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Characterization of zebrafish phf6 CRISPR mutant phenotypes

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Characterization of zebrafish phf6 CRISPR mutant phenotypes

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Characterization of Zebrafish phf6 CRISPR Mutant Phenotypes
by
Zhiyu Tian

A Thesis Presented to the
FACULTY OF THE USC Keck School of Medicine
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)

August 2022

Copyright 2022 Zhiyu Tian
ii
Acknowledgements
I'd like to thank my mentor, Dr. Ching-Ling Lien, in particular for her consistent support and
guidance during the research and writing of this thesis. Thank you for allowing me to be a part of
this incredible lab. I'd also want to thank the two members of my thesis committee, Dr. Hooman
Allayee and Dr. Ram Kumar Subramanyan, for their help and advise.
Also, I'd like to thank Yuhan Sun, Xidi Feng, Siqi Tao, David Wong, Subir Kapuria, Stanislao
Travisano, and Raquel Rodriguez for their support and assistance at Dr.Lien's lab. They helped
me a lot in finishing this project within the limited time. I am really thankful to them.
Thank you everybody for your assistance and support!

iii
Table of Contents
ACKNOWLEDGEMENTS ......................................................................................................... ii
LIST OF FIGURES ...................................................................................................................... v
ABSTRACT .................................................................................................................................. vi
CHAPTER ONE: INTRODUCTION ......................................................................................... 1
BORJESON-FORSSMAN-LEHMANN SYNDROME (BFLS) ................................................................ 1
PHF6 ........................................................................................................................................... 2
ZEBRAFISH ................................................................................................................................... 3
CRISPR ....................................................................................................................................... 4
PREVIOUS STUDIES ...................................................................................................................... 5
GENETIC COMPENSATION ............................................................................................................ 8
MATERNAL ZYGOTIC MUTANTS ................................................................................................... 9
CHAPTER TWO: MATERIALS AND METHODS ............................................................... 10
RT-QPCR .................................................................................................................................. 10
GRNA TEMPLATE ....................................................................................................................... 10
MICROINJECTION ....................................................................................................................... 10
ALCIAN BLUE STAINING ............................................................................................................ 11
GENOTYPING ............................................................................................................................. 12
KITS ........................................................................................................................................... 13
ZEBRAFISH LINES ....................................................................................................................... 13
ZEBRAFISH MAINTENANCE ........................................................................................................ 14
CHAPTER THREE: RESULTS ................................................................................................ 15
POTENTIAL MATERNAL CONTRIBUTION ...................................................................................... 15
GENOTYPING ............................................................................................................................. 16
POTENTIAL PHENOTYPE ............................................................................................................. 19
GENERATE PROMOTER-LESS CRISPR MUTANT ......................................................................... 22
CHAPTER FOUR: DISCUSSION ............................................................................................ 26
PHENOTYPIC CHARACTERIZATION OF PHF6 MUTANTS ................................................................ 26
GENERATION OF NEW PHF6 MUTANTS ........................................................................................ 26
iv
NEURAL CREST CELLS ................................................................................................................ 27
CHAPTER FIVE: FUTURE STUDY ....................................................................................... 28
REFERENCES ............................................................................................................................ 29

v
List of Figures
FIGURE 1. PHF6 PROTEIN STRUCTURE. ............................................................................................ 2
FIGURE 2. CRISPR-CAS9. SCHEMATIC SHOWING THE INV ASION OF A SGRNA INTO A DNA DOUBLE
HELIX IN CONCERT WITH CAS9. ................................................................................................ 5
FIGURE 3. SEQUENCES OF TWO PHF6 MUTANT ALLELES. ................................................................... 6
FIGURE 4. GENOTYPING RESULTS OF TWO PHF6 MUTATIONS AT 7DPF AND 90DPF (N>100). .............. 7
FIGURE 5. EXPRESSION LEVELS OF PHF6 IN WILD TYPE CONTROL, HETEROZYGOUS AND
HOMOZYGOUS PHF6 MUTANTS AT 1DPF. ................................................................................... 8
FIGURE 6. MATERNAL ZYGOTIC BREEDING SCHEME ......................................................................... 9
FIGURE 7. MICROINJECTION. .......................................................................................................... 11
FIGURE 8. EXPRESSION LEVELS OF PHF6 IN WT AT DIFFERENT TIMEPOINTS. .................................. 16
FIGURE 9. PHF6 C43Y GENOTYPING PCR RESULT. ........................................................................ 17
FIGURE 10. PHF6 8BP GENOTYPING PCR RESULT. .......................................................................... 18
FIGURE 11. ALCIAN BLUE STAINING RESULT AT 5DPF. ..................................................................... 20
FIGURE 12. QUANTIFICATION AND ANALYZE OF POTENTIAL PHENOTYPES. ..................................... 21
FIGURE 13. DESIGNED DNA TEMPLATES OF GRNAS. .................................................................... 22
FIGURE 14. SCHEME OF T7E1 ASSAY TO ASSESS CRISPR SGRNA EFFICIENCY. ............................ 23
FIGURE 15. SGRNAS EFFICIENCY DETERMINED BY T7E1. ............................................................. 24
FIGURE 16. PRIMERS AND METHODS USED TO TEST THE DELETION ................................................. 24
FIGURE 17. PCR RESULTS AFTER 3GRNAS INJECTION. .................................................................. 25

vi
Abstract
Borjeson-Forssman-Lehmann syndrome (BFLS) is caused by disruptions or mutations of the
PHF6 gene and characterized by intellectual disability, developmental delay, epilepsy and
obesity. PHF6 (PHD Finger Protein 6) is a member of the plant homeodomain (PHD)-like finger
(PHF) protein family. CRISPR associated protein 9 (Cas9) is an efficient, convenient,
economical and rapid gene editing technology emerging in recent years. With the gene editing
technology, mutant zebrafish may be generated to study loss-of-function phenotypes.
CRISPR/Cas9 technology was used to create zebrafish mutations that resemble the patient alleles.
However, the phf6 mutants previously generated do not show obvious phenotypes and are adult
viable. To determine if the lack of phenotypes is due to maternal transcript contribution, I
analyzed phf6 maternal zygotic mutant phenotypes. I am also in the process of generating a new
phf6 CRISPR mutant by completely removing the promoter and coding region to avoid genetic
compensation triggered by nonsense mediated decay of mutant phf6 mRNAs.

1
Chapter One: Introduction
Borjeson-Forssman-Lehmann syndrome (BFLS)
Borjeson-Forssman-Lehmann syndrome (BFLS) is an extremely rare disease caused by
disruptions or mutations of the PHF6 gene (Cheng et al., 2018, Jahani-Asl et al., 2016). BFLS is
characterized by cognitive disability, epilepsy, narrow forehead, hypogonadism, hypometabolism,
obesity, swelling of subcutaneous tissue of the face, thick calvarium, protruding chin, broad jaw,
narrow palpebral fissures, and large ears. These symptoms are variable, even among members of
the same family (Gécz et al. 2006). The disorder is fully expressed predominantly in males
because this mutation is usually transmitted as an X-linked recessive trait (Borjeson et al., 1962).
Male patients are hemizygous. Heterozygote females may have milder phenotypes such as
hypothyroidism, and many carrier females seem unaffected. Most females with known PHF6
mutations have skewed X-inactivation in their blood leucocytes (Crawford, 2005). So far, the
majority of discovered mutations occurring in exon 2 and one recurring mutation at p. C45Y
(Baumstark, 2003). This recurring mutation is at p. C45Y in human, and its counterpart in
zebrafish is at p. C43Y. BFLS is extremely rare and there is no cure for BFLS, but its symptoms
can be managed with surgery and medication. We hypothesize that zebrafish CRISPR mutants
can mimic BFLS disease phenotypes. As a result, we may research BFLS disease using zebrafish
models to better understand its mechanisms and identify potential therapeutic approaches for it.
2
PHF6
PHF6 (PHD Finger Protein 6) is a member of the plant homeodomain (PHD)-like finger (PHF)
family. The PHF6 gene encodes a 365-amino acid protein, which contains four nuclear
localization signals and two imperfect PHD zinc finger domains with a proposed role in gene
transcription regulation. PHF6 is a tumor suppressor gene as well. PHF6 levels also have impact
on the development of human hematopoietic progenitor cells into distinct blood cell lineages,
with lymphoid and erythroid differentiation having the greatest impact (Loontiens et al. 2020).
PHF6 is found on the X chromosome in humans. While in zebrafish, phf6 is located on
chromosome 14. It is not on sex chromosomes in zebrafish because zebrafish do not have
distinguishable heteromorphic sex chromosomes, and their sex is determined by multiple genes
and some environmental effect (Nagabhushana and Mishra. 2016).

Figure 1. PHF6 protein structure.
NLS (Nuclear localization signals), PHD (Plant Homeodomain) PHD domains contain two zinc
fingers and Extended PHD domains contain three zinc fingers.
3
Zebrafish
Zebrafish (Danio Rerio) is a member of the Cypriniformes order's minnow family. These tropical
freshwater fish are commonly utilized in developmental biology research and disease models.
Zebrafish grow quickly, needing just around 72 hours to mature and hatch into free-swimming
larvae with fully developed organs and tissues. Because they are vertebrates, they have numerous
parallels with humans; in fact, the zebrafish and human genomes are 70 percent identical and 84
percent of human genes known to be involved with human diseases have an equivalent in
zebrafish (Howe et al. 2013). As a result, the zebrafish has become a well-known animal model
in the field of biology research.
Zebrafish have several advantages for developmental research. Zebrafish can generate ten times
more offspring from each parental cross than mice and may be mated once a week. In addition,
the growing embryos are translucent, making them easier to investigate. Larval zebrafish offer
several benefits as a model for studying face cartilage. The most noticeable is that the
development of face tissue in living embryos may be seen constantly throughout time. Because
fish and humans are both vertebrates, their cartilage structures are comparable.

4
CRISPR
CRISPR (clustered regularly interspaced short palindromic repeats) - associated protein 9 (Cas9)
is an efficient, convenient, economical and rapid gene editing technology emerging in recent
years (Liu et al., 2019). The CRISPR/Cas9 system uses an RNA-protein complex consisting of
two essential components: a Cas9 effector protein and a single guide RNA (sgRNA) containing a
targeting sequence which around twenty nucleotides. After introducing the RNA-protein
complex into a cell, the sgRNA identifies a complementary target DNA site with a canonical
NGG and non-canonical NGA or NAG protospacer adjacent motif (PAM) sequence and directs
the Cas9 endonuclease to induce DNA double-stranded breaks (DSBs). Cells can only ensure
normal operation by restoring DSBs by either non-homologous end joining (NHEJ) or
homology-directed repair (HDR). With the aid of this gene editing technology, mutated zebrafish
may be constructed (Wu et al. 2014, Liu et al., 2019).
5

Figure 2. CRISPR-Cas9. Schematic showing the invasion of a sgRNA into a DNA double
helix in concert with Cas9.
Cas9 protein cuts the DNA at the PAM sequence causing a double-strand break. This can be
repaired either by the NHEJ (Non-Homologous End Joining) pathway or, in conjunction with a
template oligo, by HDR (Homology-Directed Repair).
Previous Studies
In previous studies, our lab member, Dr. Yuhan Sun, constructed two mutants (Figure 3). One is
on ePHD1 (extended plant homeodomain1) and another is right in front of ePHD2. On ePHD1,
she generated a C43Y mutant, because cysteine is an important amino acid on extended PHD
finger. Many cancer patients also have this mutation (Meacham et al. 2015). Another allele is an
8bp deletion before ePHD2. It causes frame shift and lead to dominant negative function (Liu et
al. 2014).
6

Figure 3. Sequences of two phf6 mutant alleles.
A. Phf6 C43Y deletion on ePHD1. B. Phf6 8 bp deletion before ePHD2. (Data provided by Dr.
Yuhan Sun)

However, the phf6 mutants Dr. Sun generated do not show obvious phenotypes and are adult
viable. Dr. Sun and I did genotyping on day 7 and day 90 after fertilization (Figure 4) and the
results show that the genotype of the offspring’s population is 25% wild type, 50% heterozygous,
and 25% homozygous (a ratio of 1:2:1) which follows the Mendelian ratios and do not have
developmental phenotype.
7

Figure 4. Genotyping results of two phf6 mutations at 7dpf and 90dpf.
Statistic performed by Chi-square test, and ns (not significant) indicates P > 0.05 (Data in this
figure are from experiments done by Dr.Yuhan Sun and me in collaboration).

We also determined the expression levels of phf6 in wild type control embryos and phf6 mutants
by RT-qPCR (Figure 5). The expression level of phf6 RNA in 8bp homozygous, heterozygous
and C43Y homozygous are all reduced. However there are still some Phf6 expressions, so we
possibly did not completely deleted Phf6. Therefore I tried to generate phf6 CRISPR mutants by
completely removing the promoter and coding region to avoid genetic compensation triggered by
nonsense mediated decay of mutant phf6 mRNAs. At the same time, there might be some
potential phenotypes we did not discovered. So I also characterize those mutants while I am
generating the promoter-less mutants.
8

Figure 5. Expression levels of phf6 in wild type control, heterozygous and homozygous Phf6
mutants at 1dpf.
Two different primer sets targeting different regions of phf6 were used to perform RT-qPCR.

Genetic Compensation
Genetic compensation occurs when an organism with a disease-causing mutation does not
develop the expected undesirable phenotype due to the compensatory action of another or
functionally compensating gene for the loss-of-function genotype, leading to restoring normal
physiological function. (Buglo et al. 2020, Sztal et al. 2018) This phenomenon was discovered
due to the weak phenotypes of some zebrafish CRISPR mutants compared to morphants. In
addition to the off-target effects of morpholinos, genetic compensation triggered by nonsense
mediated decay of mutant mRNA might account for the weaker phenotypes for some zebrafish
CRISPR mutants.
9
Maternal Zygotic Mutants
During oogenesis, maternal genes are transcribed and stored as maternal RNA. These maternal
mRNAs are translated after oocyte maturation or fertilization. After fertilization, zygotic genes
are transcribed (Tora and Vincent, 2021).
A considerable number of RNAs and proteins are deposited in the yolk of zebrafish in maternal
form, termed maternal gene-encoded maternal factors, which are often required for early
embryonic development (Dosch et al., 2004; Wagner et al., 2004). In order to explore the
function of maternal factors in zebrafish, it is frequently essential to create maternal-zygotic (MZ)
mutants (Reim and Brand, 2006, Veil et al., 2017). A maternal zygotic mutant is generated when
a homozygous female is used in the cross with a heterozygous male to produce a homozygote as
shown in Figure 6.

Figure 6. Maternal zygotic breeding scheme
10
Chapter Two: Materials and Methods
RT-qPCR
Real-time reverse transcription quantitative PCR (RT-qPCR) is a sensitive and repeatable
method for analyzing gene expression patterns. Total RNA was extracted from embryos using
TRIzol (Invitrogen) according to the manufacturer's procedure. RT-qPCR was carried out using a
one-step SYBR Green RT-qPCR Master Mix (Qiagen, Valencia, CA). Triplicate samples from
each genotype were evaluated on the target genes.
gRNA template
To design gRNA targeting the phf6 gene, I used SnapGene software to create and annotate DNA
sequence files. Then using crispor.tefor.net website to design gRNAs. Depending on their
specificity score, guides are colored green, yellow, or red. Green means high-scoring and
recommended, and red guides are low-scorings that you should avoid. To generate Cas9 mRNA
and gRNAs, using kits to do in vitro transcription.
Microinjection
At the one-cell stage, embryos were injected using microinjection needles as shown in Figure 7. I
injected about 1.5 nl of injection mixture per embryo in this experiment. To signify successful
injection into the cell, the mixture I used contained phenol red as a dye. Cas9 protein
11
concentration was around 150 ng/ul. Since higher gRNA concentrations do not appear to produce
any further malformations or death. I injected 300 ng/ul in my experiment.

Figure 7. Microinjection.
Using the microinjection needle to inject the reagent into embryo at the one-cell stage.
Alcian Blue Staining
Zebrafish were sacrificed 5 days after fertilization (dpf) and fixed for one hour in 2%
paraformaldehyde (PFA). The zebrafish were then rinsed with 100 mM Tris (pH 7.5), 10 mM
MgCl2 before being placed in an alcian blue solution (100 mM Tris (pH 7.5), 10 mM MgCl2, 80%
ethanol, 0.04 percent alcian blue from Anatech LTD.) and incubated overnight in the dark at
room temperature. The larval zebrafish were then bleached with 30% hydrogen peroxide for ~30
minutes. After that, the specimens were washed with 25% glycerol/0.1% KOH. Following these
methods, larval zebrafish were stored in 75% glycerol for later use.

12
Genotyping
Clipped tails or collected embryos are immersed in 20 ul 50mM NaOH for 30 minutes at 95
o
C
to lysis the tissue. After lysis, add 2 ul Tris-HCl (pH 7.5-8.0) to neutralize the pH. The mutations
were then identified using PCR. We use GoTaq® DNA Polymerase (Promega) to replicate DNA.
The primer sequences I used are:
phf6 (C43Y) WT: Forward: 5’-GCAGTTCTGAAGGCAAAAGGG-3’
Reverse: 5’-TTGTACAGCTAACATACCATGCAC-3’
Mutant: Forward: 5’-CCTCCATGTGCACCTCAGTT-3’
Reverse: 5’-TTGTACAGCTAACATACCATGCTG-3’
phf6 (8bp deletion): Forward: 5’-GGTCAAGTCATTCACATATG-3’
Reverse: 5’-TTGTAAATTCCTCTCGCCAT-3’

13
Kits
1. MinElute®Gel Extraction Kit (by QIAGEN) was used for gel extracting.
2. MAXIscript® T7 Kit and MEGAscript® T7 Kit (by Invitrogen) was used for in vitro
transcription of gRNAs.
3. SuperScript® III First-Strand Synthesis System for RT-PCR (by Invitrogen) was used to
synthesize cDNA from purified total mRNA.
4. One-step SYBR Green RT-qPCR Master Mix (Qiagen, Valencia, CA) was used for
RT-qPCR
5. EnGen
®
Spy Cas9 NLS and NEBuffer™ 3.1 were used for CRISPR.
6. Anatech LTD's Alcian blue was used to make Alcian blue stock.
7. Syndel USA's TRICAINE-S was used to make 0.04% Tricaine to anesthesia fish.

Zebrafish lines
AB outcross wild type

14
Zebrafish Maintenance
All procedures described have been approved by CHLA IACUC committee. Zebrafish are
housed in a circulating system that continually filters and aerates the system water, and the
temperature in the room is usually kept between 26-28.5 °C. Zebrafish would be anesthetized in
0.04% Tricaine-S.
Fertilized eggs are incubated in a 28.5 °C incubator for 72 hours until the larvae hatch. Larvae
can be housed in round dishes for the first week with 50 percent or more of E3 medium changed
daily. After one week, larvae can be carefully transferred into a tank with a 400-micron baffle
and shelved into the system.

15
Chapter Three: Results
Potential maternal contribution
Zebrafish phf6 is expressed more at early stages. The expression level significantly decreased
after 24hpf. I incrossed wild types and collected 25 embryos at each different time points (8hpf,
12hpf, 18hpf, 24hpf and 48hpf). After collecting embryos, I extracted mRNAs from embryos for
later RT-qPCR experiment. I did triplicate experiments and calculated the fold change of phf6
expression comparing to its level at 48 hpf (Figure 8). This suggested that there might be
maternal phf6 contribution at the early stages. Therefore, we want to test this hypothesis and
examine maternal zygotic phenotypes of phf6 mutants by crossing homozygous female fish and
heterozygous male fish and vice versa.
16

Figure 8. Expression levels of phf6 in WT at different timepoints.
RT-qPCR of phf6 to determine its expression compared to the level at 48hpf. Y-axis represents
the fold change of phf6 expression level. Statistic performed by unpaired t-test, ns indicates P >
0.05, * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001.)

Genotyping
We need to genotype the offspring we have before we can cross the homozygous female/male
with heterozygous male/female. For C43Y mutant, we have two sets of primers. One of them is
C43Y primers used to detect if they have C43Y band in gel electrophoresis. If they have C43Y
band as shown in figure 9 lane 5, then they are either homozygous or heterozygous. Otherwise,
17
they are wild type as shown in figure 9 lane 4. Another set of primer is WT primers used to
determine whether it is homozygous or heterozygous. If it has both WT band and C43Y band,
then it is a heterozygous. If it only has C43Y band but no WT band, then it is a homozygous.

Figure 9. phf6 C43Y Genotyping PCR result.
1. WT using WT primers. 2. C43Y Heterozygous using WT primers. 3. C43Y Homozygous
using WT primers. 4. WT using C43Y primers. 5. C43Y homo- or heterozygous using C43Y
primers.

For 8bp allele genotyping, we only use one pair of primers. Heterozygous will have two bands,
and homozygous will only have a lower band. Wild type will have a higher WT band as shown in
figure 10 lane 1. Unlike C43Y using 2% agarose, we run 4% agarose gel overnight to distinguish
the bands.
18

Figure 10. phf6 8bp Genotyping PCR result.
1. Wild type 2. 8bp heterozygous mutant 3. 8bp homozygous mutant

19
Potential Phenotype
We obtained homozygous male and heterozygous female after genotyping. I crossed them and
collected the larvae at day 5 post fertilization. I first used Tricaine to euthanize them, and then I
fixed their head and genotyped their tails. After that I divided them into homozygous or
heterozygous, I used alcian blue to stain the craniofacial cartilage then take pictures under
microscope. Comparing the phenotypes of phf6 fish and wild type fish (Figure 11), the phf6
mutants' eyes and jaws seem normal, but they look like they have a smaller head than wild type.
To quantify the results I observed, I used image J to measure the straight-line distance from the
most protruding part of their forebrain to the eye and the straight-line distance from the most
protruding part of the midbrain to the eye with same angle for each fish. I also measured the
length the longest side of their eyes. After comparing the average length of the forebrain and
midbrain between different genotypes and wild type control, the phf6 mutants have smaller head
than wild type, no matter C43Y allele or 8bp allele (Figure 12 A). All groups have p-value less
than 0.05 except 8bp heterozygous group. And phf6 mutants' eyes seem as large as the wild type
(Figure 12 B). All groups have p-value more than 0.05 indicate that there is no significant
different between phf6 mutants' eyes’ length and wild type’s eyes’ length.
20

Figure 11. Alcian blue staining result at 5dpf.
A. 8bp Heterozygous (n=5) B. 8bp Homozygous (n=5) C. C43Y Heterozygous (n=6) D. C43Y
Homozygous (n=10) E. Wild Type control (n=3). Red arrows indicate the location where I
measured the length of forebrain, midbrain, and eyes.

21
A.

B.

Figure 12. Quantification and analyze of potential phenotypes.
A. Compare the average length of the forehead and midhead of 5dpf zebrafish between different
genotypes and wild type control. B. Compare average length the longest side of eyes between
different genotypes and wild type control. Y-axis represents the fold change of phf6 expression
level. Statistics performed by unpaired t-test, ns indicates P > 0.05, * indicates P ≤ 0.05, **
indicates P ≤ 0.01, *** indicates P ≤ 0.001.
22
Generate Promoter-less CRISPR Mutant
Our previous study shows that the expression levels of phf6 mutants are all reduced but there are
still some phf6 expressions. So I tried to generate phf6 CRISPR mutants by completely removing
the promoter and coding region to avoid genetic compensation triggered by nonsense mediated
decay of mutant phf6 mRNAs.
I designed 6 gRNAs and the sequences are shown in Figure 13.

Figure 13. Designed DNA templates of gRNAs.
The DNA template sequence I used to generate gRNAs is: [T7 promoter]- [Target Sequence]-
[gRNA Sequence Start], which in my experiment is 5'-aattaatacgactcactata- [20bp Target
Sequence] -gtttagagctagaatagc-3'.
In order to completely knock out the phf6 gene, I tried to design 6 gRNAs before the promoter
and at the end of the phf6 gene (Figure 13). There is no study indicating the location of the phf6
gene promoter, thus I assumed it is at 1000bp upstream of phf6. I don't want to disrupt other
23
genes since there is another reverse gene located at 3000bp upstream of phf6. Therefore, I also
designed the gRNA 100bp upstream.
After generating guide RNAs, I need to test their efficiency. CRISPR single guide RNAs’
efficiency can be measured using the T7 Endonuclease Assay (Figure 14). The T7E1 assay bases
on the generation of homoduplexes and heteroduplexes between genomic wild type and mutant
DNA fragments amplified by PCR. The mutant will have mismatches that the T7E1 enzyme will
recognize and cleave, resulting in either cleaved or full-length fragments that may be seen and
differentiated by gel electrophoresis.

Figure 14. Scheme of T7E1 Assay to assess CRISPR sgRNA efficiency.
(Figure created using BioRender and adapted from Luo et al. 2019)

Figure 15 shows the result of efficiencies of single guide RNAs. As can be seen from the figure,
lane 1 and lane 5 have darker cleaved bands, indicating that they are more efficient. Lane 6 is
wild type control without any injections and only shows full-length fragments band.
24

Figure 15. SgRNAs efficiency determined by T7E1.
1-5. Different sgRNAs. 6. WT control.
After determining the efficiency of single guide RNAs, I select the RNAs with the highest
efficiency and inject them together to generated promoter-less mutants. I injected phf6 gRNA 1,
5, 6 as a group and 3,5,6 as another group together to generate fish with a deletion of phf6. To
determine whether there is deletion or not, I use different primers to test it (Figure 16). If there is
no deletion, we will get a 155bp size band for group 1 gRNAs and a 260bp size band for group 2
gRNAs after PCR and gel electrophoresis. If there is deletion, then we will get a 240bp size band
for group 1 and a 215bp band for group 2 gRNAs.

Figure 16. Primers and methods used to test the deletion
25
Lane 1 and 3 of figure 17 represents wild type embryos using forward primer and reverse primer
2, which are primers for mutant bands, from figure 16. Lane 2 and 4 represents wild type
embryos using forward primer and reverse primer 1, which are primers for wild type bands.
Lanes 1, 2, and 5 are using primers designed for gRNA 1+5+6. Lanes 3, 4, and 6 are using
primers designed for gRNA 3+5+6, which is second row of figure 16. From lane 5 and lane 6 we
can see bands we are looking for, which are 240bp and 215bp. However, there are also many
nonspecific bands that might indicate partial repaired of deletion.

Figure 17. PCR results after 3gRNAs injection.
1-4. WT control with different pair of primers. 5&6. Injected embryos.

26
Chapter Four: Discussion
Phenotypic characterization of phf6 mutants
In RT-qPCR of phf6 at different time points, there is a significant decrease of phf6 expression after
24 hpf while the expression level in early embryos is high. This result suggests that there might be
maternal phf6 transcripts contribution at early stage. I should test it by crossing homozygous
female with heterozygous male and heterozygous female with homozygous male. However, we do
not have homozygous females at appropriate age that give birth. And according to the alcian blue
staining results, both heterozygous and homozygous from heterozygous mother show potential
phenotypes. So far we cannot conclude if indeed there is maternal contribution. But we can
consider the smaller head as a possible phenotype to continue mating different pairs of fish to
confirm that finding. For eyes’ length of phf6 mutants, they seem as same as the eyes of wild type.
And for ears, phf6 patients usually have large and fleshy earlobes. However, we cannot observe
that phenotype in zebrafish. Because zebrafish do not have outer or middle ears, they only have
inner ears. So zebrafish might not be the appropriate animal model for observing ears’ phenotypes.
Generation of new phf6 mutants
The PCR results after 3gRNAs injection show that there are mutant bands I expected, but there
are also plenty of nonspecific bands that might represent partial repairs of the deletion. So
generating full deletion of phf6 CRISPR mutant can be practicable but we might need to inject
27
more different combination of gRNAs to find the most efficient and accurate combination. I also
have a few injected larvae left for them to grow up and then we can do genotyping to see if they
have deletions. If the mutant bands are not clear and the efficiency is low, we can cross those
phf6 deletion carriers with wild type fish to see if we can get stable mutant line. Then we can
send them to sequencing to confirm we have the mutant we interested.
Neural crest cells
Neural crest cells originate in the neural ectoderm and migrate to generate cartilage, bone,
connective tissue, sensory neurons, glia, and pigment cells in many other cell and tissue types
(Trainor 2010). Neural crest cells play an important role in craniofacial development, and
zebrafish has grown in popularity as a model for studying neural crest biology and skull
development (Mork and Crump 2015). Many symptoms of BFLS patients are showed in tissues
that were developed from neural crest cells, such as narrow forehead, broad jaw, protruding chin,
large ears, and thick calvarium. Therefore, we can do in situ hybridization of neural crest
markers such as hand2, crestin, and foxd3 to examine if phf6 has functions in neural crest cells.

28
Chapter Five: Future study
There are a few more experiments that need to be done in the future.
First, keep crossing different pairs of phf6 8bp and C43Y mutant fish and perform phenotypic
characterization to confirm if there is maternal zygotic mutant phenotype or not. Also using
alcian blue to stain their craniofacial cartilage to provide further evidence of that smaller head is
a phenotype of phf6 mutant zebrafish.
In situ hybridization with phf6 probe and other early developmental markers will also be used to
corroborate these findings. To ensure that the phenotypes are caused by the mutations, a rescue
can also be performed.
Finally, I would raise up injected larvae and screen for the phf6 deletion carriers. Then cross
them with wild type to get more stable line.

29
References
[1] Baumstark, A. “Novel PHF6 Mutation p.D333del Causes Borjeson-Forssman-Lehmann
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Abstract (if available)

Abstract Borjeson-Forssman-Lehmann syndrome (BFLS) is caused by disruptions or mutations of the PHF6 gene and characterized by intellectual disability, developmental delay, epilepsy and obesity. PHF6 (PHD Finger Protein 6) is a member of the plant homeodomain (PHD)-like finger (PHF) protein family. CRISPR associated protein 9 (Cas9) is an efficient, convenient, economical and rapid gene editing technology emerging in recent years. With the gene editing technology, mutant zebrafish may be generated to study loss-of-function phenotypes. CRISPR/Cas9 technology was used to create zebrafish mutations that resemble the patient alleles. However, the phf6 mutants previously generated do not show obvious phenotypes and are adult viable. To determine if the lack of phenotypes is due to maternal transcript contribution, I analyzed phf6 maternal zygotic mutant phenotypes. I am also in the process of generating a new phf6 CRISPR mutant by completely removing the promoter and coding region to avoid genetic compensation triggered by nonsense mediated decay of mutant phf6 mRNAs.

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Asset Metadata

Creator Tian, Zhiyu (author)

Core Title Characterization of zebrafish phf6 CRISPR mutant phenotypes

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2022-08

Publication Date 07/22/2022

Defense Date 06/14/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag BFLS,craniofacial,CRISPR,OAI-PMH Harvest,phf6,zebrafish

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Lien, Ching-Ling (committee chair), Allayee, Hooman (committee member), Subramanyan, Ram Kumar (committee member)

Creator Email tianzhiyu0531@gmail.com,zhiyutia@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111373922

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Source 20220722-usctheses-batch-961 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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