Crispr

11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 1
WELCOME

Seminar
on

Jennifer Doudna

INTRODUCTION:
Abundance of genetic information but underutilized.
 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) has
opened new era in biotechnology.
 Poised to transform developmental biology with single solution to many
problems.
Provides simple, easy, cost effective and efficient access to manipulate
virtually any part of the genome of any organism.
Widely accepted by academics and research organizations- led to CRISPR
Craze.

HISTORY: Key Events
 1987- CRISPR sequences were first discovered in Escherichia coli. (Ishino et al., 1987)
 2002- Identification of Cas genes that are associated with DNA repeats in prokaryotes. (Jansen et al.,2002)
 2007- CRISPR provides acquired resistance against viruses in prokaryotes. (Barrangou et al., 2007)
 October 2011 CARIBOU BIOSCIENCES, Berkeley, California. Focus: explicitly interested in agricultural applications of
CRISPR technology. Raised:$11 MILLION
 2012- Idea of using CRISPR- Cas9 as a genome engineering tool was published by Jennifer Doudna and
Emmanuelle Charpentier.
Emmanuelle Charpentier
Umea University, Sweden
Emmanuelle Charpentier awarded the 2015
Louis- Jeantet Prize for Medicine for her
contribution in harnessing an ancient mechanism
of bacterial immunity into a powerful technology
for editing genomes.
Jennifer Doudna
UC, Berkeley
Breakthrough Prize Awards, 2015

 January 2013 - CRISPR is used in mouse and human cells, fuelling rapid uptake of the
technique by researchers. (Zhang et al., 2013)
 April 15, 2014 - First CRISPR patent was granted to Feng Zhang for “CRISPR-Cas SYSTEMS AND METHODS FOR
ALTERING EXPRESSION OF GENE PRODUCTS”
 November 2013 - EDITAS MEDICINE, Cambridge, Massachusetts Focus: Therapeutics, Raised: $43 MILLION
 November 2013 - CRISPR THEAPEUTICS, Basel, Switzerland, Focus: Therapeutics, Raised: $89 MILLION
 November 2014 - INTELLIA THEAPEUTICS, Cambridge, MA, Focus: Therapeutics, Raised: $5MILLION
Companies with an interest in using CRISPR for applications related to gene therapy have raised over $600 million in
venture capital and public markets since the beginning of 2013. (Paul et al., 2015)
(Feng Zhang)

The CRISPR Craze
a race forever….
National Library of medicine
5 6 12 22
32 45
79
127
277
587
1141
2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015
No. of CRISPR paper published year wise

The CRISPR
Craze
a race forever….
Heidi Ledford, Nature, June, 2015
Main Actors in CRISPR/Cas9 patent war
WIPO Patents for CRISPR 2015 by country

The CRISPR
Craze
a race forever….
CRISPR era begins
here
Heidi Ledford, Nature, June, 2015

What makes CRISPR system the ideal genome engineering technology

CORE CONCEPT:
Basic logic behind this technology is to induce cells own DNA repair mechanism at precise locus in its
genome by inducing the double strand break in the DNA.

Contd.
11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 12Amber Dance, 2015
Cas9 complexed with gRNA and target DNA 3D view of the complex

Different CRISPR-Cas system in Bacterial Adaptive Immunity
Class 1- type I (CRISPR-Cas3) and type III (CRISPR-
Cas10)
uses several Cas proteins and the crRNA
Class 2- type II (CRISPR-Cas9) and type V (CRISPR-
Cpf1)
employ a large single-component Cas-9 protein in
conjunction with crRNA and tracerRNA.
11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 13(Zetsche et al., 2015)
functioning of type II CRISPR system

Different Cas proteins and their function
Protein Distribution Process Function
Cas1 Universal Spacer acquisition DNAse, not sequence specfic, can bind RNA; present in all Types
Cas2 Universal Spacer acquisition specific to U-rich regions; present in all Types
Cas3 Type I signature Target interference DNA helicase, endonuclease
Cas4 Type I, II Spacer acquisition RecB-like nuclease with exonuclease activity homologous to RecB
Cas5 Type I crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas6 Type I, III crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas7 Type I crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas8 Type I crRNA expression Large protein with McrA/HNH-nuclease domain and RuvC-like nuclease; part of
CASCADE
Cas9 Type II signature Target interference Large multidomain protein with McrA-HNH nuclease domain and RuvC-like
nuclease domain; necessary for interference and target cleavage
Cas10 Type III signature crRNA expression
and interference
HD nuclease domain, palm domain, Zn ribbon; some homologies with CASCADE
elements
Devaki Bhaya et al., 2011

BACTERIAL IMMUNE SYSTEM:

Jiang et al., 2015
Currently used CRISPR technology is based on the type II
adaptive immune system
of Streptococcus pyogenes

Combining crRNA and tarcerRNA into sgRNA was the crucial
step for the development of CRISPR technology
Joung et al., 2012

Discovery of new version of Cas9
Engineered Cas9 with altered PAM specificity
Development of photoactivable CRISPR system
RECENT ADVANCES

Cpf1 (CRISPR from Prevotella and Francisella 1) at Broad Institute of MIT
and Harvard, Cambridge.
CRISPR-Cpf1 is a class 2 CRISPR system
Cpf1 is a CRISPR-associated two-component RNA programmable DNA
nuclease
Does not require tracerRNA and the gene is 1kb smaller
Targeted DNA is cleaved as a 5 nt staggered cut distal to a 5’ T-rich PAM
Cpf1 exhibit robust nuclease activity in human cells
(Zetsche et al., October 22, 2015, Cell 163, 1–13)
New Version of Cas9:

Cpf1 makes staggered cut at 5’
distal end from the PAM
Organization of two CRISPR loci found in Francisella
novicida .The domain architectures of FnCas9 and FnCpf1
are compared
This discovery expands the potential of CRISPR
toolbox for treating genetic diseases in humans

11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 21Benjamin et al., 2015
Structural and functional roles of D1135, R1335, E1219, G1218 and T1337 in PAM recognition by SpCas9.
In order to alter the PAM specificity of wild Cas9
we have to replace these PAM interacting amino
acids with different amino acids

11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 22Benjamin et al., 2015
B
Characterization of SpCas9 variants with altered PAM specificities.
NGG
>69 m
NGCG
>3 m
NGAT
>15 m
NGAG
>21 m
NGAC
>10 m
NGAA
>23 m
Representation of the number of sites in
the human genome with 20 nucleotide
spacers potentially targetable by wild-
type, VQR, and VRER SpCas9
Total targetable sites = 143 m
These effort towards increasing the targeting range of
Cas9 has given an alternative ways to overcome the
problem of limited choices for PAM sequences

Photoactivatable CRISPR-Cas9 for Optogenetic Genome Editing
Yuta et al., July, 2015
Based on a recently developed photoinducible dimerization system named Magnets
Light-induced reporter activity in HEK293T cells using
N713 and C714 fragments of Cas9 fused with
photoinducible dimerization domains
Optogenetic control of targeted genome editing
will facilitate improved understanding of complex
gene networks and will be helpful in dissection of
causal gene function in diverse biological processes.

Applications of Genome Engineering
Patrick et al., 2014
CRISPR technology has got its application in
all fields of genome engineering

Versatile Nature of CRISPR Technology
11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 25Jeffry et al., 2014

EDITING OUT DISEASES: CRISPR Therapy
Publications demonstrating use of CRISPR-Cas9 for targeting diseases
Disease Summary Reference
Cataracts Rescue of a dominant mutation in the Crygc gene that causes cataracts Wu et al., 2013
Cystic fibrosis Correction of the CFTR locus by HDR in cultured intestinal stem cells from
patients with cystic fibrosis
Schwank et al.,
2013
β-thalassemia Correction of the human hemoglobin beta (HBB) gene in induced pluripotent
stem cells from thalassemia patients using CRISPR technology
Xie et al., 2014
HPV-associated
cervical cancer
Targeting of promoters of human papillomavirus oncogenes; inhibited
tumorigenesis
Zhen et al.,
2014
Hereditary
tyrosinemia type I
Correction of the Fah mutation in hepatocytes of a mouse model of hereditary
tyrosinemia
Yin et al., 2014
HIV Generation of homozygous CCR5 deletion mutations in iPSCs; proposed
approach toward a functional cure of HIV-1 infection. Targeting of LTR
sequences in the HIV-1 genome; inactivated viral gene expression and
replication in latently infected cells and prevented new HIV-1 infection
Yi et al., 2014
Malaria High (50–100%) gene disruption of the Plasmodium falciporum genome.
Potential to generate transgenic parasites to prevent malaria
Hu et al., 2014

High-throughput functional genomics using CRISPR–Cas9
dCas9-mediated Multiplexed Transcriptional Modulation Ophir Shalem et al., 2015,
Nature Reviews Genetics
Knockout, knockdown and activation screens are
complementary methods for forward and reverse genetic study
which can be multiplexed by dCas9 fusion protein based
genetic screening. This strategy has also extended screening
opportunities beyond coding genes.

Experimental parameters of recent Cas9-mediated large scale genetic screens
Cas9 delivery Cas9
protein
sgRNA
library size
bp
Number of
targeted genes
Coverage
(sgRNAs per
gene)
Cell lines Species Ref.
Clonal isolation of
stably integrated cells
Cas9 nuclease 73,151 7,114 10 KBM7; HL60 Human Wang et al., 2014
Delivery with the
sgRNA library
Cas9 nuclease 64,751 18,080 3 or 4 on average A375;
HUES62
Human Shalem, et al., 2014
Clonal isolation of
Cas9 nuclease 87,897 19,150 4 on average mESC Mouse Koike-Yusa et al.,
2014
Clonal isolation of
Cas9 nuclease 873 291 3 HeLa Human Zhou, Y. et al., 2014
Polyclonal selected cell
population
dCas9
repression
complex
206,421 15,977 10 per TSS K562 Human Gilbert et al., 2014
population
dCas9
repression
complex
198,810 15,977 10 per TSS K562 Human Gilbert et al., 2014
population
dCas9
repression
complex
70,290 23,430 3 per TSS A375 Human Konermann et al.,
2015

The Mutagenic Chain Reaction: A method for converting heterozygous
mutation to homozygous mutations
Scheme outlining the mutagenic chain reaction (MCR) A to F Valentino et al., 2015

Experimental demonstration of MCR in Drosophila
11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 30Valentino et al., 2015

Gene Drive: stimulating biased inheritance of a desired gene
MCR can be used to propagate a genetic modification rapidly through generations.
It might be used to eradicate a population of disease-carrying mosquitoes.
11/8/2015 Department of Plant Biotechnology, GKVK, UAS, Bengaluru 31Heidi Ledford, 2015
CRISPR-Cas9 gene drive approach to invasive species control would be
based on a laboratory strain with a deleterious trait (e.g., distorted sex
ratio, reduced fertility, chemical sensitivity) being mass-reared and
released into the field in sufficient numbers for the engineered mutation to
spread and control the target population within a desired time frame.

DNAi-Targeted DNA degradation
 Once an engineered organism completes its task, it
is useful to degrade the associated DNA to reduce
environmental release and protect intellectual
property.
 Here is a genetically encoded device (DNAi) that
responds to a transcriptional input and degrades
user-defined DNA.
 This enables engineered regions to be obscured
when the cell enters a new environment.
 DNAi is based on type-IE CRISPR biochemistry
and a synthetic CRISPR array defines the DNA
target.
 When the genome is targeted, this causes cell
death, reducing viable cells by a factor of 10^8
Brian J. et al., 2015
 Once an engineered organism completes its task, it
is useful to degrade the associated DNA to reduce
environmental release and protect intellectual
property.
 Here is a genetically encoded device (DNAi) that
responds to a transcriptional input and degrades
user-defined DNA.
 This enables engineered regions to be obscured
when the cell enters a new environment.
 DNAi is based on type-IE CRISPR biochemistry
and a synthetic CRISPR array defines the DNA
target.
 When the genome is targeted, this causes cell
death, reducing viable cells by a factor of 10^8
A schematic of the device (dashed box)
control

CRISPR for Farm
Can be used to create high degree of genetic variability at precise locus in the
genome of the crop plants.
Potential tool for multiplexed reverse and forward genetic study.
Precise transgene integration at specific loci.
Developing biotic and abiotic resistant traits in crop plants.
Potential tool for developing virus resistant crop varieties.
Can be used to eradicate unwanted species like herbicide resistant weeds, insect
pest, pathogen through the use of MCR.
Potential tool for improving polyploid crops like potato and wheat.

Disease symptoms of wild-type (WT) and tamlo-aabbdd mutant plants 7 d after inoculation in planta
Micrographs of microcolony formation of Bgt on the surfaces of leaves

Transgenic N. benthamiana and
Arabidopsis plants develop resistance to
beet severe curly top virus (BSCTV)
Cas9 gene
gRNA gene targeting viral DNA

Examples of crops modified with CRISPR technology
CROPS DESCRIPTION REFERNCES
Corn Targeted mutagenesis Liang et al. 2014
Rice Targeted mutagenesis Belhaj et al. 2013
Sorghum Targeted gene modification Jiang et al. 2013b
Sweet orange Targeted genome editing Jia and Wang 2014
Tobacco Targeted mutagenesis Belhaj et al. 2013
Wheat Targeted mutagenesis Upadhyay et al. 2013, Yanpeng et
al. 2014
Potato
Soybean
Targeted mutagenesis
Gene editing
Shaohui et al., 2015
Yupeng et al., 2015
Harrison et al., 2014

Some pitfalls of this technology
Proper selection of gRNA
Use dCas9 version of Cas9 protein
Make sure that there is no mismatch within the seed sequences(first 12 nt adjacent
to PAM)
Use smaller gRNA of 17 nt instead of 20 nt
Sequence the organism first you want to work with
Use NHEJ inhibitor in order to boost up HDR
Solutions
Off target indels
Limited choice of PAM sequences

sgRNA designing tools
Optimized CRISPR Design (Feng Zhang's Lab at MIT/BROAD, USA)
sgRNA Scorer (George Church's Lab at Harvard, USA)
sgRNA Designer (BROAD Institute)
ChopChop web tool (George Church's Lab at Harvard, USA)
E-CRISP (Michael Boutros' lab at DKFZ, Germany)
CRISPR Finder (Wellcome Trust Sanger Institute, Hinxton, UK)
RepeatMasker (Institute for Systems Biology) to double check and avoid selecting target sites
with repeated sequences

Case Study
Cell Reports 12, 1668–1677, September 8, 2015

Introduction
Mutations of the Janus family kinase JAK3 gene cause severe combined
immunodeficiency (SCID).
In this case C> T nucleotide substitution in exon 14 of the JAK3 gene replaces a
CGA codon (arginine at 613) with a TGA stop codon.

JAK3 deficiency in humans is characterized by the absence of circulating T cells
and natural killer (NK) cells with normal numbers of poorly functioning B cells
(T–B+NK–).
Allogeneic hematopoietic stem cell transplantation is currently the only
established therapy for SCID; however, delayed immune recovery and graft-
versus-host disease present significant risks.

Contd..
Treatment by retroviral-based gene therapy has been successfully demonstrated
for X-linked SCID but severe adverse effects of insertional mutagenesis have
been observed.
Alternative therapeutic strategy is one in which patient-specific induced
pluripotent stem cells (iPSCs) are derived, and disease-causing mutations are
corrected by gene targeting.
These corrected iPSCs could then be differentiated into hematopoietic
progenitors for transplantation into patients to treat the disease.
The recent development of CRISPR/Cas9 enhanced gene targeting dramatically
advances the practicality of this strategy.

Experimental Plan

Material and method
Patient Information– Eight month old patient was enrolled in
institutional review board-approved study, and parents signed consents, in
accordance with the Declaration of Helsinki. The family history was
negative for immune deficiencies.
Human iPSC Reprogramming and Characterization- iPSC induction
from primary keratinocytes with 1ml of virus supernatant and 1 ml of
human keratinocyte medium containing polybrene.
Generation of CD34+ Cells and T Cells- from iPSC with OP9 Co-
culture following the protocol of Chang et al., 2014.

contd..
T Cell Stimulation - In vitro derived T cells from hiPSCs were stimulated
by incubation with CD3/28 beads.
Flow Cytometry
Vector - Lenti-hDL4-mCherry plasmid
Gene Targeting
Whole-Genome Sequencing and Analysis
 Identification of Potential Off-Target Sites

In Vitro Differentiation of JAK3 C1837T Patient iPSCs Recapitulates
SCID Phenotypes

Correction of the JAK3 Deficiency in SCID Human Induced Pluripotent Stem
Cells by CRISPR/ Cas9-Enhanced Gene Targeting
Results

Cre-recombinase mediated selection marker excision from
corrected iPSC clones
Each lane indicates individual heterozygous corrected
iPSC clones

In Vitro Differentiation of JAK3- Corrected Patient iPSCs Produces NK Cell
and T Cells with Phenotypic and Functional Characteristics of Mature T Cells

Specificity of CRISPR/Cas9-Directed JAK3 Correction
Whole-genome sequencing of the one homozygous and two
heterozygous corrected iPSC lines demonstrated that no mutations
(SNVs nor indels) were introduced into the 1,450 potential off-target
sites .
These results demonstrate the specificity of CRISPR/Cas9-directed
gene correction.

Discussion
These studies describe an approach for the study of human lymphopoiesis
and provide a foundation for gene correction therapy in humans with
immunodeficiencies and other monogenic disorders
These results shows that there are no intrinsic defects in lineage
specification of early hematopoietic progenitors produced in vitro after
correction.
Phase 1 clinical trials will be required to determine whether these early
progenitors are capable of engraftment and sustained reconstitution of
multilineage hematopoiesis in human patients.

Conclusion
CRISPR technology has emerged as a powerful and universal technology
for genome engineering with wide-ranging innovative implications across
biology and medicine.
This technology has proved its potential by being user friendly and has
shown its practicality in ensuring health as well as food security of the
future.
The tool itself do not pose a threat and we hope that the CRISPR
technology will live up to its promise by being used responsibly and
carefully.

Future Prospects
Realizing the promise of gene therapy
Development of personalized therapeutics
Presenting the new face of GE

Eugenics lurk in the shadow of CRISPR
Jennifer Sills, 2015

A view of international regulations
suggests
where in the world a CRISPR baby
could be born.
Heidi Ledford, October 15, 2015

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