Targeting Drive Associated Genes using CRISPR-Cas9David Akanonu & Dr. Josie Reinhardt

Teleopsis Dalmanni (also known as Stalk-eyed Flies) are

creatures that have these long stalks growing out of their

heads. Attached to these stalks are their eyes which is not

noticeable at first glance. My focus is on selfish gene these

flies carry known as a meiotic drive gene. According to

Mendel’s Law of segregation in species where

chromosomes determine sex, the chances of receiving a

male or a female is half and half. However, in this case

that half and half is now 10 and 90. Meiotic drive is a

caused by a selfish gene on the X-chromosome that also

causes specific tradeoffs in the sexes: males have reduced

fertility whereas in females have higher fertility. In

addition, the way these flies are attracted to each other are

due to their stalks. Female stalk eyed flies are attracted to

long stalks on males. Males with the drive associated X-

chromosome will have shorter stalks and therefore reduce

their chances of reproducing with other females. However,

almost nothing is known about the genetic causes of any of

these consequences of carrying a meiotic drive

chromosome. In my project, we plan on modifying a

standard stalk-eyed fly and hope to replicate meiotic drive.

In addition, we look at impacts on the other traits described

above. What we want to do with these stalk-eyed flies is

modify candidate genes using CRISPR-Cas9. We’ll use

CRISPR to induce a frameshift mutation, and that is an

insertion or a deletion of a nucleotide in a DNA sequence.

All things considered, modifying genomes to flies is just a

small portion of the project.

A a

A AA Aa

a aA aa

Dad (sperm): Aa

Mo

m (eggs)

:Aa

Methods

Figure 1. Effect of meiotic drive on gene inheritance

Introduction

Ligate gRNA insert Transform into Bacteria

Digest Plasmid

Plasmid DNA Prep

Design and order gRNAs as well as PCR primers

Find Genes that are associated with meiotic drive

Generate Hypothesis… What Gene Do I need to Target? What

Phenotype?

Results

Figure ?. Colony PCR Results

Unfortunately Colony PCR did not work

for the Chiffon Gene. However, the

ladders set in place showed bands.

The Yuri transformation was

unsuccessful however, the Chiffon

transformation was successful.

Moving forward, we want to re-do

our Yuri ligation and transformation,

sequence colonies from the chiffon

transformation. We need to

troubleshoot our PCR approach and

hopefully be ready to inject modified

plasmids into fly embryos!

Conclusion

A a*

A X Aa*

a X aa*

g1 c

hif_

1620_L

eft&

Rig

ht

“Mini-Prep” Plasmid DNA, then sequence the DNA

Strategy 1: Gel extraction, ligation, transformation

“Mini-Prep” Plasmid DNA, then sequence the DNA

Colony PCR

Strategy 2: No gel extraction, ligation, transformation

Inject plasmid and cas9 into fly embryos

Figure 2. Effect of meiotic drive on sex ratio

Figure 3. Effect of meiotic drive on fertility

Figure 4. Plasmid Map

Figure 8. Nano drop

concentration graph Pre-

Digest

Figure 9. Nano drop

concentrations Post

Digest.

Figure 5. gRNA Mock Design

References• Reinhardt, J. A., Brand, C. L., Paczolt, K. A., Johns, P. M., Baker, R. H., & Wilkinson, G. S. (n.d.). Meiotic Drive Impacts Expression and Evolution of

X-Linked Genes in Stalk-Eyed Flies

• David, P., Bjorksten, T., Fowler, K., Pomiankowski, A. (2000). Condition-dependent signalling of genetic variation in stalk-eyed flies. Nature, 406, 186-

188.

• Wilkinson, Gerald & S Presgraves, D & Crymes, Lili. (1998). Male eye span in stalk-eyed flies indicates genetic quality by meiotic drive suppresion.

Nature. 391. 276-279. 10.1038/34640.

• Port, F., Chen, H., Lee, T., & Bullock, S. L. (2014). Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

Proceedings of the National Academy of Sciences,111(29). doi:10.1073/pnas.1405500111

• Warren, I. A., Fowler, K., & Smith, H. (2010). Germline transformation of the stalk-eyed fly, Teleopsis dalmanni. BMC Molecular Biology,11(1), 86.

• Wilkinson, G. S., Johns, P. M., Kelleher, E. S., Muscedere, M. L., & Lorsong, A. (2006). Fitness effects of X chromosome drive in the stalk-eyed fly,

Cyrtodiopsis dalmanni. Journal of Evolutionary Biology, 19(6), 1851-1860.

Figure 12. PCR

Primer Gel

“g” means DNA with

master mix and “h”

means DNA w/o master

mix

Figure 7.

DAVID

Functional

Annotation

Tool

Results on

the Yuri

and

Chiffon

Gene

Figure 6. Selected Genes

From Gene Pool

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h2 c

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g3yu

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h3 y

uri_

52_L

eft&

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g4yu

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eft&

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h4 y

uri_

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g5 1

6S

1F

+ 1

6S

1 R

g5 1

6S

1F

+ 1

6S

1 R

Lad

der

Lad

der

Lad

der

Chiffon B colonies

m13 rev/spacer

Chiffon F colonies

m13 rev/for

Chiffon B

colonies

m13

rev/spacer

Chiffon

F

colonies

m13

rev/for

Chiffon B colonies

m13 rev/for

Chiffon F colonies

m13 rev/spacer

+

r

e

v

/

f

o

r

+

r

e

v

/

s

p

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Figure 11. Colony PCR on m13 rev/for

& m13 rev/ spacer primers for Chiffon

colonies

X

Figure 13. Digest of PCFD3 using BbS1

and Pst1

Dav

id P

st1

Dav

id B

bS

1

A B

Figure 10. Bacterial Transformations

Unsuccessful Yuri Gene Transformation (A) and

Successful Chiffon Gene Transformation (B)