Multiple loci of small effect confer wide variability in efficiency and resistance rate of CRISPR gene drive

ABSTRACTGene drives could allow for control of vector-borne diseases by directly suppressing vector populations or spreading genetic payloads designed to reduce pathogen transmission. CRISPR homing gene drives work by cleaving wild-type alleles, which are then converted to drive alleles by homology-directed repair, increasing the frequency of the drive in a population. However, resistance alleles can form when end-joining repair takes place in lieu of homology-directed repair. Such alleles cannot be converted to drive alleles, which would halt the spread of a drive through a population. To investigate the effects of natural genetic variation on resistance formation, we developed a CRISPR homing gene drive in Drosophila melanogaster and crossed it into the genetically diverse Drosophila Genetic Reference Panel (DGRP) lines, measuring several performance parameters. Most strikingly, resistance allele formation post-fertilization in the early embryo ranged from 7% to 79% among lines and averaged 42±18%. We performed a Genome-Wide Association Study (GWAS) using our results in the DGRP lines and found that the resistance and conversion rates were polygenic, with several genetic polymorphisms showing relatively weak association. RNAi knockdown of several of these genes confirmed their effect, but their small effect sizes implies that their manipulation will yield only modest improvements to the efficacy of gene drives.

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Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400985 ◽

2019 ◽

Vol 10 (2) ◽

pp. 827-837 ◽

Cited By ~ 13

Author(s):

Nikolay P. Kandul ◽

Junru Liu ◽

Anna Buchman ◽

Valentino M. Gantz ◽

Ethan Bier ◽

...

Keyword(s):

Disease Transmission ◽

Target Genes ◽

High Efficiency ◽

Population Control ◽

Natural Populations ◽

Pathogen Transmission ◽

Gene Drive ◽

Guide Rnas ◽

Cas9 Protein ◽

Gene Drives

Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of ‘shadow drive’ generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.

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Molecular safeguarding of CRISPR gene drive experiments

eLife ◽

10.7554/elife.41439 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 39

Author(s):

Jackson Champer ◽

Joan Chung ◽

Yoo Lim Lee ◽

Chen Liu ◽

Emily Yang ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Natural Population ◽

Early Embryo ◽

Drive System ◽

Target Site ◽

Gene Drive ◽

Gene Drives ◽

Synthetic Target

CRISPR-based homing gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target site drives as well as split drives as flexible safeguarding strategies for gene drive experiments by showing that their performance closely resembles that of standard homing drives in Drosophila melanogaster. Using our split drive system, we further find that maternal deposition of both Cas9 and gRNA is required to form resistance alleles in the early embryo and that maternally-deposited Cas9 alone can power germline drive conversion in individuals that lack a genomic source of Cas9.

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Assessment of a split homing based gene drive for efficient knockout of multiple genes

10.1101/706929 ◽

2019 ◽

Cited By ~ 1

Author(s):

Nikolay P. Kandul ◽

Junru Liu ◽

Anna Buchman ◽

Valentino M. Gantz ◽

Ethan Bier ◽

...

Keyword(s):

Target Gene ◽

Tissue Specificity ◽

Target Genes ◽

High Efficiency ◽

Population Control ◽

Natural Populations ◽

Pathogen Transmission ◽

Gene Drive ◽

Guide Rnas ◽

Gene Drives

AbstractHoming based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host-encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate host encoded genes has not been thoroughly explored. To test this approach, here we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of its target gene during super-Mendelian propagation of split-HGD. However, maternal deposition and embryonic expression of Cas9 resulted in the generation of drive resistant alleles which can accumulate and limit the spread of such a drive. Alternative design principles are discussed that could mitigate the accumulation of resistance alleles while incorporating a GME.

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Analysis of off-target effects in CRISPR-based gene drives in the human malaria mosquito

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2004838117 ◽

2021 ◽

Vol 118 (22) ◽

pp. e2004838117

Author(s):

William T. Garrood ◽

Nace Kranjc ◽

Karl Petri ◽

Daniel Y. Kim ◽

Jimmy A. Guo ◽

...

Keyword(s):

Mendelian Inheritance ◽

Gene Drive ◽

Guide Rna ◽

Human Malaria ◽

Homology Directed Repair ◽

Cas9 Nuclease ◽

Set Up ◽

Gene Drives ◽

Target Effects ◽

Important Milestone

CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Anopheles gambiae. Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair (“homing”). Understanding whether CRISPR-induced off-target mutations are generated in Anopheles mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations’ exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels.

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Computational and experimental performance of CRISPR homing gene drive strategies with multiplexed gRNAs

10.1101/679902 ◽

2019 ◽

Cited By ~ 8

Author(s):

Samuel E. Champer ◽

Suh Yeon Oh ◽

Chen Liu ◽

Zhaoxin Wen ◽

Andrew G. Clark ◽

...

Keyword(s):

Large Scale ◽

Optimal Number ◽

Activity Level ◽

Gene Drive ◽

Factors Affecting ◽

Homology Directed Repair ◽

Scale Population ◽

And Performance ◽

Using Data ◽

Gene Drives

ABSTRACTCRISPR homing gene drives potentially have the capacity for large-scale population modification or suppression. However, resistance alleles formed by the drives can prevent them from successfully spreading. Such alleles have been found to form at high rates in most studies, including those in both insects and mammals. One possible solution to this issue is the use of multiple guide RNAs (gRNAs), thus allowing cleavage by the drive even if resistance sequences are present at some of the gRNA target sequences. Here, we develop a high-fidelity model incorporating several factors affecting the performance of drives with multiple gRNAs, including timing of cleavage, reduction in homology-directed repair efficiency due to imperfect homology around the cleavage site, Cas9 activity saturation, variance in the activity level of individual gRNAs, and formation of resistance alleles due to incomplete homology-directed repair. We parameterize the model using data from homing drive experiments designed to investigate these factors and then use it to analyze several types of homing gene drives. We find that each type of drive has an optimal number of gRNAs, usually between two and eight, dependent on drive type and performance parameters. Our model indicates that utilization of multiple gRNAs is insufficient for construction of successful gene drives, but that it provides a critical boost to drive efficiency when combined with other strategies for population modification or suppression.

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Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

10.1101/362558 ◽

2018 ◽

Cited By ~ 16

Author(s):

Hannah A. Grunwald ◽

Valentino M. Gantz ◽

Gunnar Poplawski ◽

Xiang-ru S. Xu ◽

Ethan Bier ◽

...

Keyword(s):

Dna Cleavage ◽

Mendelian Inheritance ◽

Early Embryo ◽

Gene Drive ◽

Dna Breaks ◽

Homology Directed Repair ◽

Double Strand Dna Breaks ◽

Repair Mechanisms ◽

Island Communities ◽

Random Assortment

AbstractA gene drive biases the transmission of a particular allele of a gene such that it is inherited at a greater frequency than by random assortment. Recently, a highly efficient gene drive was developed in insects, which leverages the sequence-targeted DNA cleavage activity of CRISPR/Cas9 and endogenous homology directed repair mechanisms to convert heterozygous genotypes to homozygosity. If implemented in laboratory rodents, this powerful system would enable the rapid assembly of genotypes that involve multiple genes (e.g., to model multigenic human diseases). Such complex genetic models are currently precluded by time, cost, and a requirement for a large number of animals to obtain a few individuals of the desired genotype. However, the efficiency of a CRISPR/Cas9 gene drive system in mammals has not yet been determined. Here, we utilize an active genetic “CopyCat” element embedded in the mouse Tyrosinase gene to detect genotype conversions after Cas9 activity in the embryo and in the germline. Although Cas9 efficiently induces double strand DNA breaks in the early embryo and is therefore highly mutagenic, these breaks are not resolved by homology directed repair. However, when Cas9 expression is limited to the developing female germline, resulting double strand breaks are resolved by homology directed repair that copies the CopyCat allele from the donor to the receiver chromosome and leads to its super-Mendelian inheritance. These results demonstrate that the CRISPR/Cas9 gene drive mechanism can be implemented to simplify complex genetic crosses in laboratory mice and also contribute valuable data to the ongoing debate about applications to combat invasive rodent populations in island communities.

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A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles

10.1101/2021.05.27.446071 ◽

2021 ◽

Author(s):

Emily Yang ◽

Matthew Metzloff ◽

Anna M. Langmüller ◽

Andrew G. Clark ◽

Philipp W Messer ◽

...

Keyword(s):

Conversion Efficiency ◽

Target Gene ◽

Gene Drive ◽

Wild Type ◽

Homology Directed Repair ◽

High Drive ◽

Target Sites ◽

Cage Population ◽

Gene Drives ◽

Population Suppression

Gene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster that utilized multiplexed gRNAs to inhibit the formation of functional resistance alleles in its female fertility target gene. The gRNA target sites were placed close together, preventing reduction in drive conversion efficiency. The construct reached a moderate equilibrium frequency in cage populations without apparent formation of resistance alleles. However, a moderate fitness cost prevented suppression of the cage population. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing type suppression gene drives.

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Natural genetic variation in Drosophila melanogaster reveals genes associated with Coxiella burnetii infection

Genetics ◽

10.1093/genetics/iyab005 ◽

2021 ◽

Author(s):

Rosa M Guzman ◽

Zachary P Howard ◽

Ziying Liu ◽

Ryan D Oliveira ◽

Alisha T Massa ◽

...

Keyword(s):

Association Study ◽

Candidate Genes ◽

Coxiella Burnetii ◽

Genome Wide Association Study ◽

Q Fever ◽

Host Susceptibility ◽

Genome Wide Association ◽

Natural Genetic Variation ◽

Genome Wide ◽

A Genome

Abstract The gram-negative bacterium Coxiella burnetii is the causative agent of Query (Q) fever in humans and coxiellosis in livestock. Host genetics are associated with C. burnetii pathogenesis both in humans and animals; however, it remains unknown if specific genes are associated with severity of infection. We employed the Drosophila Genetics Reference Panel to perform a genome-wide association study to identify host genetic variants that affect host survival to C. burnetii infection. The genome-wide association study identified 64 unique variants (P < 10−5) associated with 25 candidate genes. We examined the role each candidate gene contributes to host survival during C. burnetii infection using flies carrying a null mutation or RNAi knockdown of each candidate. We validated 15 of the 25 candidate genes using at least one method. This is the first report establishing involvement of many of these genes or their homologs with C. burnetii susceptibility in any system. Among the validated genes, FER and tara play roles in the JAK/STAT, JNK, and decapentaplegic/TGF-β signaling pathways which are components of known innate immune responses to C. burnetii infection. CG42673 and DIP-ε play roles in bacterial infection and synaptic signaling but have no previous association with C. burnetii pathogenesis. Furthermore, since the mammalian ortholog of CG13404 (PLGRKT) is an important regulator of macrophage function, CG13404 could play a role in host susceptibility to C. burnetii through hemocyte regulation. These insights provide a foundation for further investigation regarding the genetics of C. burnetii susceptibility across a wide variety of hosts.

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Natural Genetic Variation in Drosophila melanogaster Reveals Genes Associated with Coxiella burnetii Infection

10.1101/2020.05.21.109371 ◽

2020 ◽

Author(s):

Rosa M. Guzman ◽

Zachary P. Howard ◽

Ziying Liu ◽

Ryan D. Oliveira ◽

Alisha T. Massa ◽

...

Keyword(s):

Coxiella Burnetii ◽

Genome Wide Association Study ◽

Q Fever ◽

Nucleotide Polymorphisms ◽

Natural Genetic Variation ◽

Genome Wide ◽

A Genome ◽

Important Regulator ◽

Host Genetic ◽

Or Gene

ABSTRACTThe gram-negative bacterium Coxiella burnetii is the causative agent of Query (Q) fever in humans and coxiellosis in livestock. Association between host genetic background and Coxiella burnetii pathogenesis has been demonstrated both in humans and animals; however, specific genes associated with severity of infection remain unknown. We employed the Drosophila Genetics Reference Panel to perform a genome-wide association study and identify host genetic variants that affect Coxiella burnetii infection outcome. The analysis resulted in 64 genome-wide suggestive (P < 10−5) single nucleotide polymorphisms or gene variants in 25 unique genes. We examined the role of each gene in Coxiella burnetii infection using flies carrying a null mutation or RNAi knockdown of each gene and monitoring survival. Of the 25 candidate genes, 15 validated using at least one method. For many, this is the first report establishing involvement of these genes or their homologs with Coxiella burnetii susceptibility in any system. Among the validated genes, FER and tara play roles in the JAK-STAT, JNK, and decapentaplegic/TGF-β signaling pathways that are associated with the innate immune response to Coxiella burnetii infection. Two other two validated genes, CG42673 and DIP-ɛ, play roles in bacterial infection and synaptic signaling but no previous association with Coxiella burnetii pathogenesis. Furthermore, since the mammalian ortholog of CG13404 (PLGRKT) is an important regulator of macrophage function, CG13404 could play a role in Coxiella burnetii susceptibility through hemocyte regulation. These insights provide a foundation for further investigation of genetics of Coxiella burnetii susceptibility across a wide variety of hosts.

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Development of a confinable gene drive system in the human disease vector Aedes aegypti

eLife ◽

10.7554/elife.51701 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 25

Author(s):

Ming Li ◽

Ting Yang ◽

Nikolay P Kandul ◽

Michelle Bui ◽

Stephanie Gamez ◽

...

Keyword(s):

Aedes Aegypti ◽

Field Trials ◽

Pathogen Transmission ◽

Control Measures ◽

Mosquito Vector ◽

Gene Drive ◽

Wild Populations ◽

Linked Gene ◽

Pathogen Effector ◽

Gene Drives

Aedes aegypti is the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti. To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.

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