CRISPR-Cas9 targeted disruption of the<i>yellow</i>ortholog in the housefly identifies the<i>brown body</i>locus

Heinze, Svenia D.; Kohlbrenner, Tea; Ippolito, Domenica; Meccariello, Angela; Burger, Alexa; Mosimann, Christian; Saccone, Giuseppe; Bopp, Daniel

doi:10.1101/105023

Cited by 7 publications

(9 citation statements)

References 28 publications

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“…However, we propose a repair mechanism that involves HDR of one of the targeted chromatids at the 2n stage of the cell cycle (Fig. 4B), which is consistent with all phenotypes and genotypes encountered in this study as well as in previous studies involving hemizygous introduction of DSBs (9,26,27,30).…”

Section: Resultssupporting

confidence: 91%

“…Chromosome-arm LOH has previously been reported upon introduction of a DSB in one of two homologous chromosomes, but was considered rare and has not been described as disruptive to gene-editing approaches (9,26,27). To investigate the extent and nature of the LOH caused by Cas9-editing of heterozygous loci, a strain with approximately four heterozygous SNPs or INDELs per kbp was generated by mating IMX1555 (CEN.PK genetic background, expressing Cas9, mRuby2 and mTurquoise2 from chromosome V) with S288C (Table S6).…”

Section: Resultsmentioning

confidence: 99%

“…While the HDR machinery is well conserved in eukaryotes (11,12), further research is required to determine if LOH occurs at similar rates in eukaryotes other than S. cerevisiae , and if it impedes gene editing. While DSB-mediated LOH was observed in S. cerevisiae, C. albicans, Drosophilia melanogaster and Mus musculus (9,26,27,30), relative contributions of HR, HDR and NHEJ to DSB repair vary across species. However, since integration of a repair fragment and repair by LOH both involve HDR (33,34), targeting heterozygous loci likely causes gene-editing efficiencies and off-target mutations in other eukaryotes as well, regardless of the efficiency of NHEJ and HR.…”

Section: Discussionmentioning

confidence: 99%

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Allele-specific genome editing using CRISPR-Cas9 causes off-target mutations in diploid yeast

Vries

Couwenberg

Broek

et al. 2018

Preprint

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7 Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic 8 modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate 9 gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous 10 genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less 11 amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when 12 editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a 13 DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb that resulted in 14 introduction of nearly 1700 off-target mutations, due to replacement of sequences on the targeted 15 chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of 16 loss of heterozygosity were consistent with homology directed repair. The extent and frequency of 17 loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, 18 which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the 19 limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be 20 particularly deleterious for human gene therapy, as loss of heterozygous functional copies of anti-21 proliferative and pro-apoptotic genes is a known path to cancer.22 30 DSB facilitates genome editing by increasing the rate of repair by homologous recombination (6).31 When a repair fragment consisting of a DNA oligomer with homology to regions on both sides of the 32 introduced DSB is added, it is integrated at the targeted locus by homologous recombination, 33 resulting in replacement of the original sequence and repair of the DSB (2-4). In S. cerevisiae, double 34 stranded DNA oligomers with 60 bp of homology are sufficient to obtain accurate gene-editing in 35 almost 100% of transformed cells (3). By inserting sequences between the homologous regions of the 36 1 repair oligonucleotide, heterozygous sequences of up to 35 Kbp could be inserted at targeted loci (7). 37While such gene editing approaches have been very efficient in haploid and homozygous diploid 38 yeasts, the accurate introduction of short DNA fragments can be tedious in heterozygous yeast. In 39 homozygous diploid and polyploid eukaryotes, CRISPR-Cas9 introduces DSBs in all alleles of a 40 targeted sequence (8). In heterozygous genomes, gRNAs can be designed for allele-specific targeting 41 if heterozygous loci have different PAM motifs and/or different 5' sequences close to a PAM motif 42 (8,9), enabling allele-specific gene editing using Cas9. In such cases, a DSB is introduced in only one 43 of the homologous chromosomes while the other homolog remains intact. However, the presence of 44 intact homologous chromosomes facilitates repair of DSBs by homologous recombination (HR), 45 homology-directed repair (HDR) or break-induced repair (BIR) in eukaryotes (10-1...

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Allele-specific genome editing using CRISPR-Cas9 causes off-target mutations in diploid yeast

Vries

Couwenberg

Broek

et al. 2018

Preprint

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“…Similarly, we identified five new putative prophenoloxidases, which may play key roles in the process of eggshell melanization in mosquitoes (39). Insect dopachrome conversion enzymes (DCE) have been studied in A. aegypti (20), D. melanogaster (41), Tribolium castaneum (42), and Musca domestica (43), since they are responsible for catalyzing melanization and sclerotization reactions. We now know that at least five extracellular DCE are present to form intact eggshells in A. aegypti and that they may exhibit specific functions ( Fig.…”

Section: Ultrastructural Analysis Of Defective Eggshellsmentioning

confidence: 99%

Characterization of essential eggshell proteins from Aedes aegypti mosquitoes

Oscherwitz

et al. 2020

Preprint

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Up to 40% of the world population now live in areas where dengue mosquito vectors coexist with humans. Aedes aegypti are vectors for zoonotic diseases that affect hundreds of millions of individuals per year globally. We recently identified the eggshell organizing factor 1 (EOF1) protein using systematic RNA interference (RNAi) screening of mosquito lineage-specific genes. It was shown that eggs deposited by RNAi-EOF1 A. aegypti and A. albopictus mosquitoes were non-melanized, fragile, and contained nonviable embryos. Motivated by this discovery, we performed RNAi screening of eggshell proteins to determine putative downstream target proteins of intracellular EOF1. We identified several eggshell proteins as essential for eggshell formation in A. aegypti and characterized their phenotypes in detail by molecular and biochemical approaches. We found that Nasrat, Closca, and Polehole structural proteins, together with the Nudel serine protease, are indispensable for eggshell melanization and egg viability. While all four proteins are predominantly expressed in ovaries of adult females, Nudel mRNA expression is highly upregulated in response to blood feeding. Furthermore, we identified four secreted eggshell enzymes as important factors for controlling the processes of mosquito eggshell formation and melanization. These enzymes included three dopachrome converting enzymes and one cysteine protease. All eight characterized eggshell proteins were required for intact eggshell formation. However, their surface topologies in response to RNAi did not phenocopy the effect of RNAi-EOF1. Still, it remains unclear how EOF1 influences eggshell formation and melanization. The use of proteomic analysis of eggshell proteins from RNAi-EOF1 assisted in the identification of additional proteins that could be regulated in EOF1 deficient eggshells.

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“…To overcome high costs and workload, and embryo lethality associated with injection, new methods of delivery directly into a gravid female are now being developed [31,32]. Since its introduction as a gene editing tool in 2012, CRISPR/Cas9 system has advanced research in many insect species, including flies [33][34][35][36][37][38], sandflies [39], mosquitoes [40][41][42][43], moths [13,[44][45][46][47], butterflies [48,49], crickets [50], locusts [51], planthoppers [52], honeybees [53,54], wasps [55], ants [11,12], beetles [56], aphids [57] and psyllid bugs [31].…”

Section: Directed Mutagenesis By Zfns Talens and Crispr/cas9mentioning

confidence: 99%

Progress in the use of genetic methods to study insect behavior outside Drosophila

Mansourian

Fandino

Riabinina

2019

Current Opinion in Insect Science

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CRISPR-Cas9 targeted disruption of theyellowortholog in the housefly identifies thebrown bodylocus

Abstract: The classic brown body (bwb) mutation in the housefly

Cited by 7 publications

References 28 publications

Allele-specific genome editing using CRISPR-Cas9 causes off-target mutations in diploid yeast

Allele-specific genome editing using CRISPR-Cas9 causes off-target mutations in diploid yeast

Characterization of essential eggshell proteins from Aedes aegypti mosquitoes

Progress in the use of genetic methods to study insect behavior outside Drosophila

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