CRISPR-CAS 9 – A NEW GENOME EDITING TOOL

Anuradha C1and Lakshmi K2

1
ICAR-National Research Centre for Banana, Trichy, Tamil Nadu, India
2
ICAR-Sugarcane Breeding Institute, Coimbatore, Tamil Nadu, India.

Technologies for making and manipulating DNA have resulted in many advances
over past years and the discovery of DNA double helix, DNA synthesis and polymerase chain
reaction (PCR) has revolutionised molecular biology. The advent of genomic sequencing
technologies and rapid generation of whole-genome sequencing data for large numbers of
organisms has been one of the singular advances of the past two decades. This follows
several attempts over the years to manipulate gene function, including homologous
recombination and RNA interference (RNAi). The development of efficient and reliable ways
to make precise, targeted changes to the genome of living cells is a long-standing goal for
researchers. The ability to engineer genomic DNA in cells and organisms easily and precisely
will have major implications for basic biology, medicine and biotechnology. Recently, a new
tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus
pyogenes is transforming biology by providing a genome engineering tool based on the
principles of Watson-Crick base pairing. Other recent approaches to targeted genome
modification – zinc-finger nucleases [ZFNs] and transcription-activator like effector
nucleases [TALENs] – enable us to generate permanent mutations by introducing double
stranded breaks to activate repair pathways. These approaches are costly and time-consuming
to engineer, limiting their widespread use, particularly for large scale, high-throughput
studies.
CRISPR/Cas9, a genome editing technology
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and
CRISPR- associated (Cas) system is the latest addition to the genome editing toolbox,
offering a simple, rapid, and efficient solution. It is derived from components of a simple
bacterial immune system and is composed of a short noncoding guide RNA (gRNA) that has
two molecular components: a target-specific CRISPR RNA (crRNA) and an auxiliary trans-
activating crRNA (tracrRNA). The gRNA unit guides the Cas9 protein to a specific genomic
locus via base pairing between the crRNA sequence and the target sequence.
History of CRISPR/Cas system
Ishino et al. in 1987 first described a pattern of short, palindromic repeats of DNA
interspersed with short, non-repettive “spacers” of DNA in E. coli. Jansen et al. in 2002,
named the pattern CRISPR, short for “clustered regularly interspaced short palindromic
repeats.” Barrangou et al., 2007 showed that CRISPR, mediated by Cas proteins, provides
bacterial immunity against viruses by matching DNA in spacer sequences with DNA from
viruses. Garneau et al. in 2010 also proved that the CRISPR/Cas system can acquire new
spacers from foreign DNA. In 2012, Jinek et al. developed CRISPR/Cas9, which can be
programmed to recognize and target any DNA sequence. In 2013, Cong et al. showed that
CRISPR/Cas9 can precisely edit DNA in human and mouse cells, and that a single
CRISPR/Cas9 array can be programmed to edit several sites simultaneously. Tan et al. in
2013 used CRISPR/Cas9 in pig, goat, and cattle cells. Ran et al. in 2013 reported a technique
called “double nicking,” which breaks both strands of DNA. In 2014, Fu et al. reported that
truncated guide RNAs can reduce CRISPR/Cas9 to-targeting by 5,000-fold or more. Shalem
et al. In 2014 used CRISPR/Cas9 for genome-scale screening of cancer-related genes in
human cells. Niu et al. (2014) reported the birth of twin monkeys that have been genetically
engineered with CRISPR/Cas9. Hu et al. (2014) used CRISPR/Cas9 to eradicate HIV from
human immune cell lines. In 2015, Wu et al., used CRISPR/Cas9 to correct genetic disease in
mice germ cells. Hilton et al. (2015) created a CRISPR/Cas9-based system that can edit
the epigenome, a set of chemical “switches” that can turn genes on and off. Liang et al.
(2015) reported gene-editing of non-viable human embryos with limited success using
CRISPR/Cas9.
Diversity, ecology and evolution of the CRISPR-Cas systems
The length and sequence of repeats and the length of spacers are well conserved
within a CRISPR locus, but may vary between CRISPRs in the same or different genomes.
Repeat sequences are in the range of 21 bp to 48 bp, and spacers are between 26 bp and 72
bp. The number of spacers within a CRISPR locus vary widely; from a few to several
hundreds. Genomes can have single or multiple CRISPR loci and in some species these loci
can make up a significant part of the chromosome. Not all CRISPR loci have
adjacent cas genes and instead rely on trans-encoded factors. Another feature associated with
CRISPR loci is the presence of a conserved sequence, called leader, located upstream of the
CRISPR with respect to direction of transcription.
The Cas proteins are a highly diverse group. Many are predicted or identified to
interact with nucleic acids; eg. as nucleases, helicases and RNA-binding proteins. The Cas1
and Cas2 proteins are involved in adaptation and are virtually universal for CRISPR-Cas
systems. Other Cas proteins are only associated with certain types of CRISPR-Cas systems.
The diversity of Cas proteins, presence of multiple CRISPR loci and frequent horizontal
transfer of CRISPR-Cas systems make classification a complex task. The most adopted
classification identifies Type I, II and III CRISPR-Cas systems, with each having several
subgroups. Different types of CRISPR-Cas systems can co-exist in a single organism.
Recently, a Type IV system was proposed, which contain several Cascade genes but no
CRISPR, cas1 or cas2. Type IV complex would be guided by protein-DNA interaction, not
by crRNA, and constitutes an innate immune system preset to attack certain sequences.
The Type I systems are defined by the presence of the signature protein Cas3, a
protein with both helicase and DNase domains responsible for degrading the target.
Currently, six subtypes of the Type I system are identified (Type I-A through Type I-F) that
have a variable number of cas genes. Apart from cas1, cas2 and cas3, all Type I systems
encode a Cascade-like complex. Cascade binds crRNA and locates the target, and most
variants are also responsible for processing the crRNA. Cascade also enhances spacer
acquisition in some cases. In the Type I-A system, Cas3 is a part of the Cascade complex.
The Type II CRISPR-Cas systems encode Cas1 and Cas2, the Cas9 signature protein
and sometimes a fourth protein (Csn2 or Cas4). Cas9 assists in adaptation, participates in
crRNA processing and cleaves the target DNA assisted by crRNA and an additional RNA
called tracrRNA. Type II systems have been divided into subtypes II-A and II-B but recently
a third, II-C, has been suggested. The csn2 and cas4 genes, both encoding proteins involved
in adaptation, are present in Type II-A and the Type II-B, respectively, while Type II-C lacks
a fourth gene.
The Type III CRISPR-Cas systems contain the signature protein Cas10 with unclear
function. Most Cas proteins are destined for the Csm (in Type III-A) or Cmr (in Type III-B)
complexes, which are similar to Cascade. Interestingly, while all Type I and II systems are
known to target DNA, Type III systems target DNA and/or RNA. So far, the Type II systems
have been exclusively found in bacteria while the Type I and Type III systems occur both in
bacteria and archaea.
The large number of genomes with detected CRISPRs shows its importance as
defense mechanism. However, the CRISPR-Cas systems are probably mobile genetic
elements that frequently transfers horizontally, which also contributes to their high
prevalence. It has been hypothesized that the Type IV system is similar to an ancestral innate
immune system that gained adaptive ability by associating with a transposon-like element
containing cas1 and cas2. The transposon was domesticated but retained the terminal
inverted repeats that formed the ancestral CRISPR repeats. Repeats were then duplicated and
spacers added by the action of Cas1. The process eventually resulted in the formation of Type
I and III CRISPR-Cas systems. The Type II systems are suggested to have been formed by a
replacement of the Cascade genes by cas9. Cas9 is also linked to mobile genetic elements as
it resembles transposon-encoded proteins
CRISPR – CAS mechanism
CRISPR activity requires the presence of a set of CRISPR-associated (cas) genes,
usually found adjacent to the CRISPR, that code for proteins essential to the immune
response. Since the genome is modified in the process of spacer acquisition, offspring inherit
the protection. New spacers are usually added at one side of the CRISPR, making the
CRISPR a chronological record of the viruses, the cell and its ancestors have encountered.
The CRISPR-Cas mediated defense process is divided into three stages. The first
stage, adaptation, leads to insertion of new spacers in the CRISPR locus. In the second stage,
expression, the system gets ready for action by expressing the cas genes and transcribing the
CRISPR into a long precursor CRISPR RNA (pre-crRNA). The pre-crRNA is subsequently
processed into mature crRNA by Cas proteins and accessory factors. In the third and last
stage, interference, target nucleic acid is recognized and destroyed by the combined action of
crRNA and Cas proteins
The key step in editing an organism’s genome is selective targeting of a specific
sequence of DNA. Two biological macromolecules, the Cas9 protein and guide RNA,
interact to form a complex that can identify target sequences with high selectivity. The Cas9
protein is responsible for locating and cleaving target DNA, both in natural and in artificial
CRISPR/Cas systems. The Cas9 protein has six domains, REC I, REC II, Bridge Helix, PAM
Interacting, HNH and RuvC.
The REC I domain is the largest and is responsible for binding guide RNA. The role
of the REC II domain is not yet well understood. The arginine-rich bridge helix is crucial for
initiating cleavage activity upon binding of target DNA. The PAM-Interacting domain
confers PAM specificity and is therefore responsible for initiating binding to target. The
HNH and RuvC domains are nuclease domains that cut single-stranded DNA. They are
highly homologous to HNH and RuvC domains found in other.
The Cas9 protein remains inactive in the absence of guide RNA. In engineered
CRISPR systems, guide RNA is comprised of a single strand of RNA that forms a T-shape
comprised of one tetraloop and two or three stem loops. The guide RNA is engineered to
have a 5′ end that is complimentary to the target DNA sequence. This artificial guide RNA
binds to the Cas9 protein and, upon binding, induces a conformational change in the protein.
The conformational change converts the inactive protein into its active form. The mechanism
of the conformational change is not completely understood, but Jinek and colleagues
hypothesize that steric interactions or weak binding between protein side chains and RNA
bases may induce the change. Once the Cas9 protein is activated, it stochastically searches for
target DNA by binding with sequences that match its protospacer adjacent motif (PAM)
sequence. A PAM is a two- or three-base sequence located within one nucleotide downstream
of the region complementary to the guide RNA. PAMs have been identified in all CRISPR
systems, and the specific nucleotides that define PAMs are specific to the particular category
of CRISPR system. When the Cas9 protein finds a potential target sequence with the
appropriate PAM, the protein will melt the bases immediately upstream of the PAM and pair
them with the complementary region on the guide RNA. If the complementary region and the
target region pair properly, the RuvC and HNH nuclease domains will cut the target DNA
after the third nucleotide base upstream of the PAM.
CRISPR applications
 CRISPR-Cas systems have been developed for programmable gene regulation. Both
Cascade and a nuclease-deficient Cas9 mutant (dCas9) can be used for gene silencing by
interfering with RNA polymerase binding or elongation. By fusing dCas9 with a
transcriptional activation domain or a repressor, transcriptional activation or repression
can be achieved. By adding multiple activating domains strong induction can be achieved.
Genome-wide application of this approach can be used for screening, as demonstrated by
the identification of genes that allow melanoma cancer cells to escape treatment
 The use of CRISPR-Cas as a direct antimicrobial tool has been studied recently. Artificial
CRISPR arrays have been designed to kill pathogenic bacteria by targeting antibiotic
resistance or virulence genes. This will aim for harmful strains in a bacterial population
and allows non-pathogenic strains to overgrow the pathogens.
 Cas9 has been developed as an antimicrobial agent that can be used to specifically target
antibiotic-resistant and/or highly virulent strains of bacteria.
 The CRISPR-Cas9 technique is used for genome-scale screens. These screens will help in
identification of genes that are involved in certain metabolic or pathogenetic processes by
abolishing gene function and studying the resulting phenotype. Using this approach, genes
that are involved in tumour growth or convey susceptibility towards bacterial toxins could
be identified.
 Gene therapy applications have also been demonstrated by repairing the cftr gene in
cultured cells from human cystic fibrosis patients, by curing dominant cataract disorder
 and Duchenne muscular dystrophy by altering DNA in mouse germ-line cells, and by
curing hereditary tyrosinemia in adult mice.
 Cas9 also holds potential for treatment of viral infections, as demonstrated for HIV and
hepatitis B. Cas9 has been demonstrated to be deliverable by Adeno-associated virus,
greatly facilitating its use in somatic gene therapy.
 It also helps in precise genetic modification of primates by gene editing in embryos. The
finding allows for development of disease models in animals very similar to humans. A
similar approach could be used to alter DNA in human embryos to prevent non-complex
hereditary diseases.