Publications by authors named "Guan-Heng Zhu"

10 Publications

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Expanding the Toolkit for Genome Editing in a Disease Vector, Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis.

CRISPR J 2021 Jan 15. Epub 2021 Jan 15.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA.

CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, , a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, (), and the juvenile hormone receptor, (). PiggyBac transformation vectors were prepared to express sgRNAs targeting and under the control of the U6 promoter. Transgenic expressing -sgRNA or -sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in -sgRNA and -sgRNA groups, respectively, showed mutations in their target genes.
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http://dx.doi.org/10.1089/crispr.2020.0052DOI Listing
January 2021

Genome editing in the fall armyworm, Spodoptera frugiperda: Multiple sgRNA/Cas9 method for identification of knockouts in one generation.

Insect Biochem Mol Biol 2020 07 7;122:103373. Epub 2020 Apr 7.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, USA. Electronic address:

The CRISPR/Cas9 system is an efficient genome editing method that can be used in functional genomics research. The fall armyworm, Spodoptera frugiperda, is a serious agricultural pest that has spread over most of the world. However, very little information is available on functional genomics for this insect. We performed CRISPR/Cas9-mediated site-specific mutagenesis of three target genes: two marker genes [Biogenesis of lysosome-related organelles complex 1 subunit 2 (BLOS2) and tryptophan 2, 3-dioxygenase (TO)], and a developmental gene, E93 (a key ecdysone-induced transcription factor that promotes adult development). The knockouts (KO) of BLOS2, TO and E93 induced translucent mosaic integument, olive eye color, and larval-pupal intermediate phenotypes, respectively. Sequencing RNA isolated from wild-type and E93 KO insects showed that E93 promotes adult development by influencing the expression of the genes coding for transcription factor, Krüppel homolog 1, the pupal specifier, Broad-Complex, serine proteases, and heat shock proteins. Often, gene-edited insects display mosaicism in which only a fraction of the cells are edited as intended, and establishing a homozygous line is both costly and time-consuming. To overcome these limitations, a method to completely KO the target gene in S. frugiperda by injecting the Cas9 protein and multiple sgRNAs targeting one exon of the E93 gene into embryos was developed. Ten percent of the G0 larvae exhibited larval-pupal intermediates. The mutations were confirmed by T7E1 assay, and the mutation frequency was determined as >80%. Complete KO of the E93 gene was achieved in one generation using the multiple sgRNA method, demonstrating a powerful approach to improve genome editing in lepidopteran and other non-model insects.
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http://dx.doi.org/10.1016/j.ibmb.2020.103373DOI Listing
July 2020

Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, .

Proc Natl Acad Sci U S A 2019 10 30;116(43):21501-21507. Epub 2019 Sep 30.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY 40546;

The yellow fever mosquito, , vectors human pathogens. Juvenile hormones (JH) control almost every aspect of an insect's life, and JH analogs are currently used to control mosquito larvae. Since RNA interference does not work efficiently during the larval stages of this insect, JH regulation of larval development and mode of action of JH analogs are not well studied. To overcome this limitation, we used a multiple single guide RNA-based CRISPR/Cas9 genome-editing method to knockout the () gene coding for a JH receptor. The knockout larvae exhibited a black larval phenotype during the L3 (third instar larvae) and L4 (fourth instar larvae) stages and died before pupation. However, knockout did not affect embryonic development or the L1 and L2 stages. Microscopy studies revealed the precocious synthesis of a dark pupal cuticle during the L3 and L4 stages. Gene expression analysis showed that , a key transcription factor in JH action, was down-regulated, but genes coding for proteins involved in melanization, pupal and adult cuticle synthesis, and blood meal digestion in adults were up-regulated in L4 mutants. These data suggest that, during the L3 and L4 stages, mediates JH suppression of pupal/adult genes involved in the synthesis and melanization of the cuticle and blood meal digestion. These results help to advance our knowledge of JH regulation of larval development and the mode of action of JH analogs in .
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http://dx.doi.org/10.1073/pnas.1905729116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815201PMC
October 2019

CRISPR/Cas9 mediated gene knockout reveals a more important role of PBP1 than PBP2 in the perception of female sex pheromone components in Spodoptera litura.

Insect Biochem Mol Biol 2019 12 24;115:103244. Epub 2019 Sep 24.

Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China. Electronic address:

Three different pheromone binding proteins (PBPs) can typically be found in the sensilla lymph of noctuid moth antennae, but their relative contributions in perception of the sex pheromone is rarely verified in vivo. Previously, we demonstrated that SlitPBP3 plays a minor role in the sex pheromone detection in Spodoptera litura using the CRISPR/Cas9 system. In the present study, the roles of two other SlitPBPs (SlitPBP1 and SlitPBP2) are further verified using the same system. First, by co-injection of Cas9 mRNA/sgRNA into newly laid eggs, a high rate of target mutagenesis was induced, 51.5% for SlitPBP1 and 46.8% for SlitPBP2 as determined by restriction enzyme assay. Then, the homozygous SlitPBP1 and SlitPBP2 knockout lines were obtained by cross-breeding. Finally, using homozygous knockout male moths, we performed electrophysiological (EAG recording) and behavioral analyses. Results showed that knockout of either SlitPBP1 or SlitPBP2 in males decreased EAG response to each of the 3 sex pheromone components (Z9,E11-14:Ac, Z9,E12-14:Ac and Z9-14:Ac) by 53%, 60% and 63% (for SlitPBP1 knockout) and 40%, 43% and 46% (for SlitPBP2 knockout), respectively. These decreases in EAG responses were similar among 3 pheromone components, but were more pronounced in SlitPBP1 knockout males than in SlitPBP2 knockout males. Consistently, behavioral assays with the major component (Z9,E11-14:Ac) showed that SlitPBP1 knockout males responded in much lower percentages than SlitPBP2 knockout males in terms of orientation to the pheromone, along with reduction in close range behaviors such as hairpencil display and mating attempt. Taken together, this study provides direct functional evidence for the roles of SlitPBP1 and SlitPBP2, as well as their relative importance (SlitPBP1 > SlitPBP2) in the sex pheromone perception. This information is valuable in understanding mechanisms of sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.
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http://dx.doi.org/10.1016/j.ibmb.2019.103244DOI Listing
December 2019

A Δ9 desaturase (SlitDes11) is associated with the biosynthesis of ester sex pheromone components in Spodoptera litura.

Pestic Biochem Physiol 2019 May 27;156:152-159. Epub 2019 Feb 27.

Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, College of Plant Protection, Nanjing Agricultural University, Nanjing, China. Electronic address:

Sex pheromone biosynthesis in moths relies on the activity of multiple enzymes, including Δ9 desaturase, which plays an important role in catalyzing desaturation at the Δ9 position of the carbon chain. However, the physiological function of moth Δ9 desaturase has not been elucidated in vivo. In this study, we used the CRISPR/Cas9 system to knockout the Δ9 desaturase gene (SlitDes11) of Spodoptera litura to analyze its role in sex pheromone biosynthesis. First, through the direct injection of SlitDes11-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, gene editing was induced in around 30% of eggs 24 h after injection and was induced in 20.8% of the resulting adult moths. Second, using a sibling-crossing strategy, insects with mutant SlitDes11 (bearing a premature stop codon) were selected, and homozygous mutants were obtained in the G5 generation. Third, pheromone gland extracts of adult female homozygous SlitDes11 mutants were analyzed using Gas chromatography (GC). The results showed that titers of all three ester sex pheromone components; Z9, E11-14:Ac, Z9,E12-14:Ac, and Z9-14:Ac; were reduced by 62.40%, 78.50%, and 72.50%, respectively. This study provides the first direct evidence for the role of SlitDes11 in sex pheromone biosynthesis in S. litura, and indicates the gene could be as potential target to disrupt sexual communication in S. litura for developing a new pollution-free insecticide.
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http://dx.doi.org/10.1016/j.pestbp.2019.02.018DOI Listing
May 2019

Expression Profile and Functional Characterization Suggesting the Involvement of Three Chemosensory Proteins in Perception of Host Plant Volatiles in Chilo suppressalis (Lepidoptera: Pyralidae).

J Insect Sci 2018 Sep 1;18(5). Epub 2018 Sep 1.

Department of Entomology, College of Plant Protection, Nanjing Agricultural University/ Key Laboratory of Integrated Management of Crop Diseases and Pests (Nanjing Agricultural University), Ministry of Education, Nanjing, China.

The high sensitivity of the olfactory system is essential for feeding and oviposition in moth insects, and some chemosensory proteins (CSPs) are thought to play roles in this system by binding and carrying hydrophobic odorants across the aqueous sensillar lymph. In this study, to identify the olfactory CSPs from a repertoire of 21 CSP members in the notorious rice pest Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), tissue expression patterns were firstly examined by quantitative real-time polymerase chain reaction (qPCR). It showed that CSP2 was antennae specific and seven more CSPs (CSP1, 3, 4, 6, 15, 16, and 17) were antennae biased in expression, suggesting their olfactory roles; while other CSPs were multiple-tissue expressed and non-antennae biased, suggesting other functions for these genes. To further determine the ligand binding specificity, three putative olfactory genes (CSP1-3) were expressed in Escherichia coli cells, and binding affinity of these three recombinant CSP proteins were measured for 35 plant volatiles by the ligand binding assays. CSP1 and CSP2 exhibited high binding affinities (Ki ≤ 10.00 µM) for four (2-tridecanone, benzaldehyde, laurinaldehyde and 2-pentadecanone) and two (2-heptanol and (+)-cedrol) host plant volatiles, respectively; the three CSPs also showed moderate binding affinity (Ki = 10.01-20.00 µM) for 16 plant volatiles. Our study suggests that the three CSPs play essential roles in the perception of host plant volatiles, providing bases for the elucidation of olfactory mechanisms in this important pyralid pest.
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http://dx.doi.org/10.1093/jisesa/iey088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159316PMC
September 2018

CRISPR/Cas9-mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis.

Insect Sci 2019 Jun 7;26(3):388-399. Epub 2017 Dec 7.

College of Plant Protection, Nanjing Agricultural University/Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China.

Pheromone-binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo. In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target-gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1-sgRNA injected eggs and 19.5% for PBP3-sgRNA injected eggs. Second, by an in-crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA-injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.
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http://dx.doi.org/10.1111/1744-7917.12544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7379591PMC
June 2019

CRISPR/Cas9 mediated BLOS2 knockout resulting in disappearance of yellow strips and white spots on the larval integument in Spodoptera litura.

J Insect Physiol 2017 11 18;103:29-35. Epub 2017 Sep 18.

Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address:

The custom-design bacterial CRISPR/Cas9 system has been recently used in some insects, indicating a powerful technique for studies on gene function and transgenic insects. However, its use in lepidopteran pests is scarce. Here, we reported a CRISPR/Cas9 system mediated mutagenesis of biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene in a noctuid pest Spodoptera litura. A fragment of SlitBLOS2 was identified by analyzing a S. litura transcriptome database by local basic BLAST, and the full length cDNA was acquired by RACE strategy. To clarify the function of SlitBLOS2, CRISPR/Cas9 based target mutagenesis of SlitBLOS2 was achieved, displaying a mosaic translucent integument in 62.3-70.6% larvae of G0 generation. Further PCR-based genotype analysis demonstrated various mutations occurred at the SlitBLOS2 specific target site. A homozygote mutant individual was obtained in G1 generation, in which the yellow strips and white spots on the larval integument completely disappeared. Our study clearly demonstrates the function of SlitBLOS2 in the integument coloration, and thus provides a useful marker gene for genome editing based gene functional study and pest control strategy in S. litura as well as other lepidopteran pests.
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http://dx.doi.org/10.1016/j.jinsphys.2017.09.008DOI Listing
November 2017

Functional characterization of PBP1 gene in Helicoverpa armigera (Lepidoptera: Noctuidae) by using the CRISPR/Cas9 system.

Sci Rep 2017 08 16;7(1):8470. Epub 2017 Aug 16.

Key Laboratory of Integrated Pest Management in Crops in Eastern China (Ministry of Agriculture of China), College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.

Pheromone binding proteins (PBPs) are thought to play crucial roles in perception of the sex pheromones particularly in noctuid moths, but this is rarely in vivo evidenced due to lacking an effective technique. Here, we reported an in vivo functional study of PBP1 in the important lepidopteran pest Helicoverpa armigera (HarmPBP1), by using the CRISPR/Cas9 system. Efficient and heritable mutagenesis was achieved by egg injection of mixture of Cas9-mRNA and HarmPBP1-sgRNA. The TA cloning and sequencing revealed various insertion and/or deletion (indel) mutations at the target site. Among those, one mutation resulted in a premature stop codon at the target site, which led to a highly truncated protein with only 10 amino acids. The HarmPBP1 with this mutation would completely loss its function, and thus was used to select the homozygous mutant insects for functional analysis. The electroantennogram recording showed that the mutant male adults displayed severely impaired responses to all three sex pheromone components (Z11-16:Ald, Z9-16:Ald and Z9-14:Ald). Our study provides the first in vivo evidence that HarmPBP1 plays important role in perception of female sex pheromones, and also an effective methodology for using CRISPR/Cas9 system in functional genetic study in H. armigera as well as other insects.
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http://dx.doi.org/10.1038/s41598-017-08769-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559583PMC
August 2017

Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing.

Insect Biochem Mol Biol 2016 08 15;75:1-9. Epub 2016 May 15.

Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address:

Functional gene analysis by using genome editing techniques is limited only in few model insects. Here, we reported an efficient and heritable gene mutagenesis analysis in an important lepidopteran pest, Spodoptera litura, using the CRISPR/Cas9 system. By using this system, we successfully obtained the homozygous S. litura strain by targeting the pheromone binding protein 3 gene (SlitPBP3), which allowed us to elucidate the role of this gene in the olfaction of the female sex pheromones. By co-injection of Cas9 mRNA and sgRNA into S. litura eggs, highly efficient chimera mutation in SlitPBP3 loci was detected both in injected eggs (39.1%) and in the resulting individual moths (87.5%). We used the mutant moths as parents to obtain the G1 offspring and the homozygous mutant strain in G2. The function of SlitPBP3 was explored by Electroantennogram (EAG) recordings with a homozygous mutant strain. The result showed that the EAG responses were significantly decreased in mutant males than in control males when treated with the major sex pheromone component (Z9,E11-14:Ac) and a minor component (Z9-14:Ac) at higher dosages. The results demonstrate that s SlitPBP3 gene plays a minor role in the perception of the female sex pheromones. Furthermore, our study provides a useful methodology with the CRISPR/Cas9 system for gene in vivo functional study, particular for lepidopteran species in which the RNAi approach is not efficient.
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http://dx.doi.org/10.1016/j.ibmb.2016.05.006DOI Listing
August 2016