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

2017 ◽  
Vol 284 (1860) ◽  
pp. 20170799 ◽  
Author(s):  
Thomas A. A. Prowse ◽  
Phillip Cassey ◽  
Joshua V. Ross ◽  
Chandran Pfitzner ◽  
Talia A. Wittmann ◽  
...  
Keyword(s):  
Female Sterility ◽  
Gene Drive ◽  
End Joining ◽  
Guide Rna ◽  
Deleterious Alleles ◽  
Precise Knowledge ◽  
Animal Populations ◽  
Individual Based Models ◽  
Non Homologous End Joining ◽  
Gene Drives

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

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population

2020 ◽  
Vol 117 (39) ◽  
pp. 24377-24383 ◽  
Author(s):  
Jackson Champer ◽  
Emily Yang ◽  
Esther Lee ◽  
Jingxian Liu ◽  
Andrew G. Clark ◽  
...  
Keyword(s):  
Gene Drive ◽  
End Joining ◽  
Large Cage ◽  
Vector Borne Diseases ◽  
Guide Rnas ◽  
Genetic Modifications ◽  
New Strategy ◽  
Cage Population ◽  
Vector Borne ◽  
Gene Drives

Engineered gene drives are being explored as a new strategy in the fight against vector-borne diseases due to their potential for rapidly spreading genetic modifications through a population. However, CRISPR-based homing gene drives proposed for this purpose have faced a major obstacle in the formation of resistance alleles that prevent Cas9 cleavage. Here, we present a homing drive in Drosophila melanogaster that reduces the prevalence of resistance alleles below detectable levels by targeting a haplolethal gene with two guide RNAs (gRNAs) while also providing a rescue allele. Resistance alleles that form by end-joining repair typically disrupt the haplolethal target gene and are thus removed from the population because individuals that carry them are nonviable. We demonstrate that our drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no functional resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals within a few generations. These results show that a haplolethal homing drive can provide an effective tool for targeted genetic modification of entire populations.

scholarly journals Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

2018 ◽  
Author(s):  
Wannaporn Ittiprasert ◽  
Victoria H. Mann ◽  
Shannon E. Karinshak ◽  
Avril Coghlan ◽  
Gabriel Rinaldi ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Genome Editing ◽  
Human Macrophage ◽  
Wild Type ◽  
End Joining ◽  
Chromosomal Breakage ◽  
Knock Out ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

scholarly journals Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing

Gene Therapy ◽  
2021 ◽  
Author(s):  
Jessica D. Gumerson ◽  
Amal Alsufyani ◽  
Wenhan Yu ◽  
Jingqi Lei ◽  
Xun Sun ◽  
...  
Keyword(s):  
Expression Vectors ◽  
End Joining ◽  
Reading Frame ◽  
Guide Rna ◽  
Domain Specific ◽  
Somatic Gene ◽  
Non Homologous End Joining ◽  
Cone Opsins ◽  
Inherited Retinal Degeneration

AbstractMutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.

scholarly journals Evolution of Resistance Against CRISPR/Cas9 Gene Drive

2016 ◽  
Author(s):  
Robert L. Unckless ◽  
Andrew G. Clark ◽  
Philipp W. Messer
Keyword(s):  
De Novo ◽  
Natural Populations ◽  
Resistance Mechanisms ◽  
Target Site ◽  
Pest Species ◽  
Gene Drive ◽  
Guide Rna ◽  
Deleterious Alleles ◽  
Laboratory Populations ◽  
Potential Applications

AbstractThe idea of driving genetically modified alleles to fixation in a population has fascinated scientists for over 40 years1,2. Potential applications are broad and ambitious, including the eradication of disease vectors, the control of pest species, and the preservation of endangered species from extinction3. Until recently, these possibilities have remained largely abstract due to the lack of an effective drive mechanism. CRISPR/Cas9 gene drive (CGD) now promise a highly adaptable approach for driving even deleterious alleles to high population frequency, and this approach was recently shown to be effective in small laboratory populations of insects4–7. However, it remains unclear whether CGD will also work in large natural populations in the face of potential resistance mechanisms. Here we show that resistance against CGD will inevitably evolve unless populations are small and repair of CGD-induced cleavage via nonhomologous end joining (NHEJ) can be effectively suppressed, or resistance costs are on par with those of the driver. We specifically calculate the probability that resistance evolves from variants at the target site that are not recognized by the driver's guide RNA, either because they are already present when the driver allele is introduced, arise byde novomutation, or are created by the driver itself when NHEJ introduces mutations at the target site. Our results shed light on strategies that could facilitate the engineering of a successful drive by lowering resistance potential, as well as strategies that could promote resistance as a possible mechanism for controlling a drive. This study highlights the need for careful modeling of CGD prior to the actual release of a driver construct into the wild.

scholarly journals Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs

2018 ◽  
Vol 115 (40) ◽  
pp. E9343-E9352 ◽  
Author(s):  
Georg Oberhofer ◽  
Tobin Ivy ◽  
Bruce A. Hay
Keyword(s):  
Homing Endonuclease ◽  
Marker Gene ◽  
Specific Gene ◽  
Gene Drive ◽  
Loss Of Function ◽  
End Joining ◽  
Complex Activity ◽  
Guide Rna ◽  
Type Gene ◽  
Population Suppression

A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work inDrosophilaand mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs inDrosophila melanogastertargeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.

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

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.

scholarly journals Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing

2021 ◽  
Author(s):  
Ya-Wen Fu ◽  
Xin-Yue Dai ◽  
Wen-Tian Wang ◽  
Zhi-Xue Yang ◽  
Juan-Juan Zhao ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Trichostatin A ◽  
Fold Increase ◽  
Cell Types ◽  
End Joining ◽  
Guide Rna ◽  
Human Ipscs ◽  
Virus Serotype ◽  
Non Homologous End Joining ◽  
Kinetics Of

Abstract Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.

scholarly journals Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mollie S. Schubert ◽  
Bernice Thommandru ◽  
Jessica Woodley ◽  
Rolf Turk ◽  
Shuqi Yan ◽  
...  
Keyword(s):  
Design Parameters ◽  
Optimized Design ◽  
End Joining ◽  
Exogenous Dna ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Mammalian Cell Lines ◽  
Non Homologous End Joining ◽  
Dna Template ◽  
Donor Template

AbstractCRISPR–Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during repair allows for the intentional, precise incorporation of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR–Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein (RNP) complexes. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: https://www.idtdna.com/HDR

scholarly journals Daisy-chain gene drives for the alteration of local populations

2019 ◽  
Vol 116 (17) ◽  
pp. 8275-8282 ◽  
Author(s):  
Charleston Noble ◽  
John Min ◽  
Jason Olejarz ◽  
Joanna Buchthal ◽  
Alejandro Chavez ◽  
...  
Keyword(s):  
Drive System ◽  
Chain Gene ◽  
Gene Drive ◽  
Rna Sequences ◽  
Guide Rna ◽  
Highly Active ◽  
Wide Range ◽  
Fitness Parameters ◽  
Drive Systems ◽  
Gene Drives

If they are able to spread in wild populations, CRISPR-based gene-drive elements would provide new ways to address ecological problems by altering the traits of wild organisms, but the potential for uncontrolled spread tremendously complicates ethical development and use. Here, we detail a self-exhausting form of CRISPR-based drive system comprising genetic elements arranged in a daisy chain such that each drives the next. “Daisy-drive” systems can locally duplicate any effect achievable by using an equivalent self-propagating drive system, but their capacity to spread is limited by the successive loss of nondriving elements from one end of the chain. Releasing daisy-drive organisms constituting a small fraction of the local wild population can drive a useful genetic element nearly to local fixation for a wide range of fitness parameters without self-propagating spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionarily stable daisy drive as well as self-propagating CRISPR-based gene drive. Especially when combined with threshold dependence, daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.

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