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