosate-sensitive (S) and glyphosate-resistant (R) horseweed, Conyza canadensis (L.) Cronq., show significantly more accumulation of glyphosate within the R biotype vacuole.



Selective sequestration of glyphosate into the vacuole confers the observed horseweed resistance to glyphosate. This observation represents the first clear evidence for the glyphosate resistance mechanism in C. canadensis.

title: The Deadly Chytrid Fungus: A Story of an Emerging Pathogen
abstract: Emerging infectious diseases present a great challenge for the health of both humans and wildlife. The increasing prevalence of drug-resistant fungal pathogens in humans  and recent outbreaks of novel fungal pathogens in wildlife populations  underscore the need to better understand the origins and mechanisms of fungal pathogenicity. One of the most dramatic examples of fungal impacts on vertebrate populations is the effect of the amphibian disease chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). 
Amphibians around the world are experiencing unprecedented population losses and local extinctions . While there are multiple causes of amphibian declines, many catastrophic die-offs are attributed to Bd [4],[5]. The chytrid pathogen has been documented in hundreds of amphibian species, and reports of Bd's impact on additional species and in additional geographic regions are accumulating at an alarming rate (e.g., see http://www.spatialepidemiology.net/bd). Bd is a microbial, aquatic fungus with distinct life stages. The motile stage, called a zoospore, swims using a flagellum and initiates the colonization of frog skin. Within the host epidermal cells, a zoospore forms a spherical thallus, which matures and produces new zoospores by dividing asexually, renewing the cycle of infection when zoospores are released to the skin surface . Bd is considered an emerging pathogen, discovered and described only a decade ago [6],[7]. Despite intensive ecological study of Bd over the last decade, a number of unanswered questions remain. Here we summarize what has been recently learned about this lethal pathogen.

title: Engineering the Caenorhabditis elegans Genome Using Cas9-Triggered Homologous Recombination
abstract: Study of the nematode Caenorhabditis elegans has provided important insights in a wide range of fields in biology. The ability to precisely modify genomes is critical to fully realize the utility of model organisms. Here we report a method to edit the C. elegans genome using the clustered, regularly interspersed, short palindromic repeats (CRISPR) RNA-guided Cas9 nuclease and homologous recombination. We demonstrate that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites and that these breaks can be repaired efficiently by homologous recombination. By supplying engineered homologous repair templates, we generated gfp knock-ins and targeted mutations. Together our results outline a flexible methodology to produce essentially any desired modification in the C. elegans genome quickly and at low cost. This technology is an important addition to the array of genetic techniques already available in this experimentally tractable model organism.

title: Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells.
abstract: RNA-based regulation and CRISPR/Cas transcription factors (CRISPR-TFs) have the potential to be integrated for the tunable modulation of gene networks. A major limitation of this methodology is that guide RNAs (gRNAs) for CRISPR-TFs can only be expressed from RNA polymerase III promoters in human cells, limiting their use for conditional gene regulation. We present new strategies that enable expression of functional gRNAs from RNA polymerase II promoters and multiplexed production of proteins and gRNAs from a single transcript in human cells. We use multiple RNA regulatory strategies, including RNA-triple-helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing. Using these tools, we efficiently modulate endogenous promoters and implement tunable synthetic circuits, including multistage cascades and RNA-dependent networks that can be rewired with Csy4 to achieve complex behaviors. This toolkit can be used for programming scalable gene circuits and perturbing endogenous networks for biology, therapeutic, and synthetic biology applications.