Verifying polymorphisms associated with long and short sleep using polycistronic CRISPR coupled with extreme QTL mapping
Akanksha Singh
Terese R. Tansey
Carey J. Fagerstrom
Nasser M. Rusan
Susan T Harbison
10.6084/m9.figshare.12148713.v1
https://tagc2020.figshare.com/articles/poster/Verifying_polymorphisms_associated_with_long_and_short_sleep_using_polycistronic_CRISPR_coupled_with_extreme_QTL_mapping/12148713
Artificial selection
and genome-wide association mapping have isolated unprecedented numbers of
candidate polymorphisms putatively involved in sleep. One of the greatest
challenges in functional genomics is to understand how these polymorphic
variants affect sleep. CRISPR technology offers the possibility of replacing
alleles to directly observe their effects; however, the task can be daunting
for traits having large numbers of predicted polymorphic targets. Previously, we
identified 126 polymorphisms for long and short night sleep using artificial
selection. Here we apply a new approach to verify these polymorphisms. Using a polycistronic CRISPR gRNA design, we expressed multiple gRNAs per polycistronic construct to
create indels near target polymorphisms in a long-sleeping line of the Sleep
Inbred Panel, SIP_L1_9. We cloned four gRNAs into a pCFD5 plasmid and injected the
plasmid into SIP_L1_9. We allowed the resulting transformants to mate randomly for
two generations in order to recombine the transformed chromosomes. We then
measured sleep in the transformed populations. Night sleep ranged from 136.7
min. ± 25.2 to 706.3 min. ± 7.4 for progeny from one polycistronic construct and
from 266.9 min. ± 103.0 to 696.9 min. ± 9.7 for a second polycistronic
construct, exceeding the range of night sleep in the unperturbed SIP_L1_9
background. This suggests that efficient transformation occurred in both populations.
We collected the 10% shortest-sleeping and 10% longest-sleeping flies for each
construct and extracted their DNA. We are currently sequencing the genomic DNA from
each of the high/low 10% and will associate the sleep phenotypes with the
number and combination of genomic breaks. In principle, more breaks should be
present in the short sleeping flies than in the long sleepers as long sleepers
represent the unperturbed background of the injected strain. In this way, we can
quickly verify sleep-relevant polymorphisms with the greatest effects,
screening out any false positives.
2020-04-20 22:59:43
Polycistronic CRISPR
Extreme QTL mapping
Polymorphisms
Drosophila melanogaster sleep
Quantitative Genetics (incl. Disease and Trait Mapping Genetics)
Molecular Biology
Genomics