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Marcé Lorenzen
NC State University
PacBio User Group Meeting
September 20, 2018
Why low-coverage PacBio?
Major Savings!
?
Genetic Pest Management
Using molecular genetics to render pest
species “less pesky”.
Genetically Engineered Autocidal Control
Population Suppression
Pathogen competent
Population Replacement
Population Replacement
Do not transmit disease
HOW?
Transposon-based Engineering
http://www.genecopoeia.com/product/donor-gene/
CRISPR/Cas9-based Engineering
Enzyme
“cuts” DNA
GFP-marked donor
“inserts” into chromosome
Presumptive Germ Cells
X
G1 beetles
Image from: Wimmer 2003 Nature Reviews
Green fluorescence = transgenic
Expressing Transgenes
DNA sequence of the Promoter directs expression of the
transgene
3xP3 “Universal” Eye-specific Promoter
Horn C, Jaunich B and Wimmer EA. Dev Genes Evol. 2000
Wild-type Tribolium eye color vs. mutant eye color
Affect of Eye Pigmentation
Tribolium Wild Type (Black eye pigment)
Tribolium Eye color mutant (no eye pigment)
VS.
7x copies of GFP!
3xP3-DsRed WCR
WT Transgenic – 3xP3-DsRed
• DsRed expression in adult eyes may be delayed by as much as five days.
WCR white eye strain
CRISPR/Cas9 knockout of WCR white eye-color gene
WT eye color/ DsRed White eye color/ DsRed
Model Organisms
Some of the Arthropod Species We Currently Work With
Alpha Tubulin Promoters
WT WT
WT WT
Heterologous promoters
Transgenic Tenebrio molitor
Have successfully used Tribolium
castaneum aTub promoter in a closely related
beetle, Tenebrio molitor
Heterologous promoters
Tribolium Actin Promoter
BlastN of an actin promoter vs. all the nucleotide sequences at NCBI
Promoter sequence only matches itself. There is little to no conservation of the DNA sequence of the actin promoter between species.
BlastP of an actin protein vs. all the protein sequences at NCBI
MANY matches! 100% conservation of the amino-acid sequence of actin proteins between species.
Transcriptome
Illumina MiSeq
The Lorenzen Method
Genome
1-2x PacBio
New Lorenzen Method???
Genome
1-2x PacBio
Transcriptome
PacBio IsoSeq?
How to Locate a Native Promoter
http://preuniversity.grkraj.org/html/10_MOLECULAR_BIOLOGY_files/image015.jpg
How to Locate a Native Promoter
http://preuniversity.grkraj.org/html/10_MOLECULAR_BIOLOGY_files/image015.jpg
How to Locate a Native Promoter
http://preuniversity.grkraj.org/html/10_MOLECULAR_BIOLOGY_files/image015.jpg
1 5’ UTR (full-length transcript)
How to Locate a Native Promoter
2 Genomic sequence to align transcript to
http://preuniversity.grkraj.org/html/10_MOLECULAR_BIOLOGY_files/image015.jpg
How to Locate a Native Promoter
http://preuniversity.grkraj.org/html/10_MOLECULAR_BIOLOGY_files/image015.jpg
5’ UTR aligned to PacBio read
Published Tribolium promoter
Lorenzen et al 2003
Matches PacBio read!
Can verify PacBio via comparison to published genome
PacBio read vs. Sanger genome
Not just for flies!
Enter sequence here
Results from promoter prediction
Lorenzen Lab: Dr. Nathaniel Grubbs Dr. Fu-Chyun “Clay” Chu Will Klobasa Terri O’Leary Pei-Shan Wu Sofia Pinzi Lauren Slayton Neelan Patel
Tenebrio project: Dr. Brenda Oppert (USDA-ARS) Dr. Aaron Dossey (All Things Bugs)
Diabrotica Genome Project: Dr. Hugh Robertson (University of Illinois) Dr. Brad Coates (USDA-ARS, Iowa) Dr. Tom Sappington (USDA-ARS, Iowa) Dr. Blair Siegfried (University of Florida)
Corn Rootworm Knowledge Research Program
Acknowledgments