CRISPR

cas-9 Gene Editing

Erina Shan

I. Background on CRISPR cas-9:

Clustered Regularly interspaced short palindromic repeats (CRISPR) are

segments of prokaryotic DNA that contain repetitions of short base sequences.

Each repetition is separated by a spacer DNA, stemming from exposure to viruses

or plasmids. Plasmids are additional genetic information containing DNA. More

specifically, the cas system, or CRISPR associated system is an immune system,

which relates to resistance to plasmids and phages. Cas-9 is a type of protein that

works with CRISPR repeats to inhibit the replication of viral DNA by cutting out

pieces of the DNA and replacing it with either a random sequence or a desired

base pair sequence, which result in a mutation in the viral DNA, demonstrating an

adaptive immunity in select bacteria and archaea. (Wikipedia)

a. How CRISPR cas-9 works in Bacteria

This bacterial defense mechanism has been replicated to create methods of

inhibiting the expression of specific genes or to replacing mutated genes for

sequences that promote proper function. Currently, there are three known types

of CRISPR cas-9 systems. Because of simplicity sake, we will only be focusing on

type II CRISPR cas-9 systems as they only require 3 components. (New England
Labs 2014) Type I and type III are more complex and have fewer recent

advancements.

II. History of CRISPR cas-9

The CRIPSR locus was first discovered in 1993 by Francisco Mojica, and its

function was further explored between 1993 and 2005. In 2005, Alexander

Bolotin of the French National Institute for Agricultural Research identified the

section of the CRISPR locus which coded for the cas-9 nuclease, and that each

photo spacer contained the same sequence of base pairs, thereby identifying the

PAM sequence which was required for target identification and cas-9 binding.

(Bolotin 2005) In 2006, Eugene Koonin of the US National Center for

Biotechnology Information identified that that photo spacers matched the DNA

sequence of the new phage DNA, where it was previously believed that cas-9

comprised of a novel DNA repair system. (Koonin 2006) 2008 through 2010, the

transcription of spacers into RNA and the interaction with DNA was better

understood by biologists John van der Oost of University of Wageningen and

Sylvain Moineau of the University of Laval. (Oost and Moineau 2008) 2010

through 2012, the mode of action for cas9 was further researched and only in

2013, was Feng Zhang of the Broad Institute of MIT and Harvard able to harness

CRISPR cas-9 for genome editing. Zhang and his team successfully adapted
genome editing in human and mouse cells initiating a new period of rapid

experimentation with CRISPR cas-9. (Broad Institute 2013)

III. Advantages of CRISPR cas-9 over existing gene editing tools

CRISPR cas-9 has substantial benefits to other gene editing techniques such

as zinc finger nuclease (ZFN) and transcription activator-like effector nucleases

(TALEN) in a number of ways. For one, the target sequence can easily be adapted

to any genomic sequence by changing the 20-bp photospacer of the guide RNA

with the cas-9 protein component remaining unchanged. Additionally, the

protein-guided DNA nuclease for ZFN and TALEN is much more difficult to

engineer, and does not have the same ability to make simultaneous genomic

modifications at multiple independent sites. (Ozbiosciences 2013) There has been

a lot of research focusing around the efficiencies of cas-9 versus existing methods,

most which have deemed CRISPR cas-9 systems the more accurate and targeted

method of cleaving DNA. Where ZFNs and TALENs have efficiencies ranging from

1% to 50%, cas-9 has efficiencies much higher at above 70% in many systems.

(Hwang et al. 2013) This has been tested in zebra fish, plants and even pluripotent

stem cells.

In addition to these conveniences of the CRISPR cas-9 system, this gene

editing tool provides huge potential for gene editing in human genetic disease.
The idea that one can administer CRISPR cas-9 containing the correct genetic

sequence to removing and replacing mutations could be the answer to all future

treatments, both genetic and viral. All living systems have DNA. Therefore, the

ability to edit and modify it, seems to hold the key to curing many incurable

diseases. (Chemistry World 2016) Additionally, the injection of CRISPR cas-9 into a

zygote or early stage embryo allows for the modification of the genome in cells of

all organisms, resulting in permanent changes which are passed down from

generation to generation, offering the possibility of eliminating genetic disease

from an entire family. (Savic and Schwank 2016)

IV. How CRISPR cas-9 functions in nature

The way CRISPR edits genes in bacteria is through the cutting of CRISPR

DNA into small pieces. Through transcription CRISPR RNA or crRNA is formed.

crRNA combines with trans-activating or tracrRNA to form a chimeric guide RNA

which bonds with the cas9 nuclease to form a unit, which can edit and cut genes.

(Savic and Schwank 2016) The guide RNA is the key to CRISPR cas 9s gene editing

abilities as the gene sequence in the spacer DNA, which corresponds to a section

in the viral DNA. The series of base pairs in the spacer DNA is what allows for viral

immunity. (Sander and Joung 2014) When a guide RNA finds a matching base pair,

CRISPR cas9 binds to the PAM site and unzips the viral DNA. The chimeric RNA
(fusion of crRNA/tracrRNA) binds to the viral DNA and begins the cutting process

of the DNA, breaking the sequence into two pieces. This is called a double-strand

break (DSB). This double strand break can then be filled by a random sequence of

nucleotides, resulting in a mutation in the viral gene.

V. Three variants of CRISPR cas-9

To date, only three variants of cas-9 have been used in gene editing

protocols. These include the wild-type cas-9, mutant cas-9 also known as

Cas9D10A, and a nuclease deficient cas-9. Each of these variations have

advantages and disadvantages, which will be discussed in the sections ahead.

a. Wild- type CRISPR cas-9 used in targeted gene editing

The first is the wild-type cas-9. The difference between the bacterial CRISPR

cas-9 and engineered or wild type CRISPR cas-9 used in targeted gene editing is

the shorter tracrRNA sequence and attached to crRNA via fusion as opposed to a

hybridization of two RNA strands. The hybridization process is the simple base

pairing of crRNA and tracrRNA, whereas fusion consists of creating a chimeric RNA

through trans-splicing which the simple joining of RNA or DNA sequences by the

sugar backbone, a process used in eukaryotes in exon ligating. (Wikipedia) These

differences between the natural occurring and engineered CRISPR cas-9 is shown

in Figure 1. The resulting strand of RNA, which binds to the pathogen DNA is
called the guide RNA (gRNA) and is known as a chimeric variant. The benefit for

this modification is the greater resistance to environmental disturbances as

covalent bonds are much stronger than hydrogen bonds. Additionally, engineered

CRISPR cas-9 differs from naturally occurring CRISPR cas-9 by allowing for at least

two different pathways that operate in nearly all cell types, one is non-

homologous end-joining (NHEJ) and the other is homology-directed repair (HDR).

NHEJ is the insertion/deletion of base pairs that disrupt the original function of

the DNA chain, inhibiting its desired effect. HDR is the insertion of desired

sequences through recombination of the target locus with exogenously supplied

DNA. This ability to modify genes has huge implications in inhibiting gene altering

diseases and modifying the biological foundation of humans that is DNA.

Figure 1: a) Natural occurring CRISPR cas-9 b) Engineered CRISPR cas-9

 [image taken from Sander and Joung 2014]

b. Mutant Form cas-9D10A and cas-9H840A

The mutant form differs from the wild-type by only expressing nickase

activity. Nickase is an enzyme that cuts DNA in only one of the two strands

leading to a single strand break. There are two types, D10A and H840A. D10A

creates a single break on the side of the gRNA, whereas H840A cleaves the DNA at

the strand, which is not bonded to the gRNA. In addition, instead of being able to

either delete or insert base pairs through to deactivate the gene through NHEJ,

the mutant form of cas-9 is only able to conduct modifications through the HDR

pathway, resulting in reduced indel mutations (or mutations stemming from

insertions and deletions). This is particularly useful when nicks or cuts need to be

made in adjacent base pairs, in which case a paired cas-9 nickase system allows

for the cutting of two regions simultaneously and is able to attach them together

via the HDR pathway. (Cong et al. 2013)

c. Nuclease deficient cas-9 (dcas-9)

Nuclease deficient cas-9 is a cas-9 protein which is unable to cleave the

DNA it binds to. However, it can still interact with adjacent DNA through various

effector domains that can activate, repress or highlight a specific section of DNA.

The guide RNA is therefore an adjacent sequence to the targeted region and can
therefore still interact without having to create any breaks in DNA. The

interaction of the effector domains allows for gene silencing, activation or

inducing fluorescence used as a visual tool. Recent advances in each of these

CRISPR cas-9 systems will be discussed as well. (Qi et al. 2013)

VI. Reducing off-target mutations with CRISPR cas-9

Even though CRISPR cas-9 sounds miraculous and incredible, it still has its

faults. Much of these are focused around off-target mutations, which occur when

cas-9 binds to a sequence of DNA that is off by 1-5 base pairs in the gRNA and

next to a PAM sequence. (Hsu et al. 2o13) Sometimes off-target mutations will

even occur for up to 1 base pair difference in the PAM sequence. Detection of

these off-target mutations is extremely difficult. Therefore, a lot of research

conducted now is focused around minimizing these mutations.

One method in which off-target mutations are minimized is through

truncation of the guide RNA at the 5 end. Truncated guide RNAs of less than 20

nucleotides have a 5,000-fold decrease in undesired mutagenesis. This technique

can also be combined with other off-target mutation prevention strategies, such

as nickases. (Fu et al. 2014) Another method by which off-target mutations were

prevented was by using mutated cas-9D10A and two guide RNA which matched

adjacent bases. This method uses paired nickases, where two single strand breaks
are made individually instead of a single double strand break. Mutated cas-9D10A

is much more specific and can serve the same function as wild type cas-9. The two

adjacent guide RNA prevent off-target mutations by requiring more correct base

pair matches in order for cleaving to occur. (Ran et al. 2013) This method is highly

effective by inducing chromosomal deletions without any translocation side

effects. (Cho et al. 2013)

VII. CRISPR cas-9 in curing genetic disease

Recent research has focused around applying CRISPR cas-9 in eukaryotic

cells and curing hereditary diseases. The experiments surrounding CRISPR cas-9

are both in vivo (in living systems) or ex vivo (outside of living systems) and tests

have been conducted mostly in animals such as mice and zebra fish.

a. Type I Tyrosinemia

The first successfully corrected disease was conducted on Type I

Tyrosinemia, which is caused by the deficiency of an enzyme called

fumarylacetoacetate hydrolase (FAH), which leads to cytotoxic metabolite

accumulation, specifically Tyrosine, leading to death of hepatocytes or liver cells.

Yin et al injected a viral vector for targeted editing of the inhibited gene,

correcting the mutations that prevent the correct expression of FAH. Yin was able

to successfully improve symptoms of Tyrosinemia in mice.

 Tyrosinemia often induces weight loss due to inhibited metabolism.

Therefore, when one subset of mice with Tyrosinemia were injected with CRISPR

cas-9, they did not experience any weight loss compared to weight loss of less

than 20% their original mass of untreated mice after 30 days. Additionally, there

was a significant decrease in liver damage and indication that FAH was being

produced through serum markers like aspartate aminotransferase (AST) and

alanine aminotransferase (ALT) enzymes used in later stages of metabolized

tyrosine. (Yin et al. 2014)

b. Treating Hepatitis B

Another study using CRISPR cas-9 in curing disease is Lin et al study which

suggests the use of cas-9 in curing hepatitis B virus (HBV). HBV is a chronic disease

causing liver cirrhosis and hepatocellular carcinoma. Lin showed that the

combination of CRISPR cas-9 with antiviral therapies reduced the the serum

hepatitis B surface antigen. Experiments in duck also indicated the potential of

complete HBV eradication through genome editing. (Lin et al. 2014) Even though

this is promising research, existing pharmaceutical companies such as Gilead have

developed nucleotide analog Sovaldi as a cure for HBV. CRISPR cas-9 may be a

superfluous method of curing this disease unless it significantly undercuts it.

c. Gene editing of patient-derived stem cells for beta-thalassemia

 CRISPR cas-9 can be used in induced pluripotent stem cells (iPSC), which are

stem cells derived from differentiated cells. These cells can be differentiated to

any desired cell type in the body. One of the first times gene editing was

employed in iPSCs was in tackling the blood disorder beta-thalassemia, which is

caused by a mutation of the hemoglobin beta gene. Beta-thalassemia is a blood

condition, where patients fall extremely anemic and as a result, experience

deformation of the face and body. Beta thalassemia is caused by a single

nucleotide exchange, specifically the replacement of a Guanine for an Adenine,

forming a protein much shorter than the normal length of 146 amino acids,

therefore caused by a nonsense mutation at the junction between an exon and

intron. Using CRISPR cas-9, this nonsense mutation can be targeted, and

successfully was ex vivo. Xie et al created iPSC from fibroblasts of a patient

homozygous of beta-thalassemia and transfected them with CRISPR cas-9 using

the HDR pathway to correct the mutation. These iPSC were then allowed to

differentiate into regular red blood cells and could be used for future transplants.

(Xie 2014)

d. Gene editing of patient-derived stem cells for Cystic Fibrosis

Another study aimed at curing hereditary disease was lead by Clevers et al

which used gene editing in primary somatic stem cells, specifically the intestinal
organoids to correct an allele which causes cystic fibrosis. Cystic Fibrosis is a

mutation that effects the protein channels in the cell membrane and inhibit

chlorine transfer into the cell. The study focuses on the F508 mutation, which is a

deletion of three nucleotides that comprise the codon for phenylalanine at

position 508. It is one of the more common mutations in cystic fibrosis. This study,

conducted in 2013, was successful in correcting the F508 mutation. It also had

very few off-target mutations, and concluded unlikely adoption into adult stem

cell therapy with much greater potential in iPSC. (Schwank 2013) Additionally,

Vertex pharmaceuticals is currently working on a treatment in combination with

one of their approved drugs, Klaydeco, which targets a different mutation.

e. Using CRISPR cas-9 to better tackle HIV

Another way CRISPR cas-9 can be used is in combating latent human

immunodeficiency virus (HIV). Because the latent virus resides in memory cells,

they are not destroyed and therefore persist in the human body even after

antiretroviral drugs. There are two ways in which CRISPR cas-9 can combat these

viruses. One way is to target the viral DNA in T-cells, introducing a mutation so

that it cannot further replicate. (Liao et al. 2015) The second way is to modify the

chemokine receptor (CCR5) needed for HIV T-cell infection. The efficiency of this

process is approximately 30%. (Mandal et al. 2014)

 VIII. Looking Forward

Despite this recent research, there are many obstacles CRISPR cas-9 has yet to

overcome. Like many new treatments, there is a question of safety and efficacy.

However, the nature of CRISPR cas-9 is so fundamentally different to the

traditional small molecule treatments that people and policy makers may be

hesitant to adopt it into clinical practice without thoroughly examining their

effects. Additionally, the efficiency of CRISPR cas-9 has to be improved upon

before clinical adoption. (Cox et al. 2015) Even though there has been a gene

therapy approved in Europe for lipoprotein lipase deficiency (LPLD), a rare fat-

processing disorder, which causes acute pancreatitis, the US is still working

towards commercializing CRISPR cas-9 gene editing techniques. (Bloomberg 2012)

However, progress is being made. The UK recently received approval to conduct

CRISPR cas-9 gene editing on human embryos and Chinese scientists have

successfully used gene editing to introduce HIV resistance, having successfully

edited embryos for the second time. (Nature 2016) Even though this is a huge

leap toward modifying genes before they are even expressed, it also raises ethical

questions on gene modification. With the ability to alter genes in embryos, can

families eventually decide how children will look in the future, and can embryo

gene editing commercialized? These are the key questions to be asked moving
forward. Until then, CRISPR cas-9s seemingly unlimited potential will be further

explored by industry and academia, only growing further in its potential

applications.

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