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Adhikari 1
Utilizing the CRISPR/Cas9 method of genome engineering to
generate unc-119 mutants in C. briggsae
By
Anand Adhikari
Student #: 1450551
Under Supervision of Dr. Bhagwati P. Gupta
Molecular Biology 3I03
McMaster University
Submitted: April 23, 2015
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Table of Contents
Abstract …………………………………………………………………………………..…… 3
List of Abbreviations………………………………………………………………………..
4
Introduction………………………………………………………………………………….
5-8
Models of Genetic Engineering……………………………………………….…..5
Overview of CRISPR/Cas9…………………………………………….………….5-6
Plasmids and sgRNAs…………………………………………………….....…….6
CRISPR/Cas in C. elegans…………………………………………..………...7
Sister Species: C. elegans & C.briggsae…………………………………….……7-8
Brief Overview…………………………………………………………………….8
Materials & Methods…………………………………………………………………………. .9-11
Creating ssODN & Related Genomics……………………………………….……9
Mutagenesis & Transformation……………………………………………………9
Restriction Digest &DNA Prep……………………………………………………10
Microinjection, Cloning, Screening……………………………………….………10
gDNA Isolation, Sequencing, & Alignment………………………………………11
Results…………………………………………………………………………. ….…………12-16
Trial #1…………………………………………………………………………….12
Trial #2 ………………………………………………………………………..…..13-16
Target Sequence, Restriction Digest………………………………..13
DNA Concentrations, Mutation Results……………………………14
Cbr-unc-119 F2-10-1 DNA sequencing alignment…………………15
Cbr-unc-119 F2-13-2 DNA sequence alignment……………...……16
Discussion…………………………………………………………………………. ….……17-19
Trial #1 vs Trial #2……………………………………………………………17
Restriction Digest…………………………………………………………… 17-18
Trial #2 Additional Analysis…………………………………………………18
Sequencing Analysis…………………………………………………………18
Pitfalls and Future Experimentation…………………………………………19
Supplementary Tables……………………………………………………………….………..20-21
References……………………………………………………………….……………………22-23
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List of Abbreviations
The following list below describes the meaning of various abbreviations and acronyms used
throughout the report. The page on which each one is defined or first used is also given.
Abbreviation
Meaning
Page
ApE
A plasmid editor (software)
9
CRISPR
Clustered regularly
interspaced short palindromic
repeats
4
DSB
Double-stranded break
5
GFP
Green fluorescent protein
7
PAM
Protospacer adjacent motif
5
sgRNA
Single-guide RNA
4
TALEN
Transcription activator-like
effector nuclease
5
ZFN
Zinc-finger nuclease
5
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Abstract
The CRISPR/Cas9 system stands as one of the new developments in genetic engineering used
to modify any genomic sequence with high levels of specificity. The system first found in
bacteria allows these species to develop resistance to foreign genetic elements, providing an
acquired immunity.1 More recently, the technique has been expanded to model organisms, such
as C. elegans, in which modified sgRNAs are able to target genomic sequences expressed from a
U6 RNA promoter.2 This technique has not been explored as extensively in C. briggsae, the sister
species of C. elegans. This study was done to see whether targeted modifications of gene
sequences in C. briggsae could be acquired through the CRISPR/Cas9 system. The focus of the
study was the unc-119 gene, which has been shown to be involved with motor ability in C.
elegans and C. briggsae.3 Additionally, the unc-119-regulated pathway and gene function is
conserved to higher organisms, including humans.4
Initially, the experiment was done and yielded no mutant worms. After designing a sgRNA
site ending with a “GG” motif, the mutation was visible in the F2 generation. The cbr-unc-119
gene was able to be mutated in 6 of the 28 candidates, yielding a 19% efficiency rate. None of the
mutants were able to be rescued in the subsequent generation, illustrating that they followed a
Mendelian model of inheritance. After sequencing the DNA of two of the obtained candidates,
both illustrated a nonsense mutation resulting from an insertion. Two of the other identified
candidates were unable to produce viable offspring, potentially due to additional mutations. This
project has allowed additional insight towards the use of identifying mutant alleles through
CRISPR/Cas9 in C. briggsae, and allows future potential for understanding evolutionary
differences of development and gene function between C. elegans and C. briggsae.
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Introduction
Models of Genetic Engineering
Before CRISPR/Cas9 became an establish mode of genetic engineering, ZFN and TALEN
methods were relied upon. ZFNs work by introducing a generation of DSBs at specific genomic
target locations, which are based on the presence of chimeric proteins containing a DNA binding
domain.5 As DNA nucleases became more efficient, ZFNs improved in localizing the specificity
and efficacy of DSBs. ZFNs, however, require a very complicated recognition code and must be
selected for experimentally to ensure high rate of efficiency.6 TALENs, similar to ZFNs, consist
of a DNA-binding domain with a TALE protein fused to the FokI nuclease. Sequential repeats in
the DNA domain correspond to the DNA targeting code of Tal effectors. Restriction enzymes can
be engineered that are specific for any desired DNA sequence of choice with TALENs.7 Through
homologous recombination, both of these methods have been used to create hereditary changes as
well as sequence manipulation, but CRISPR/Cas9 has emerged as the most effective genetic
engineering mechanism with the greatest potential in biotechnology and medicine.8
Overview of CRISPR/Cas9
The CRISPR/Cas9 system is an RNA/enzyme complex composed of a targeting RNA
(sgRNA) and the Cas9 RNA-guided nuclease. Cas9 searches the genetic material of a cell until it
recognizes the specific 3-nucleotide series (NGG) known as the PAM sequence. A 20 base pair
stretch (known as the protospacer) of the sgRNA associates with the complementary DNA
adjacent to the PAM sequence and allows Cas9 to unwind the DNA double helix and create a
double stranded break in the DNA sequence 3 nucleotides down from the PAM sequence.
Changing the targeting sequence at the 5’ end of the sgRNA allows Cas9 to cause double strand
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break in virtually any DNA sequence of interest in a genome. This allows for repair enzymes to
repair gaps in the DNA through either error-prone non-homologous end joining (in the absence of
any template) or precise homologous recombination (if a homologous sequence is provided in
trans), thereby causing a deletion, insertion, or point mutation in the DNA. The Cas9 protein and
specific guide RNAs can be generated into a vector that can be inserted into the germline of the
model organism.9 The potential developments of CRISPR/Cas9 can be used in for many potential
outlooks, including human gene therapy, screening for drug target, synthetic biology engineering,
and agricultural biology.8
Plasmids and sgRNAs
Through plasmid based delivery, 20bp sgRNAs can be integrated into a template plasmid
through site-directed mutagenesis. Genomic software can be used to determine the idea location
to insert sgRNAs into a plasmid. Construction of an expression plasmid for sgRNA is quite
simple and rapid as it only requires one cloning step with a pair of partially complementary
oligonucleotides. The oligo pairs encoding the 20-nt guide sequences are annealed and ligated
into a plasmid through a common mutagenesis kit. Additionally, the transfected plasmids can be
modified to enable virus production for in vivo delivery. In addition to PCR and plasmid-based
delivery methods, Cas9 and sgRNAs can be introduced into cells as mRNA and RNA,
respectively. Commonly used plasmid cloning vector in E.coli is pUC57, which has been proven
to work with CRISPR/Cas9 engineering countless number of times.9 This plasmid (2710 bp in
length) is also bacterially resistant to ampicillin and contains many restriction sites to confirm
successful transfection.10
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CRISPR/Cas in C. elegans
In C. elegans, Cas9 activity was initially completed to screen for random mutations after
DNA injection. Researchers were able to create mutations in two genes (unc-119 and dpy-13)
that illustrated clear phenotypic defects in the F2 generation. Further studies have shown success
in this mechanism with C. elegans with heat-shock promoter to drive expression of Cas9 and use
of the U6 promoter to express the sgRNA to create random deletions in specific genes to show
visible phenotypes.2 These changes introduce either missense or nonsense mutations in the gene
of interest. PCR amplicons, like GFP reporter can be used to study in vivo expression of genes
and can serve as indication of mutation carriers in the F1 generation of worms. The GFP insertion
approach has the added advantage of disrupting the function of genes for which no obvious
visible phenotype exists. One-fourth of the F2 generation of worms from the GFP-expressing F1s
are expected to carry the mutations, following patterns of Mendelian inheritance. Expansion by
using a co-inject plasmid to replace genes with GFP by screening for fluorescence has also been
an adopted technique in CRISPR/Cas9.11 From these experiments, researchers have been able to
garner average frequencies of gene editing ranging from 7 to 32% in C. elegans. More recently,
in vivo there has been higher rates of frequency of genome editing in C. elegans with guide
RNAs ending with a GG motif at the 3’ end of the target-specific sequence.12
Sister Species: C. elegans & C.briggsae
C. briggsae and C. elegans are two very closely related species of nematodes from the
genus Caenorhabditis. Both these organisms share a similar number of chromosomes and
protein/nonprotein coding genes, interpreted as conserved genes and pathways. The wildtype
strain of C. elegans is N2 whereas the wildtype strain of C. briggsae is AF16.3 From evolutionary
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studies, it has been determined that C. briggsae diverged from C. elegans about 30 million years
ago, yet the two species have almost identical morphology. However, comparison of DNA
sequences has revealed significant number of rapidly evolving and divergent genes suggesting
alterations in underlying mechanisms of development and behaviour. Consistent with this, recent
comparative studies of vulval development between C. elegans and C. briggsae have revealed
differences in the competence of certain vulval precursor cells.13 There are many genes
responsible for regulating cell competence, or the ability of cells to respond to intercellular
signals. Additionally, the striking homology between these nematodes and humans makes them
an ideal organism for genetic and developmental studies.12
Brief Overview
This study looked at whether targeted modifications of unc-119 in C. briggsae could be
acquired through the CRISPR/Cas9 system. Through the creation of a plasmid vector that
contained an sgRNA target site of the unc-119 gene in C. briggsae and introducing it into the
AF16 worms, visible mutants were able to be seen in the F2 generation of worms. Sequencing
potential candidates allowed for alterations in gene sequences to be identified as insertions that
led to nonsense mutations, immediately preceding the chosen sgRNA target site.
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Materials & Methods:
Creating ssODN & Related Genomics
To create the single-stranded oligonucleotide for cbr-unc-119, the transcript for unc-119
gene in C. briggsae was found on Wormbase. The transcript excluding introns and untranslated
regions was copied into ZiFit in FASTA format in order to check for potential target sites.14 A
target site was chosen that had a G/C content >50%. For Trial #2, a target site was chosen that
had a similar G/C content and additionally ended with a “GG” motif.12 The chosen target site was
then entered on the NCBI BLAST software in order to confirm the identity of the chosen
targetsite on the unc-119 gene in C. briggsae.15,16 Primers were created that matched the
targetsite using ApE software.17 The forward primer included the 5’ end of the pUC57 plasmid
recognition site were ordered via MOBIX Lab Online Oligio Synthesis at McMaster University.
Mutagenesis & Transformation
Q5 Site-Directed Mutagenesis was performed to add the cbr-unc-119 sgRNA site into the
pUC57 plasmid vector.17 Differences in the protocol were that the heat shock lasted for 45
seconds, the reaction mix was incubated on ice for two minutes following heat shock, and the
SOC/bacteria mix was incubated for two hours. The mix was plated and incubated overnight at
37ºC for colony formation. Three colonies were isolated and were each placed in a separate test
tube containing 4 µl of SOC medium. Additionally a 1:1000 dilution of carbenicillin was added
to the test tubes to reduce the formation of satellite colonies. Colonies were each transferred into
a separate test tube and they were placed in the incubated shaker for 16 hours.
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Restriction Digest &DNA Prep
The plasmid was then isolated in 1.5mL tubes using the Presto Mini Prep Kit.18 DNA
Spectrophotometer was used to determine the initial concentrations of the plasmids (shown in
Table #4). Restriction enzyme digest was then performed using HindIII, EcoRI , and Acl1.
Double digest was set up using HindIII and EcoRI. Separate digest was done with Acl1.
Additionally, lin-10 was used as a control since it is cut by Acl1 at the target site. Gel results are
shown below in Image 1.19 After confirming successful mutagenesis via gel electrophoresis
(Image 1), plasmid DNA concentrations were increased using vacufuge centrifugation to create
sufficient concentrations for microinjection. The final DNA concentrations are shown below in
Table 4.
Microinjection, Cloning, Screening
Injection mix was then created by using appropriate concentrations of sgRNA, Cas9,
myo2::GFP, and dH20.20 16 parental AF16 hermaphrodites were injected in the gonad with the
injection mix, and the worms were given three days to grown F1s.21 F1s from each plate were
screened for GFP-expression. F1 worms that expressed GFP were plated 2 per plate and labeled
accordingly. In Trial #2, non-fluorescent worms from each plate that illustrate GFP expression
were also plated 2 per plate, since it was unsure whether the mutation corresponded with GFP
expression. The picked F1s were given three days to grow and give rise to an F2 generation. The
F2 generation of worms was then screened for the visible unc-119 mutant phenotype. Potential
candidates were plated one worm per plate. Primers were created for the F2 worms outside of the
range of the target site and were ordered via Mobix.14,15,16
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gDNA Isolation, Sequencing, & Alignment
gDNA of F2 worms was isolated using standard gDNA isolation protocol.22 Single worm
PCR was done on an F2 from each candidate plate, using High Fidelity protocol.23 PCR product
was run on a 1% gel electrophoresis, and successful PCR product formation was extracted via
using gel extraction.24 2µl of the extracted product was run on a gel alongside 2 µl of ladder, and
the intensity of the band compared to the ladder was used to gauge the concentration of the DNA.
5 µl of DNA was then sent for sequencing. Alignment was done on Clustal Omega program to
compare the sequenced DNA to the WT (Shown below, Figures 1 &2) and demonstrate the
location of the mutation.
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Results:
Trial #1
In Table #1 below, the target sequence used for Trial #1 is listed, as well as the designed
forward and reverse primers for that specific target site. The highlighted portion of the forward
primer matches the target sequence. The initial DNA concentrations and restriction enzyme
digest of the plasmid data are not included for Trial #1 since it ultimately did not succeed in the
progeny of the injected worms.
Table#1: Target sequence and primers in cbr-unc-119 gene for Trial #1
unc-119 sgRNA #1 GGAAGTGCTAAAACGTCGTT
GGAAGTGCTAAAACGTCGTTGTTTTAGAGCTAGAAATAGCAAG
Forward Primer
(sgRNA#1)
(GL1047)
AAACATTTAGATTTGCAATTCAATTATAT
Reverse Primer
(sgRNA#1)
(GL1048)
Table #1: Forward and reverse primer for Q5 site directed mutagenesis of pU6::cel-unc-119
sgRNA plasmid to substitute cbr-unc-119 sgRNA #1 sequence.
Below, Table #2 illustrates the mutation results for Trial #1. Initially, 14 AF16 worms
were injected with the injection mix, and 50 worms showing GFP expression were plated.
However, there were no worms in the F2 generation that illustrated the unc-119 mutation, thus
resulting in a 0% efficiency of genetic engineering.
Table #2: Mutation Results for Trial #1
Targeted Gene
cbr-unc-119
P0
14 worms
F1
50 worms (GFP)
F2
0
Efficiency
0%
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Trial #2
In Table #3 below, the target sequence used for Trial #2 is listed, as well as the designed
forward primer and same reverse primer from Trial #1
Table #3: Target sequence and primers in cbr-unc-119 gene for Trial #2
unc-119 sgRNA #2 GGGAAGGTCGCCGAGCCGGG
GGGAAGGTCGCCGAGCCGGGGTTTTAGAGCTAGAAATAGCAAG
Forward Primer
(sgRNA#2)
(GL1079)
AAACATTTAGATTTGCAATTCAATTATAT
Reverse Primer
(sgRNA#2)
(GL1048)
Table #3: Forward and reverse primer for Q5 site directed mutagenesis of pU6::cel-unc-119
sgRNA plasmid to substitute cbr-unc-119 sgRNA #2 sequence.
Image 1 below shows the restriction digest of the mutated pUC57 plasmids containing the
inserted unc-119 sgRNA #2 target site. Three colonies (labeled A-C) were either double digested
with HindIII + EcoRI or only digested with Acl1. The control for this experiment was the pUC57
plasmid containing lin-10 insertion. Bands for the double digest of the mutated plasmid appeared
at 3.1kb for all of the colonies. For the Acl1 digest, unc-119 sgRNA #2 C gave a band at 2.6kb.
Image 1: Restriction Digest of pUC57::pU6::cbr-unc-119 sgRNA #2 Plasmid DNA
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Below, Table #4 illustrates the initial and final concentrations of the plasmids containing the
inserted unc-119 sgRNA #2 target site for. DNA Spectrophotometer was used to achieve these
readings.
Table #4: Initial and Final Plasmid DNA Concentrations, Trial #2
Initial Reading (ng/µl)
Final Reading (ng/µl)
unc-119 sgRNA #2 A 168
248
unc-119 sgRNA #2 B 184
268
unc-119 sgRNA #2 C 172
N/A*
*Since unc-119 sgRNA #2 C provided a faulty digest for Acl1, it was discarded and thus not used
later on.
Below, Table #5 illustrates the mutation results of Trial #2. Initially, 16 AF16 worms
were microinjected, and 54 F1 worms that illustrated GFP expression were plated. Additionally,
84 worms that did not express GFP but resided on plates that contained GFP expressing worms
were plated.. 6 plates of the F2 generation illustrated visible phenotypic unc-119 mutations.
Additionally, both F1 plates with 1 worm ended up producing mutant F2 progeny. Two out of six
F2 candidates produced unhealthy eggs and only two out of six candidates were sequenced (See
Supplemental Table #2).
Table #5: Mutation Results for Trial #2
Targeted Gene
cbr-unc-119
P0
16 worms
F1
54 worms* (GFP)
84 worms (non-GFP)
F2
6 plates**
0
Efficiency
11-19%
0%
*Cloned 2 worms per plate, but two plates had 1 worm/plate (see Supplemental Table #1).
**Cloned 1 worm per plate.
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Trial #2: Alignment Data
Below, Figure 1 shows the alignment between the wt DNA and F2-8-10-1 candidate for
the unc-119 gene. The inserted sequence takes place 4 bp upstream of the PAM site and
introduces a frameshift that leads to a premature stop codon.The inserted sequence is 16bp long
the sequence itself matches part of the sgRNA target site. The insertion takes place immediately
after a base pair mutation (cytosine to thymine). There were a few nucleotides that were able to
be corrected by peak analysis of the DNA sequencing sheet. The key for the figure is shown
below the figure.
Figure 1: wt (AF16) and unc-10-1 DNA sequence alignment of unc-119 gene
CLUSTAL 2.1 multiple sequence alignment
GGCACCCTCTAATTACCATTTTCCTGTCACACCACACTCTTTTCCTTCTTCCACTTCTTT 60
---------------------------NNNNNNNNNNTNTTTTCNTTCTTCN-CTTCTTT 32
* ***** ****** *******
CCGTTTTCAGCCGCTTCCAACCAAACCGATATGAAAGCCGAGCAACAACAATCGATTCCA 120
CCGTTTTCAGCCGCTTCCAACCAAACCGATATGAAAGCCGAGCAACAACAATCGATTCCA 89
******* ******** * * *************************************
CCCG----------------GCTCGGCGACCTTCCCGTCGCAGGTGAGACTCAGAAAACT 164
CCCTCGGCGACCTTCCCGTCGCTCGGCGACCTTCCCGTCGCAGGTGAGACTCAGAAAACT 149
***
****************************************
AGAGAAACCGCTCAACTAACTCTTGATATCCGATTTCATTCTTTTCTCTTTTCTTTTTGT 224
AGAGAAACCGCTCAACTAACTCTTGATATCCGATTTCATTCTTTTCTCTTTTCTTTTTGT 209
************************************************************
TGAACTTTTCCATATTTCCAGATGCCACGGCCACCACCAAGCACCGAACAAGGAATC- 281
TGAACTTTTCCATATTTCCAGATGCCACGGCCACCACCAAGCACCGAACAAGGAATCA 267
*********************************************************
Key for Figure 1 & 2:
Yellow highlight = PAM Sequence
Green Highlight = sgRNA #2 Target Site
Red Text = Corrected Nucleotides
Blue Text = Inserted Sequence
wt
unc-10-1
wt
unc-10-1
wt
unc-10-1
wt
unc-10-1
wt
unc-10-1
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Figure 2: wt (AF16) and unc-13-2 DNA sequence alignment of unc-119 gene
CLUSTAL 2.1 multiple sequence alignment
GGCACCCTCTAATTACCATTTTCCTGTCACACCACACTCTTTTCCTTCTTCCACTTCTTT
-----------------------------NNNNNNNNTCTTTTCCTTCTTNN-CTTCTTT
*************
******* wt
CCGTTTTCAGCCGCTTCCAACCAAACCGATATGAAAGCCGAGCAACAACAATCGATTCCA
CCGTTTTCAGCCGCTTCCAACCAAACCGATATGAAAGCCGAGCAACAACAATCGATTCCA
******* ******** * * *************************************
CCCG--GCTCGGCGACCTTCCCGTCGCAGGTGAGACTCAGAAAACTAGAGAAACCGCTCA
CCCAAAGCTCGGCGACCTTCCCGTCGCAGGTGAGACTCAGAAAACTAGAGAAACCGCTCA
***
******************************************************
ACTAACTCTTGATATCCGATTTCATTCTTTTCTCTTTTCTTTTTGTTGAACTTTTCCATA
ACTAACTCTTGATATCCGATTTCATTCTTTTCTCTTTTCTTTTTGTTGAACTTTTCCATA
************************************************************
TTTCCAGATGCCACGGCCACCACCAAGCACCGAACAAGGAATC- 281
TTTCCAGATGCCACGGCCACCACCAAGCACCGAACAAGGAATCA 251
*******************************************
60
30
wt
unc-13-2
120
87
wt
unc-13-2
178
147
wt
unc-13-2
238
207
wt
unc-13-2
wt
unc-13-2
Above, Figure 2 shows the alignment between the wt DNA and F2-13-2-1 candidate for
the unc-119 gene. The inserted sequence takes place 4 bp upstream of the PAM site and
introduces a frameshift mutation that leads to a premature stop codon. The inserted sequence is a
two base pair insertion. The insertion takes place immediately after a base pair mutation (cytosine
to adenine). There were a few nucleotides that were able to be corrected by peak analysis of the
DNA sequencing sheet. Figure 2 follows the same key as Figure 1 above.
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Discussion
Trial #1 vs Trial #2 Efficiencies
Looking at the results, Trial #1 proved to be unsuccessful due to a faulty target sgRNA of
choice. None of the F2 progeny illustrated any mutations, and the 0% efficiency may be in part
due to inability of CRISPR/Cas9 to recognize the target sequence on the gene. The problem was
not due to a poor injection, since GFP expression was found in the F1 generation of worms
(Table #2). None of the specific data (e.g. DNA concentrations, restriction digest) from this
experiment was included in the results section since it was a faulty Trial. By changing the target
sequence and having it end with a “GG” motif for Trial #2, there was success in generating the
cbr-unc-119 mutation in six candidates in the F2 generation (Table #5). Only the forward primer
was modified, since the location of the reverse primer was far enough downstream from the
target. The efficiency had a range of 11-19% since we are unsure of whether both original F1
worms (on plates containing two F1 worms) gave rise to F2 unc-119 candidates. Although the
efficiency for Trial #2 seems low, it is comparable to the efficiency of genetic engineering with
CRISPR/Cas9 found in previous studies.2
Restriction Digest
The restriction digest shown in Image 1illustrates the three transformed bacterial colonies
that had the unc-119 sgRNA #2 inserted into the pUC57 plasmid. Looking at the Acl1 digest,
unc-119 sgRNA #3 was unable to properly be digested by Acl1, and gave a band of 2.6kb when
the digest was supposed to cut after 3.1kb. This may have been due to mutation in the plasmid, or
contamination of the enzyme. As a result, unc-119 sgRNA #2 C was discarded from that point on
and was not vacufuged (Table #4). The digest was also unsuccessful in the control, as the lin-10
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plasmid was supposed to provide a band at 3.1kb but instead cut at 2.6kb. This is likely due to the
old age of the plasmid. The HindIII and EcoR1 double digest, however, was successful for all
three of the plasmids, and illustrates that the mutagenesis was successful for both A and B.
Trial #2 Additional Analysis
Looking at Table #5, we can conclude that the unc-119 mutation in C. briggsae is linked
to GFP expression in F1 generation worms, since none of the non-GFP worms gave rise to
mutant F2s. All of the candidates for the unc-119 mutation arose from the same original parental
worm. This means that CRISPR/Cas9 mechanism may have only been successful in one of the
injected worms (see Supplemental Figure #1). Additionally, two of the mutant candidates were
unable to give rise to an F3 generation and had eggs that did not undergo proper gastrulation.
This may have been due to an additional mutation caused by the CRISPR/Cas9 mechanism.
Sequencing Analysis
Two of the six candidates were able to be successfully sequenced. Figure 1 shows the
sequencing data for F2-10-1. The inserted sequence is found in the sgRNA site, and the insertion
takes place 4bp upstream of the PAM sequence. F2-13-2 also had an insertion 4bp upstream of
the PAM sequences, illustrating that this recognition for the target site is highly conserved in C.
briggsae. Both of the insertions led to a premature stop codon. The non-mutant F2s may have
also had an altered DNA sequence that may have included silent mutations instead. In summary,
different targeted modifications of gene sequences in C. briggsae were acquired through the
CRISPR/Cas9 system of genetic engineering. An sgRNA site was chosen in the unc-119 gene
that led to different insertions causing loss of function for the mutants.
Adhikari 19
Pitfalls and Future Experimentation
The timeframe of this project spanned over three and a half months, when it could have
easily been completed in two. If it was known that an sgRNA site ending with a GG motif
increases the efficiency of CRISPR/Cas9, this project could have been expanded to multiple
genes of interest instead of just cbr-unc-119. Other barriers to completing the experiments on
time included various places of contamination (on the agar plates, as well as with some of the
reagents) as well as learning and reviewing some of the basic experimental techniques that I was
more unfamiliar with (e.g. gel extraction, restriction digest). Additionally, it took the unc-119
mutants a longer amount of time to progress through their life cycle compared to the AF16
worms, which made things a little bit slower as well.
For future experimentation, I would investigate other genes that are similar in function
between C. elegans and C. briggsae and involved with developmental pathways such as Wnt and
Ras. I would also further investigate the F2 candidates that produced sick eggs, and see whether
CRISPR/Cas9 was able to introduce side effect mutations as well as the expected insertion to
these worms. By expanding this effort towards other genes shared by C. elegans and C. briggsae,
the evolutionary divergence between these two species can be better understood and mapped
more accurately. Future studies can also focus on genes that are conserved to humans, to see if
similar effects of genetic modifications can be applied to these same pathway genes found in
humans.
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Supplementary Tables
Supplementary Table #1: F1 Plates (GFP Expression)
F1 (GFP) Plate Notes
F1 (GFP) Plate
F1-1-1
F1-8-1
F1-1-2
F1-8-2
F1-1-3
F1-8-3
F1-2-1
F1-8-4
F1-7-1
F1-8-5
F1-7-2
F1-8-6
F1-7-3
1 sick worm
F1-8-7
F1-7-4
F1-8-8
F1-7-5
F1-8-9
F1-7-6
F1-8-10
F1-7-7
F1-8-11
F1-7-8
F1-8-12
F1-7-9
F1-8-13
F1-14-1
F1-14-2
Notes
1 sick worm
1 worm
1 worm
The table above shows a more detailed description of the identity of each of the F1 GFP
plates. Each plate had 2 worms each, except for F1-8-11 and F1-8-13 Two of the plates had 1
sick worm each. The highlighted plates are the ones that produced mutant F2s. Additionally, it is
important to note that GFP-expressing worms were only found from 5 of the 14 parents
(P1,P2,P7,P8,P14), and each of the mutant candidates arose from the same injected parental
worm (P8). Supplementary Table #2 below shows the identity of each of the picked non-GFP
expressing worms.
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Supplementary Table #2: F1 Plates (Non-GFP Expression)
Notes
Notes
F1 (non-GFP)
F1 (non-GFP)
Plate
Plate
F1-1-1
F1-8-1
F1-1-2
F1-8-2
F1-1-3
F1-8-3
F1-1-4
F1-8-4
F1-1-5
F1-8-5
F1-2-1
F1-8-6
F1-2-2
F1-8-7
F1-2-3
F1-8-8
F1-2-4
F1-8-9
F1-2-5
F1-8-10
F1-7-1
F1-8-11
F1-7-2
F1-8-12
F1-7-3
F1-14-1
F1-7-4
F1-14-2
F1-7-5
F1-14-3
F1-7-6
F1-14-4
F1-7-7
F1-14-5
F1-7-8
F1-14-6
F1-7-9
F1-14-7
F1-7-10
F1-14-8
F1-7-11
F1-14-9
Supplementary Table #3 below shows each of the F2 candidates that were picked (1 per
plate). Candidates from F1-8-7 and F1-8-8 produced sick eggs and were unable to produce any
offspring. Candidates F2-8-10-1 and F2-13-2 were sequenced.
Supplementary Table #3: F2 Candidates
F2 (unc-119) Plates Notes
F2 (unc-119) Plates
F2-8-1-1
F2-8-8-4
F2-8-1-2
F2-8-10-1
F2-8-1-3
F2-8-10-2
F2-8-1-4
F2-8-10-3
F2-8-7-1
Sick Eggs
F2-8-10-4
F2-8-7-2
Sick Eggs
F2-8-11-1
F2-8-8-1
Sick Eggs
F2-8-11-2
F2-8-8-2
Sick Eggs
F2-8-13-1
F2-8-8-3
Sick Eggs
F2-8-13-2
Notes
Sick Eggs
Sequenced
Sequenced
Adhikari 22
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