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Genome-scale screening in mammalian cells using CRISPR-Cas9 system

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Genome-scale screening in mammalian cells using CRISPR-Cas9 system

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i

UNIVERSITY OF SOUTHERN CALIFORNIA
Genome-Scale screening in mammalian cells using CRISPR-Cas9 system
Sijie Wang
A Thesis Submitted in Partial Satisfaction of the
Requirements for the Degree of Master of Science
In
Chemical Engineering
2016 SPRING

Committee in charge:

Professor Pin Wang, Chair
Professor C.Ted Lee, Jr.
Professor Nicholas Graham

ii

Table of Contents:
Attribution iii
Acknowledgements iv
Abstract v
List of Abbreviations vi

1.Introduction 1
2.Backgrounds 6
3.Methods 15
4.Results 21
5.Discussion 27
6.Bibliography 29

iii

Attribution
Author Sijie Wang was responsible for the writing. The tables, data and images were
contributed under the assistance of Si Li. The project was conducted via Si Li and Sijie
Wang, under the supervision of Dr. Pin Wang.

iv

Acknowledgements
Thank the entire Wang Laboratory. Thank Prof. Pin Wang for his support and discussions
on this project. And I appreciate Dr. Si Li for all her work in all other aspects of this
project.

v

Abstract
Recently, CRISPR Cas9 system has been widely used as a genome-editing tool to
investigate genes-of-interest on their functions and roles in the whole biological network
system. Among this experiment, we conduct a genome-wide screening for novel genes
involved in the suppression of breast cancer metastasis. We are looking for the
gene-of-interest which are the key factors for cancer metastasis through the cut-ridge
technology-CRISPR Cas9. First, we reconstructed LentiCRISPR vector, and introduce
the whole sgRNA library into the mutated system. After construction of new CRISPR
carrier system, we transfected 4T1 cells with this LentiCPngfr_mt vector system, and
screened for positive cells. In the final step, we conducted in vivo experiments, using 4T1
breast cancer cells as the experimental model. After collecting cells in primary tumors
from two weeks later and cells in metastatic nodules from six weeks later, we submitted
all samples for NGS.

vi

List of Abbreviations
CRISPR clustered regularly interspaced short palindromic repeat
Cas CRISPR Associated protein
NGS Next generation sequencing
crRNA CRISPR RNAs
tracrRNA trans-activating crRNA
sgRNA single guide RNA
DSB double-strand breaks
NHEJ Nonhomologous end-joining

HDR Homology-directed repair

PAM Protospacer adjacent motif

1

Introduction
What is CRISPR?
The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is a
naturally occurring, adaptive microbial immune system for defense against invading
phages and other mobile genetic elements [10, 11, 12, 13]. Three types (I–III) of
CRISPR-Cas systems have been functionally identified across a wide range of microbial
species [14,15,16], and each contains a cluster of CRISPR-associated (Cas) genes and its
corresponding CRISPR array. These characteristic CRISPR arrays consist of repetitive
sequences (direct repeats, referred to as repeats) interspaced by short stretches of
nonrepetitive sequences (spacers) derived from short segments of foreign genetic material
(protospacers). The CRISPR array is transcribed and processed into short CRISPR RNAs
(crRNAs), which direct the Cas proteins to the target nucleic acids via Watson-Crick base
pairing to facilitate nucleic acid destruction.

2

FIG 1. CRISPR acts as immune defense system against foreign phages or other
mobile genetic elements. (A). Foreign DNA invasion and CRISPR structure (B)
Transcription od repetitive repeats, maturation of crRNAs (C). DSB and removal of
foreign elements.

3

How does CRISPR II work?
The type I and III CRISPR systems utilize ensembles of Cas proteins complexed with
crRNAs to mediate the recognition and subsequent degradation of target nucleic acids
[17]. In contrast, the type II CRISPR system recognizes and cleaves the target DNA
(Garneau et al., 2010) via the RNA-guided endonuclease Cas9 along with two noncoding
RNAs [20], the crRNA and the trans-activating crRNA (tracrRNA) [21]. The crRNA
hybridizes with the tracrRNA to form a crRNA:tracrRNA duplex, which is loaded onto
Cas9 to direct the cleavage of cognate DNA sequences bearing appropriate
protospacer-adjacent motifs (PAM) [22]. Cas9 contains two nuclease domains, HNH and
RuvC, which cleave the DNA strands that are complementary and non-complementary to
the 20 nucleotide (nt) guide sequence in crRNAs, respectively [23]. Cas9 can be easily
reprogrammed using RNA guides to generate targeted DNA double-strand breaks (DSBs),
which can stimulate genome editing via one of the two DNA damage repair pathways:
nonhomologous end-joining (NHEJ), resulting in insertions and deletions (indels), or
homology-directed repair (HDR), resulting in precise sequence substitution in the

4

presence of a repair template.
Fig 2. Basic procedures of targeting and cleaving DNA strands via Cas9 protein under
the help of crRNA:tracrRNA complex

5

What did we do here with CRISPR-Cas9 System?
CRISPR Cas9 has showed promising potential in genomic function research, and cancer
cell metabolism or new pathways are being further developed with this kind of leading
biotechnology. Large amounts of research have been demonstrated using this system in
the genome research. For this project, we used CRISPR-Cas9 system to perform a
genome-wide screening for novel genes involved in suppressing breast cancer metastasis,
and before that we also made reconstruction of CRISPR-Cas9 system, replacing the
antibiotic marker into NGFR marker.

6

Backgrounds
Comparison with other genome editing tools
Former studies showed there are mainly four kinds of programmable nucleases used as
genome editing tools [2].
1.Meganucleases derived from microbial mobile genetic elements
2.Zinc finger (ZF) nucleases based on Eukatyotic transcription factors
3.Transcription activator-like effectors (TALEs) from Xanthomonas bacteria
4. RNA-guided DNA endonuclease Cas9 from they type II bacterial adaptive immune
system CRISPR
Meganucleases have not been widely adopted as a genome-engineering platform due to
lack of clear correspondence between meganuclease protein residues and their target
DNA sequence specificity. ZF domains, on the other hand, exhibit context-dependent
binding preference due to crosstalk between adjacent modules when assembled into a

7

larger array. Although multiple strategies have been developed to account for these
limitations, assembly of functional ZFPs with the desired DNA binding specificity
remains a major challenge that requires an extensive screening process. Similarly,
although TALE DNA-binding monomers are for the most part modular, they can still
suffer from context-dependent specificity and their repetitive sequences render
construction of novel TALE arrays labor intensive and costly.
The CRISPR nuclease Cas9 is targeted by a short guide RNA that recognizes the target
DNA via Watson-Crick base pairing [1]. Multiplexed targeting by Cas9 can now be
achieved at unprecedented scale by introducing a battery of short guide RNAs rather than
a library of large, bulky proteins. The ease of Cas9 targeting, its high efficiency as a
site-specific nuclease, and the possibility for highly multiplexed modifications have
opened up a broad range of biological applications across basic research to biotechnology
and medicine.

8

Reprogramming of CRISPR-Cas9 System
Unlike other programmable nuclease systems used for genome editing, a unique
advantage of the Cas9 system is that Cas9 can be combined with multiple single-guide
RNAs(sgRNAs) to achieve efficient multiplexed genome editing in mammalian cells.

Fig 3. Reprogram of Cas9 system by sgRNA

9

Crystal Structure of Cas9
Precise structural information about Cas9 will thus not only enhance our understanding of
how this elegant RNA-guided, adaptive microbial immune system functions, but will also
facilitate further improvements in the Cas9 targeting specificity, the in vitro and in vivo
delivery, and the engineering of Cas9 for novel functions and optimized features.
Below is the report of crystal structure of S. pyogenes Cas9 in complex with sgRNA and
its target DNA at 2.5 A resolution. [6]

10

Fig 4. The crystal structure revealed that Cas9 consists of two lobes: a recognition (REC)
lobe and a nuclease (NUC) lobe (A– D). The REC lobe can be divided into three regions,
a long a helix referred to as the bridge helix (residues 60–93), the REC1 (residues 94–179
and 308–713) domain, and the REC2 (residues 180–307) domain (A–D). The NUC lobe
consists of the RuvC (residues 1–59, 718–769, and 909–1098), HNH (residues 775–908),
and PAM-interacting (PI) (residues 1099–1368) do- mains (A–D). The negatively
charged sgRNA:target DNA heteroduplex is accommodated in a positively charged
groove at the interface between the REC and NUC lobes (E). In the NUC lobe, the RuvC
domain is assembled from the three split RuvC motifs (RuvC I–III) and interfaces with
the PI domain to form a positively charged surface that interacts with the 30 tail of the
sgRNA (E). The HNH domain lies between the RuvC II–III motifs and forms only a few
contacts with the rest of the protein.

11

The REC lobe includes the REC1 and REC2 domains. REC1 adopts an elongated,
a-helical structure comprising 25 a helices (a2–a5 and a12–a32) and two b sheets (b6 and
b10 and b7–b9), whereas REC2 adopts a six-helix bundle structure (a6–a11) (Figures 4A).
A Dali search [25] revealed that the REC lobe does not share structural similarity with
other known proteins, indicating that it is a Cas9-specific functional domain.
As expected, a Cas9 mutant lacking the REC2 domain (D175–307) retained $50% of the
wild-type Cas9 activity (Figure 4B), indicating that the REC2 domain is not critical for
DNA cleavage. The lower cleavage efficiency may be attributed in part to the reduced
expression levels of the D175–307 mutant relative to that of the wild-type protein (Figure
4C). In striking contrast, the deletion of either the repeat-interacting region (D97–150) or
the anti-repeat-interacting region (D312–409) of the REC1 domain abolished the DNA
cleavage activity (Figure 4B), indicating that the recognition of the repeat:anti-repeat
duplex by the REC1 domain is critical for the Cas9 function [6].
The NUC lobe contains the PAM-interacting (PI) domain, which forms an elongated
structure comprising seven a helices (a46–a52), a three-stranded antiparallel b sheet

12

(b18–b20), a five-stranded antiparallel b sheet (b21–b23, b26, and b27), and a
two-stranded antiparallel b sheet (b24 and b25) (Figures 4D). PI domain is positioned to
recognize the PAM sequence on the non-complementary DNA strand. SpCas9 and
St3-SpCas9, but not St3Cas9, cleaved the target DNA with 50-NGG-30 PAM (Figure 4E),
indicating that the PI domain of SpCas9 is required for the recognition of 50-NGG-30
PAM and is sufficient to alter the PAM specificity of St3Cas9. Sp-St3Cas9 retained the
cleavage activity for the target DNA with 50-NGG-30 PAM, albeit at a lower level than
that of SpCas9 (Figure 4E). Additionally, the deletion of the PI domain (D1099–1368)
abolished the cleavage activity (Figure 4E), indicating that the PI domain is critical for
the Cas9 function. These results revealed that the PI domain is a major determinant of the
PAM specificity.

13

Fig 5. (A)Crystal structure of REC1 and REC2 (B) cleavage activity expression of Cas9
mutants compared with wide type Cas9 and (C) protein expressions of Cas9 mutants.
Both (B) and (C) indicate that REC2 is not as a critical key in Cas9 cleavage ability as
REC1.

14

Fig 5. (D) NUC domain structure (E) cleavage activity expression of wide type SpCas9,
St3-SpCas9 and St3-SpCas9, showed SpCas9 and St3-SpCas9, but not St3Cas9, cleaved
the target DNA with 50-NGG-30 PAM, indicating that the PI domain of SpCas9 is
required for the recognition of 50-NGG-30 PAM and is sufficient to alter the PAM
specificity of St3Cas9.

15

Methods
Reconstruction of the selection marker
PCR the whole vector without Puro and PCR tNGFR fragment, and combine them to
construct a new vector via Gibson assembly. Transformation and screening are performed
afterwards. Transform NEB 5-alpha Competent E. coli with 2ul Gibson Assembly
reaction. Analyze plasmid DNA from 4-8 individual colonies by colony PCR or
restriction enzyme digestion, and/or sequence the insert.
Fig 6. Replacement of Puro marker with tNGFR marker, and methods to generate and
diagnose mutated vector system.

16

Silent mutagenesis
Clone tNGFR into a 4kb vector pBlueScript, followed by the site-directed mutagenesis.
Diagnostic digestion and sequencing are used for examination.

Fig 7. Methods of diagnostic digestion and sequencing of silent mutation.

17

Clone the library into vector LentiCPngfr_mt
PCR-Amplify the sgRNA library. Digest the 2kb filler with restriction enzyme, followed
by Gibson assembly to clone the sgRNA library into the vector. Transformation and deep
sequencing are performed.

Fig 8. Introduction of sgRNA library into the LentiCPngfr system.

18

LentiCRISPR library design and construction
Here we will introduce some steps of how to build a LentiCRISPR library. Basically you
build up a sgRNA library targeting 5’ constitutive exons of thousands of genes in the
human genome with an average coverage of several sgRNAs per gene, and each target
site was selected to minimize off-target modification [2].

Fig 9. Steps for construction of LentiCRISPR library
In this experiment, we targeted 60,000 genes with 360,000 sgRNAs, giving a coverage of
6 sgRNAs per gene. The library we used is purchased from another lab.

19

Introduction of LentiCRISPR library into breast cancer cells
After construction of LentiCRISPR library, we transfect 293T cells with sgRNA plasmid
library, followed by the infection of 4T1 cells (MOI=0.3). Puromycin treatment can be
used to select the transduced cells [7].

Fig 10. Amplification of sgRNA, construction of library, transfection of 4T1 cells, and
screening for the successfully transfected cells.

20

In vivo screening of 4T1 metastasis
0.6M transduced 4T1 cells are used for injection. Primary tumor cells are collected from
mice injected after the 2
nd
week, and metastatic tumor cells are collected from mice
injected after the 6
th
week. Both the primary tumor and metastatic tumor cells are treated
by NGS (Next Generation Screening) [3,5].

Fig 11. In vivo experiments of 4T1 metastasis

21

Results
Reconstruction of selection marker
Fig 12.1 PCR vector and tNGFR insert Fig 12.2 Diagnostic digestion (BamHI/Pmel)

Fig 12.3 Sequencing Fig 12.4 Restriction enzyme digestion

Fig 12. Diagnosis and sequencing of selection marker

22

Silent mutagenesis
Fig 13.1 Restriction enzyme digestion

Fig 13.2 Diagnostic digestion (BamHI/BsmBI)

Fig 13. Diagnosis and sequencing of silent mutant

23

Diagnostic Examination of LentiCPngfr_mt
Fig 14.1 Digestion Fig 14.2 PCR Amplification

Fig 14.3 NGFR expression in 293T cells Fig 14.4 NGFR library amplification summary
after transfection

Fig 14. Digestion of vector and insert, NGFR expression after transfection using FACS
We used the FACS machine to check NGFR expression, and the compared to the control
groups, the transfected efficiency reached to 98.1%, which indicated good transfection.

24

In vivo experiment
Fig 15.1 Transformation and enrichment Fig 15.2 Tumor weight at primary site

Fig 15.3 Tumor metastasis

Fig 15. Tumor weights at primary sites and metastatic sites

25

From the Fig 14, we took Lentivector without any sgRNA as a control group, and used
two libraries- library A and library B as the experimental groups. We found out both two
library groups improve the tumor weights in metastatic sites and reduce the weights in
primary sites. Since both sgRNA library Cas9 system knock out the potential
genes-of-interest which are essential keys in suppressing metastasis, metastatic tumor
weights in experimental groups should be higher than the control groups. As expected,
the research results matched the hypothesis.

26

PCR genomic DNA samples for NGS
Fig 16.1 PCR of Genomic DNA_1 Fig 16.2 PCR of Genomic DNA_2

Fig 16.3 1
st
round of PCR to attach stagger, barcode Fig 16.4 2
nd
round of PCR to
and sequencing primer attach adaptor

Fig 16. PCR of the mice genomic DNA samples (1 and 2), and attachment to stagger,
barcode, sequencing primer and adaptors.

27

Discussion
Here we successfully replace the puro marker with NGFR marker, and we confirm that
via diagnostic digestion. We checked the NGFR expression through FACS and the results
are positively satisfactory. In addition, the metastasis tumor nodules and primary site
tumor comparisons showed the expected results. In the control group (Lentivirus vector
with Cas9 gene, but without sgRNA integral), the primary site tumor weight is larger than
other two groups (Lentiviral vector with Cas9 gene as well as sgRNA integral). And in
lung metastasis groups, control group showed more lung metastasis nodules than other
experiment groups.
After introduction of LentiCPngfr_mt library into the 4T1 cells, genes are knocked out
corresponding the sgRNA type in the 4T1 cells. In this way, we have a library of 4T1
cells, in which each of cells have been knocked out different genes. Then After NGS, we
can figure out what kind of gene-of-interest will be the factors, whose absence will lead
to even more cancer metastasis. This research will find out the novel genes involved in
the suppression of cancer metastasis, and will provide a critical step in toward

28

understanding the mechanism of metastasis of breast cancer.
According to the next step, we will submit all the samples for the next generation
sequencing in the future. Up to now, the NGS has not been finished, and we cannot
provide the sequencing result here.

29

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Abstract (if available)

Abstract Recently, CRISPR Cas9 system has been widely used as a genome-editing tool to investigate genes-of-interest on their functions and roles in the whole biological network system. Among this experiment, we conduct a genome-wide screening for novel genes involved in the suppression of breast cancer metastasis. We are looking for the gene-of-interest which are the key factors for cancer metastasis through the cut-ridge technology-CRISPR Cas9. First, we reconstructed LentiCRISPR vector, and introduce the whole sgRNA library into the mutated system. After construction of new CRISPR carrier system, we transfected 4T1 cells with this LentiCPngfr_mt vector system, and screened for positive cells. In the final step, we conducted in vivo experiments, using 4T1 breast cancer cells as the experimental model. After collecting cells in primary tumors from two weeks later and cells in metastatic nodules from six weeks later, we submitted all samples for NGS.

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Asset Metadata

Creator Wang, Sijie (author)

Core Title Genome-scale screening in mammalian cells using CRISPR-Cas9 system

School Andrew and Erna Viterbi School of Engineering

Degree Master of Science

Degree Program Chemical Engineering

Publication Date 04/15/2016

Defense Date 03/20/2016

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

Tag breast cancer,CRISPR,OAI-PMH Harvest,screening

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Pin (committee chair), Graham, Nicholas (committee member), Lee, C. Ted, Jr. (committee member)

Creator Email sijiewan@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-228844

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