Knock-out of Obesity-Related Genes in Zebrafish (Danio rerio)

Using the CRISPR-Cas9 System

Lucian F. Bloodworth III 1, Andrew Bonner 2, Dr. Bob Kesterson 3, Dr. Anil Challa 3
1. Independent Honors Research Program, Hampden-Sydney College, Hampden-Sydney VA, USA
2. Auburn University, Auburn AL, USA
3. Genetics Research Division and Transgenic & Genetically Engineered Models Core Facility,
Department of Genetics, University of Alabama at Birmingham

Methods:

Introduction:
CRISPRs, clusters of regularly interspaced short
palindromic repeats, are composed of two short
hybrid strands of RNA known as crRNA(~20bp)
and tracrRNA. This hybrid combines with the Cas9
endonuclease system and can be used to scan
DNA and cut at specific sites indicated by a specific
sequence known as the Protospacer Adjacent Motif
(PAM). When this cut takes place, the DNA can be
repaired through homologous end joining (HEJ) or
non-homologous end joining(NHEJ). NHEJ can result
in deletion mutations or insertions. The specificity of
the CRISPR-Cas9 system makes it an ideal system for
targeting specific gene locations such as the locations
of the genes targeted in this experiment.

BenchlingTM:
- Target Regions
- Guidlines
- Target Scores
CRISPRscan :
TM

Designing Gene Specific Oligonucleotide:

DrNMS-gen-F1
DrNMS-gen-R1
DrNMS-gen-F2
DrNMS-gen-R2
DrNMS-gen-F3
DrNMS-gen-R3

GTGTGTGTGTTGACATGCAG
ACTCATGATGATCAGCAGATATGTA
GATCAGATGTGTGTAGACTGACC
GGGAAGCTCAAGGTTTAATTGTATT
TGGATGGTCATCTGTGTGTTAG
GGGAAGCTCAAGGTTTAATTGTATT

Post-RNA Purification HMA & Spectrophotometer Analysis:

The genotyping map of Neuromedin-S
is 4468 base pairs in size. DrNMSgen-F1,R1 amplifies a 290 bp
amplicon including: DrNMS-C1.
DrNMS-gen-F2,R2 amplifies a 590 bp
amplicon including: DrNMS-C2, C3,
C4, C5. DrNMS-gen-F3,R3 amplifies
a 828 bp amplicon which included all
sgRNA/CRISPR targets.

Homology:
290 bp
509 bp
828 bp

Gradient PCR HMA

Optimizing Genotyping Primers:

Gene:
NMS
Parm1
Ghrl
Lepa
Lepb
Mc4r
Cnr1
Pmch
Pmchl
Scdb
Scd

C-1
203.9
358.8
373.7
392.8
185.9
250.5
222.1
237.6
280.5
123.6
255.9

Concentration CRISPR sgRNA [ng/uL]:

C-2
C-3
C-4
263.4
225.5
69.1
409.2
424.6
420.9
361.7
381
386.4
392.9
402.6
425
215.6
3
206.1
190.1
225.2
180
28.3
248.9
222.3
237.4
28.5
249.8
270.1
273.3
246.2
256.1
208
16.2
252.8
277.2
238.7

C-5
245.2

261.2
247.8

Indels from HMA analysis on 6%

polyacrylamide gels suggest the
successful synthesis of sgRNA
CRISPRs.The RNA concentrations
of each sample were recorded to
confirm these results. 48 CRISPR
sgRNAs were successfully
synthesized.

236.6

Plasmid DNA Analysis:

- Optimizing Annealing Temperature via Gradient PCR:

- Characterization of Gradient PCR through Heteroduplex
Mobility Assay (HMA):
- shows a distinct banding pattern that is specific to a
given mutation
- Heteroduplexes migrate slower in comparison to the
homoduplex species, and length of the indels in
addition to the sequence content of the
heteroduplexes determine the banding patterns.

CRISPR sgRNA Synthesis: Phase II:

Abstract:

Plasmid DNA EcoR1 Analysis:

Indels from HMA analysis post-EcoR1 digestion on 6% polyacrylamide gels suggest
successful gene knock-outs for: NMS, Mc4r, Scd, Scdb. HMA data may be made
available upon request.

Sanger Sequence DNA Analysis:

We conduct the in vivo knock-out of 11 obesity-related

genes in Danio rerio:
Name:

DrNeuromedin-S: ENSDARG00000101027

Gene Locations:

Designing CRISPR sgRNA Targets:

sgRNA Synthesis & Characterization:

Designing Genotyping Primers:

Gene IDs:

Ensembl & Bioinformatics:

Results:

Genotyping Preparation:Phase V:

CRISPR sgRNA Design: Phase I:

Select Genes of Interest:

Forming Good Men and Good Citizens in an Atmosphere of

Sound Learning

Annealing:

Genotyping F0 Modified Zebrafish Embryos: Phase VI:

DNA T4 Polymerase:
IVT:

Characterization of F0 gDNA PCR Product:

DNA Extraction:

Background:

Appetite suppressant effects

[1] NMS (Neuromedin S) >
[2] Parm1 (prostate androgen-regulated mucin-like protein 1) > Feeding regulation
Hunger Hormone
[3] Ghrl (ghrelin/obestatin prepropeptide) >
Satiety Hormones
[4] Lepa (leptin-a)
- regulate energy balance
>
- inhibit hunger
[5] Lepb (leptin-b)
- counteract Ghrelin
[6] Mc4r (melanocortin 4 receptor) > Recently linked to obesity in humans
[7] Cnr1 (Cannabinoid receptor 1)
>
Mesolimbic dopamine pathway
[8] Pmch (pro-melanin-concentrating hormone)
Produce MCH
>
[9] Pmch-l (pro-melanin-concentrating hormone, like)
- stimulate hunger
- feeding regulation
[10] Scd (stearoyl-CoA desaturase (delta-9-desaturase))
Obesity & insulin resistance
[11] Scd-b (stearoyl-CoA desaturase, b)
Affect metabolic pathways:

RNA Purification:

DNase I Treatment:

CRISPR-Cas9 Injection Solution Synthesis Phase III:

Analyzing Purified RNA:

500ng/uL Aliquots of specific RNA:
Combining Reagents:
Zebrafish Embryo Injection Phase IV:

- lipogenesis
- fatty acid oxidation

To this end, we design and

synthesize ~4 to 5 CRISPR/single guide RNAs
(sgRNA) per gene to target specific sequences. We
use a cloning-free approach to synthesize sgRNA,
and inject a Cas9 ribonucleoprotein complex into onecell stage zebrafish embryo. To test the efficiency of
nuclease activity of various CRISPR/sgRNAs, we
genotype embryos two days post-fertilization using a
PCR-based heteroduplex mobility assay (HMA). We
design pairs of genotyping primers to identify small
insertions-deletions (indels) as well as large
deletions. We collect data from HMA profiles postPCR for NMS, Parm1, Ghrl, Lepa, Mc4r, Pmch-l,
Scd, and Scd-b. Subsequently, we also obtained
sequence information of specific mutations by Sanger
sequencing cloned PCR products. F0 animals are
being raised in an effort to generate outcrossed F1
lines with successful transmission of mutant alleles.

HMA profile of DrNMS-gen-F1,R1 PCR. The PCR

was run at 65C with 0.5uL of modified gDNA from
8 different embryos, 2 days post injection. Embryo
A showed indels and was chosen for further
analysis. DrNMS-gen-F1,R1 has an amplicon size
of 290 bp, however, the HMA shows a different
amplicon size; we attribute this to the indel.

Egg Production & Collection:

Zebrafish Research
Facility (ZRF):

HMA profile of DrNMS-gen-F1,R1 Embryo A,

colonies 1-8 on plate 5. The colony PCR was run
at 65C. Colonies 5 and 6 were selected based on
indel presence.

Plating TOPO-TA PCR

Solution:
Colony PCR:

Inoculating Liquid
Bacterial Cultures:
Mini-Prep DNA Isolation:
EcoR1 Digestion:

Injecting One-Cell Stage Zebrafish Embryos:

Embryos Pre-Injection:

TOPO-TA Cloning PCR

Product:

Embryos Post-Injection:

HMA profile of an EcoR1 digestion of purified

plasmid DNA from:
[5] DrNMS-gen-F1,R1 Embryo A, colonies 9, 10,
11, 12, 13, 14, 15, and 16 from plate 5.

sgRNA ID:
DrParm1-C1
DrParm1-C2
DrParm1-C3
DrParm1-C4
DrGhrl-C1
DrGhrl-C2
DrGhrl-C3
DrGhrl-C4
DrLepa-C1
DrLepa-C2
DrLepal-C3
DrLepa-C4
DrLepb-C1
DrLepb-C2
DrLepb-C3
DrLepb-C4
DrMcr4r-C1
DrMc4r-C2
DrMc4r-C3
DrMc4r-C4
DrCnr1-C1
DrCnr1-C2
DrCnr1-C3
DrCnr1-C4
DrCnr1-C5
DrPmch-C1
DrPmch-C2
DrPmch-C3
DrPmch-C4
DrPmch-C5
DrPmchl-C1
DrPmchl-C2
DrPmchl-C3
DrPmchl-C4
DrScd-C1
DrScd-C2
DrScd-C3
DrScd-C4
DrScd-C5
DrScdb-C1
DrScdb-C2
DrScdb-C3
DrScdb-C4
DrNMS-C1
DrNMS-C2
DrNMS-C3
DrNMS-C4
DrNMS-C5

sgRNA Target sequence:

CCAGTAACCGTAACATCAGG
GGAACTAAGTATTGTGAGTG
AGTCACGTTGTGAGCTACGC
AGTAGTATGACTCTCTGAAG
GTAGCAAAGAATTAAGACTA
CGGCACCTCAGAGGCATCCT
TGTCTCGAGTCTGTGAGCGG
CAGTCCGACTCAGAAACCGC
GGATCCCCAATGATGAGCGT
CCGCTGACAAACCCATCCAA
GATGGAGCCGAGCCCTTGGA
CGAGATCTATCCACACTTCT
GACAGAACTGAGACCATCAA
AGAGACTCTCCTGAGGACAC
CCAGTTCCTCCAGTGTCCTC
GGAGGAACTGGCCGTCTCAC
AGCCAAGGAGCTCTGCCGGT
CCATGAGAGGGCTTTCCCAC
GAGCGTGAGGAAGATCTCCG
GATGTAGCGGTCCACCGCGA
ACCTCCGGCCTGCAGTACAT
GCCGATGTACTGCAGGCCGG
AGACGGCAGTCTGCAGTGCG
GATGGACACATATCGGTCGA
GAGCCTGGTCGTGCACTCGG
TGAGCTGACTACTCGCTCAA
ATATAAGTGAATTAGGACCT
AGAAGACACCCCATCATAGA
CGTAGAAGATGTCATTCCAG
TCGATGTCCCGCCTGCCCAC
GCACTCAGACAAAAGTGCAA
GCAAAGCTTTAGCGAGTCCT
TGGCTGACTCCAATCTCCTG
TGATGGCGAAATTCGGACTC
GAGTCTTCTGCACATTGCTG
GAAGGCCATGGAGTTTCCGA
GTGATGCACGCGGTGATCCC
GACCCTCACAACTCAAACCG
GCTGCTGGTTCGAAAACACC
CGATGTGACGACGACGACAG
CGATTTGTACCGGAGGACTC
GTCCTCCGGTACAAATCGTA
AGGGTTAAGGAAGAGACAAA
GTTCTCTGTGTGCTCTGCAG
ACAGAACTCACTGGGCTCGG
CTGGGCGACAGACGCATCAG
CGCCCAGACTCTCTCTGCGC
GTGTTAACACTTCATAGTAG

CC
GG
AG
AG
GT
CG
TG
CA
GG
CC
GA
CG
GA
AG
CC
GG
AG
CC
GA
GA
AC
GC
AG
GA
GA
TG
AT
AG
CG
TC
GC
GC
TG
TG
GA
GA
GT
GA
GC
CG
CG
GT
AG
GT
AC
CT
CG
GT

[sgRNA (ng/uL)]: Indels: Indel Sequence:

3588
Y
N/A
4092
Y
N/A
4246
Y
N/A
4209
Y
N/A
3737
Y
N/A
3617
Y
N/A
3810
Y
N/A
3864
Y
N/A
3928
N/A
N/A
3929
N/A
N/A
4026
N/A
N/A
4250
N/A
N/A
1859
N/A
N/A
2156
N/A
N/A
30
N/A
N/A
2061
N/A
N/A
2505
Y
Y
1901
Y
Y
2252
Y
Y
180
Y
Y
2291
N/A
N/A
283
N/A
N/A
2489
N/A
N/A
2223
N/A
N/A
2612
N/A
N/A
2376
N/A
N/A
2374
N/A
N/A
285
N/A
N/A
2498
N/A
N/A
2478
N/A
N/A
2805
N/A
N/A
2701
N/A
N/A
2733
N/A
N/A
2462
N/A
N/A
2559
NA
N
2528
NA
N
2772
Y
Y
2387
Y
Y
2366
Y
Y
1236
Y
Y
2561
Y
Maybe
208
Y
Maybe
162
Y
Y
2039
Y
Y
2634
N
N
2255
N
N
691
N
N
2452
N
N

Future Direction:
In order to create knock-out models of
the 11 genes of interest, we successfully
synthesized 48 total sgRNA CRISPRs
[Table 1]; confirmed via HMA analysis.
This data includes: off-target and on-target
scoring, sgRNA yield via concentration (ng/
uL) analysis, and various other processes
which provided relevant data. These data
sets will be studied in an effort to increase
the efficiency of sgRNA CRISPR binding
specificity to target locations. To make
knock-out models in Zebrafish (Danio rerio)
more efficient, we are developing a detailed
sgRNA Synthesis Workflow (Cloning Free
Method); available upon request. [Lucian@
UAB.edu].
Funding:

Sequencing Analysis:

We acknowledge the University of Alabama at

Birmingham, Department of Genetics, Transgenic
& Genetically Engineered Models Core Facility. We
acknowledge the University of Alabama at Birmingham,
Nutrition Obesity Research Center funded by the
NIH. We acknowledge Hampden-Sydney College,
Department of Biology. This work has benefited from
and been supported by funding through these facilities.
Availability of Data and Materials:

DrNMS-gen-F1,R1: A: [5]: Colony 15

The Sanger sequence of DrNMS-gen-F1,R1: [5] Embryo A: Colony 15. shows

a 34 bp insertion at C1; this suggests that C1 is an active CRISPR.

Each sgRNA target sequence was measured for

concentration in ng/uL for yield; the yields colored in red
represent low yields. The last column, column 6, identifies
each sequence as [Y] Active (in green), [Maybe] unable to
conclusively identify as active or inactive (in yellow), and
[N] Not Active (in red). A total of: 10 active sgRNA target
sequences, 2 possibly active sgRNA target sequences, and
4 not active target sequences were identified.

Conclusion:
We have evidence for active CRISPR/sgRNAs that
efficiently target Neuromedin S, Stearoyl-CoA desaturase
(delta-9-desaturase) - Scd, Stearoyl-CoA desaturase
(delta-9- desaturase) - Scd-b, and Mc4r to cause gene
knock-outs. This is validated by Sanger Sequence
Analysis. HMA profiles of Ghrelin and Parm1 also
suggest gene knock-out; further analysis via Sanger
Sequencing is required to confirm; a total of 10 active
sgRNA target sequences, 2 possibly active sgRNA Target
sequences, and 4 non-active sgRNA Target Sequences
were identified from the sanger sequence analysis.
Following the in vivo knock-out of the genes listed above,
F0 animals are being raised in an effort to generate
outcrossed F1 lines with successful transmission of
mutant alleles. We will continue to observe and study the
phenotypes and genotypes of future generation animals
in order to further determine the role of each gene in
obesity pathways.
Acknowledgements:
We thank the University of Alabama at Birmingham Department of
Genetics for providing us with permission to conduct our research
within their institution. We thank the University of Alabama School
of Medicine for supporting undergraduate student research with
clinical applications. We thank Dr. Bob Kesterson for providing us
with guidance and direction, and laboratory space to conduct our
research within the Transgenic & Genetically Engineered Models Core
Facility within the University of Alabama at Birmingham. We thank
the Kesterson Lab for sharing their protocols and guidance: Hayden
Bickerton, Leila Shaw, Judy Kesterson, Sami Foster, Sarah Glover,
and Rebecca Slowinski. We thank Ashley Turner and Shelly Nason
from the UAB Neurofibromatosis (NF) Program for their guidance and
support. We thank Dr. Anil Challa for providing us with our project and
for the knowledge, support, guidance, and protocols that made the
project possible. Without his involvement as a teacher, mentor, and
friend, our project would have been impossible. We thank Dr. Mike
Wolyniak and the Biology Department at Hampden-Sydney College for
the opportunity and funding to do our research.
References:
AS Rose, AR Bradley, Y Valasatava, JM Duarte, A Prli and PW Rose. Webbased molecular graphics for large complexes. ACM Proceedings of the 21st
International Conference on Web3D Technology (Web3D 16): 185-186, 2016.
doi:10.1145/2945292.2945324
AS Rose and PW Hildebrand. NGL Viewer: a web application for molecular visualization. Nucl Acids Res (1 July 2015) 43 (W1): W576-W579 first published online
April 29, 2015. doi:10.1093/nar/gkv402
Challa, Dr. Anil. Genetics and Microbiology. Kaul Human Genetics Building, Suite
230, 720 20th Street South Birmingham, Alabama 35294-0024. June-August.
2016. Lecture.

The complete protocols and data can be obtained

through request; each request will be considered and
taken into account. [Lucian@UAB.edu]. The additional
protocol, sgRNA Synthesis Workflow (Cloning Free
Method) is available upon request. [Lucian@UAB.edu].

Kesterson, Dr. Bob. Genetics and Microbiology. Kaul Human Genetics Building,
Suite 230, 720 20th Street South Birmingham, Alabama 35294-0024. JuneAugust. 2016. Lecture.

Author Details:

Schier Lab, Dept. of Molecular and Cellular Biology, Harvard University, Web. June
2016

Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of Zebrafish Embryos to

Analyze Gene Function. J. Vis. Exp. (25), e1115, doi:10.3791/1115 (2009).

Luke Bloodworth III : Biology and Business & Economics Undergraduate at Hampden-Sydney College, VA. Andrew Bonner
: Molecular Biology Undergraduate at Auburn University, AL. Bob Kesterson, Ph.D. Cell Biology, Transgenic & Genetically
Engineered Models Core Facility, Department of Genetics, University of Alabama at Birmingham School of Medicine, AL. Anil
Challa, Ph.D. Molecular Genetics, Department of Genetics, University of Alabama at Birmingham School of Medicine, AL.