CRISPR

12/20/2017 CRISPR - Wikipedia
CRISPR
CRISPR/Cas9
CRISPR (/krspr/) is a family of DNA sequences in bacteria. The sequences

contain snippets of DNA from viruses that have attacked the bacterium. These
snippets are used by the bacterium to detect and destroy DNA from similar
viruses during subsequent attacks. These sequences play a key role in a
bacterial defence system,[2] and form the basis of a technology known as
CRISPR/Cas9 that effectively and specifically changes genes within
organisms.[3]
The CRISPR/Cas system is a prokaryotic immune system that confers

resistance to foreign genetic elements such as those present within plasmids
and phages[4][5][6] that provides a form of acquired immunity. RNA harboring
the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut
exogenous DNA. Other RNA-guided Cas proteins cut foreign RNA.[7] CRISPRs
are found in approximately 40% of sequenced bacterial genomes and 90% of
sequenced archaea.[8][note 1]
CRISPR is an abbreviation of Clustered Regularly Interspaced Short
https://en.wikipedia.org/wiki/CRISPR 1/88
Diagram of the CRISPR prokaryotic antiviral defense mechanism.[1]
PalindromicRepeats.[9] The name was minted at a time when the origin and
use of the interspacing subsequences were not known. At that time the
CRISPRs were described as segments of prokaryotic DNA containing short,
repetitive base sequences. In a palindromic repeat, the sequence of nucleotides
is the same in both directions. Each repetition is followed by short segments of
spacer DNA from previous exposures to foreign DNA (e.g., a virus or
plasmid).[10] Small clusters of cas (CRISPR-associated system) genes are
located next to CRISPR sequences.
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified

to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic
guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired
location, allowing existing genes to be removed and/or new ones

added.[11][12][13] The Cas9-gRNA complex corresponds with the CAS III crRNA
complex in the above diagram.
CRISPR/Cas genome editing techniques have many potential applications,

including medicine and crop seed enhancement. The use of CRISPR/Cas9-
gRNA complex for genome editing[14][15] was the AAAS's choice for
breakthrough of the year in 2015.[16] Bioethical concerns have been raised
about the prospect of using CRISPR for germline editing.[17]
Contents
History
Repeated sequences
CRISPR-associated systems
Cas9
Cpf1
Predecessors
Locus structure
Repeats and spacers
Cas genes and CRISPR subtypes
Mechanism
Spacer acquisition
Biogenesis
Interference
Evolution
Coevolution
Rates
Identication
Use by phages
Applications
Genome engineering
Knockdown/activation
RNA editing
Disease models
Gene drive
Biomedicine
Gene function
In vitro genetic depletion
Patents and commercialization
Society and culture
Human germline modication
Policy barriers to genetic engineering
Recognition
Alternative cutters
See also
Notes
References
Further reading
External links
Cascade (CRISPR-associated
History complex for antiviral defense)
Structure of crRNA-guided E. coli Cascade

complex (Cas, blue) bound to single-stranded DNA
(orange).
Identiers
Organism Escherichia coli (https://www.ncbi.nlm.
nih.gov/Taxonomy/Browser/wwwtax.cg
i?id=&rn=1)
Symbol ?
PDB 4QYZ (http://www.rcsb.org/pdb/search/
smart.do?smartSearchSubtype_1=Stru
ctureIdQuery&structureIdList_1=4QY
Z)
The discovery of clustered DNA repeats began independently in three parts of

the world. One of the first discoveries was in 1987 at Osaka University in
Japan. Researcher Yoshizumi Ishino and colleagues published their findings
on the sequence of a gene called "iap" and its relation to E. coli. Technological
advances in the 1990's allowed them to continue their research and speed up
their sequencing with a technique called metagenomics. They were able to
collect seawater or soil samples and sequence the DNA in the sample.
Repeated sequences
The first description of what would later be called CRISPR was from Osaka
University researcher Yoshizumi Ishino in 1987, who accidentally cloned part
of a CRISPR together with the iap gene, the target of interest. The organization
of the repeats was unusual because repeated sequences are typically arranged
consecutively along DNA. The function of the interrupted clustered repeats
was not known at the time.[18][19]
In 1993 researchers of Mycobacterium tuberculosis in the Netherlands

published two articles about a cluster of interrupted direct repeats (DR) in this
bacterium. These researchers recognized the diversity of the DR-intervening
sequences among different strains of M.tuberculosis[20] and used this property
to design a typing method that was named spoligotyping, which is still in use
today.[21][22]
At the same time, repeats were observed in the archaeal organisms of

Haloferax and Haloarcula species, and their function was studied by
Francisco Mojica at the University of Alicante in Spain. Although his
hypothesis turned out to be wrong, Mojica surmised at the time that the
clustered repeats had a role in correctly segregating replicated DNA into
daughter cells during cell division because plasmids and chromosomes with
identical repeat arrays could not coexist in Haloferaxvolcanii. Transcription
of the interrupted repeats was also noted for the first time.[22][23] By 2000,
Mojica's group had identified interrupted repeats in 20 species of microbes.[24]
In 2001, Mojica and Ruud Jansen, who was searching for additional
interrupted repeats, proposed the acronym CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeats) to alleviate the confusion stemming
from the numerous acronyms used to describe the sequences in the scientific
literature.[23][25]
CRISPR-associated systems
A major addition to the understanding of CRISPR came with Jansen's
observation that the prokaryote repeat cluster was accompanied by a set of
homologous genes that make up CRISPR-associated systems or cas genes.
Four cas genes (cas 1 to 4) were initially recognized. The Cas proteins showed
helicase and nuclease motifs, suggesting a role in the dynamic structure of the
CRISPR loci.[26] In this publication the acronym CRISPR was coined as the
universal name of this pattern. However, the CRISPR function remained
enigmatic.
In 2005, three independent research groups showed that some CRISPR

spacers are derived from phage DNA and extrachromosomal DNA such as
plasmids.[27][28][29] In effect, the spacers are fragments of DNA gathered from
viruses that previously tried to attack the cell. The source of the spacers was a
sign that the CRISPR/cas system could have a role in adaptive immunity in
bacteria.[1][30] All three studies proposing this idea were initially rejected by
high-profile journals, but eventually appeared in other journals.[31]
The first publication[28] proposing a

role of CRISPR-Cas in microbial
immunity, by Mojica's group,
predicted a role for the RNA
transcript of spacers on target Simplied diagram of a CRISPR
locus. The three major components
recognition in a mechanism that
of a CRISPR locus are shown: cas
could be analogous to the RNA genes, a leader sequence, and a
interference system used by repeat-spacer array. Repeats are
eukaryotic cells. Therefore, as Ian shown as gray boxes and spacers
Wilmut became world-famous for are colored bars. The arrangement
being the scientist who cloned of the three components is not
always as shown.[1][10] In addition,
Dolly,[32][33] Koonin and colleagues
several CRISPRs with similar
extended this RNA interference
sequences can be present in a
hypothesis by proposing single genome, only one of which is
mechanisms of action for the associated with cas genes.[8]
different CRISPR-Cas subtypes
according to the predicted function
of their proteins.[34] Others hypothesized that CRISPR sequences directed Cas
enzymes to degrade viral DNA.[19][29]
Experimental work by several groups revealed the basic mechanisms of

CRISPR-Cas immunity. In 2007 the first experimental evidence that CRISPR
was an adaptive immune system was published.[19] A CRISPR region in
Streptococcus thermophilus acquired spacers from the DNA of an infecting
bacteriophage. The researchers manipulated the resistance of S.thermophilus
to phage by adding and deleting spacers whose sequence matched those found
in the tested phages.[35][36] In 2008, Brouns and colleagues identified a
complex of Cas protein that in E.coli cut the CRISPR RNA within the repeats
into spacer-containing RNA molecules, which remained bound to the protein
complex. That year Marraffini and Sontheimer showed that a CRISPR
sequence of S.epidermidis targeted DNA and not RNA to prevent conjugation.
This finding was at odds with the proposed RNA-interference-like mechanism
of CRISPR-Cas immunity, although a CRISPR-Cas system that targets foreign
RNA was later found in Pyrococcusfuriosus.[19][35] A 2010 study showed that

CRISPR-Cas cuts both strands of phage and plasmid DNA in S.
thermophilus.[37]
Cas9
Researchers studied a simpler CRISPR system from Streptococcus pyogenes
that relies on the protein Cas9. The Cas9 endonuclease is a four-component
system that includes two small RNA molecules named CRISPR RNA (crRNA)
and trans-activating CRISPR RNA (tracrRNA).[38] Jennifer Doudna and
Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a more
manageable two-component system by fusing the two RNA molecules into a
"single-guide RNA" that, when combined with Cas9, could find and cut the
DNA target specified by the guide RNA. By manipulating the nucleotide
sequence of the guide RNA, the artificial Cas9 system could be programmed to
target any DNA sequence for cleavage.[39] Another group of collaborators
comprising iknys together with Gasinas, Barrangou and Horvath showed
that Cas9 from the S.thermophilus CRISPR system can also be reprogrammed
to target a site of their choosing by changing the sequence of its crRNA. These
advances fueled efforts to edit genomes with the modified CRISPR-Cas9
system.[22]
Feng Zhang's and George Church's groups simultaneously described genome

editing in human cell cultures using CRISPR-Cas9 for the first time.[19][40][41] It
has since been used in a wide range of organisms, including baker's yeast
(Saccharomyces cerevisiae),[42][43][44] zebrafish (D. rerio),[45] fruit flies
(Drosophilamelanogaster),[46] nematodes (C.elegans),[47] plants,[48] mice,[49]
monkeys[50] and human embryos.[51]
CRISPR has been modified to make programmable transcription factors that

allow scientists to target and activate or silence specific genes.[52]
The CRIPSR/Cas9 system has shown to make effective gene edits in Human
tripronuclear zygotes first described in a 2015 paper by Chinese scientists P.
Liang and Y. Xu. The system made a successful cleavage of mutant Beta-
Hemoglobin (HBB) in 28 out of 54 embryos. 4 out of the 28 embryos were

successfully recombined using a donor template given by the scientists. The
scientists showed that during DNA recombination of the cleaved strand, the
homologous endogenous sequence HBD competes with the exogenous donor
template. DNA repair in human embryos is much more complicated and
particular than in derived stem cells.[53]
Cpf1
In 2015, the nuclease Cpf1 was discovered in the CRISPR/Cpf1 system of the
bacterium Francisella novicida.[54][55] Cpf1 showed several key differences
from Cas9 including: causing a 'staggered' cut in double stranded DNA as
opposed to the 'blunt' cut produced by Cas9, relying on a 'T rich' PAM
(providing alternate targeting sites to Cas9) and requiring only a CRISPR RNA
(crRNA) for successful targeting. By contrast Cas9 requires both crRNA and a
transactivating crRNA (tracrRNA).
These differences may give Cpf1 some advantages over Cas9. For example,
Cpf1's small crRNAs are ideal for multiplexed genome editing, as more of them
can be packaged in one vector than can Cas9's sgRNAs. As well, the sticky 5'
overhangs left by Cpf1 can be used for DNA assembly that is much more target-
specific than traditional Restriction Enzyme cloning.[56] Finally, Cpf1 cleaves
DNA 18-23 bp downstream from the PAM site. This means there is no
disruption to the recognition sequence after repair, and so Cpf1 enables
multiple rounds of DNA cleavage. By contrast, since Cas9 cuts only 3 bp
upstream of the PAM site, the NHEJ pathway results in indel mutations which
destroy the recognition sequence, thereby preventing further rounds of cutting.
In theory, repeated rounds of DNA cleavage should cause an increased
opportunity for the desired genomic editing to occur.[57]
Predecessors
In the early 2000s, researchers developed zinc finger nucleases, synthetic

proteins whose DNA-binding domains enable them to create double-stranded
breaks in DNA at specific points. In 2010, synthetic nucleases called
transcription activator-like effector nucleases (TALENs) provided an easier
way to target a double-stranded break to a specific location on the DNA strand.
Both zinc finger nucleases and TALENs require the creation of a custom
protein for each targeted DNA sequence, which is a more difficult and time-
consuming process than that for guide RNAs. CRISPRs are much easier to
design because the process requires making only a short RNA sequence.[58]
Locusstructure
Repeats and spacers

The CRISPR array comprises an AT-rich leader sequence followed by short
repeats that are separated by unique spacers.[59] CRISPR repeats typically
range in size from 28 to 37 base pairs (bps), though there can be as few as 23
bp and as many as 55 bp.[60] Some show dyad symmetry, implying the
formation of a secondary structure such as a stem-loop ('hairpin') in the RNA,
while others are predicted to be unstructured. The size of spacers in different
CRISPR arrays is typically 32 to 38 bp (range 21 to 72 bp).[60] New spacers can
appear rapidly as part of the immune response to phage infection.[61] There are
usually fewer than 50 units of the repeat-spacer sequence in a CRISPR
array.[60]
Cas genes and CRISPR subtypes

Small clusters of cas genes are often located next to CRISPR repeat-spacer
arrays. Collectively there are 93 cas genes that are grouped into 35 families
based on sequence similarity of the encoded proteins. 11 of the 35 families form
the cas core, which includes the protein families Cas1 through Cas9. A
complete CRISPR-Cas locus has at least one gene belonging to the cas core.[62]
CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of
multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a
single large Cas protein for the same purpose. Class 1 is divided into types I,
III, and IV; class 2 is divided into types II, V, and VI.[63] The 6 system types are
divided into 19 subtypes.[64] Each type and most subtypes are characterized by
a "signature gene" found almost exclusively in the category. Classification is
also based on the complement of cas genes that are present. Most CRISPR-Cas
systems have a Cas1 protein. The phylogeny of Cas1 proteins generally agrees
with the classification system.[62] Many organisms contain multiple CRISPR-
Cas systems suggesting that they are compatible and may share
components.[65][66] The sporadic distribution of the CRISPR/Cas subtypes
suggests that the CRISPR/Cas system is subject to horizontal gene transfer
during microbial evolution.
Signature genes and their putative functions for the major and minor
CRISPR-cas types.
Cas Signature
Class Function Reference
type protein
Single-stranded DNA nuclease
1 I Cas3 (HD domain) and ATP- [67][68]
dependent helicase
Cas8a,
IA Subunit of the interference
Cas5
module. Important in targeting [62]
IB Cas8b of invading DNA by recognizing
the PAM sequence
IC Cas8c
ID Cas10d contains a domain homologous
to the palm domain of nucleic [69][70]
Cse1, acid polymerases and
IE
Cse2 nucleotide cyclases
Csy1,
IF Csy2, Not determined [62]
Csy3
IU GSU0054 [62]
III Cas10 Homolog of Cas10d and Cse1 [70]
IIIA Csm2 Not Determined [62]
IIIB Cmr5 Not Determined [62]
Cas10 or [62]
IIIC
Csx11
IIID Csx10 [62]
IV Csf1
IVA
IVB
Nucleases RuvC and HNH
together produce DSBs, and
separately can produce single- [71][72]
2 II Cas9
strand breaks. Ensures the
acquisition of functional
spacers during adaptation.
IIA Csn2 Ring-shaped DNA-binding [73]

protein. Involved in primed
adaptation in Type II CRISPR
system.
IIB Cas4 Not Determined
Characterized by the absence [74]
IIC
of either Csn2 or Cas4
Cpf1,
V C2c1, Nuclease RuvC. Lacks HNH. [63]
C2c3
VI C2c2 [63]
Mechanism
CRISPR-Cas immunity is a natural
process of bacteria and archaea.
CRISPR-Cas prevents
bacteriophage infection,
conjugation and natural
transformation by degrading
foreign nucleic acids that enter the
cell.[35]
The CRISPR genetic locus provides
bacteria with a defense mechanism
Spacer acquisition to protect them from repeated
When a microbe is invaded by a phage infections.
virus, the first stage of the immune
response is to capture viral DNA
and insert it into a CRISPR locus in the form of a spacer. Cas1 and Cas2 are
found in all three types of CRISPR-Cas immune systems, which indicates that
they are involved in spacer acquisition. Mutation studies confirmed this
hypothesis, showing that removal of cas1 or cas2 stopped spacer acquisition,
without affecting CRISPR immune response.[75][76][77][78][79]
Multiple Cas1 proteins have been

characterised and their structures
resolved.[80][81][82] Cas1 proteins
have diverse amino acid sequences.
However, their crystal structures
are similar and all purified Cas1
proteins are metal-dependent
nucleases/integrases that bind to
DNA in a sequence-independent
manner.[65] Representative Cas2
proteins have been characterised
Transcripts of the CRISPR Genetic
and possess either (single strand)
Locus and Maturation of pre-crRNA
ssRNA-[83] or (double strand)
dsDNA-[84][85] specific
endoribonuclease activity.
In the I-E system of E. coli Cas1

and Cas2 form a complex where a
Cas2 dimer bridges two Cas1
dimers.[86] In this complex Cas2
performs a non-enzymatic
scaffolding role,[86] binding double- 3D Structure of the CRISPR-Cas9
Interference Complex
stranded fragments of invading
DNA, while Cas1 binds the single-
stranded flanks of the DNA and catalyses their integration into CRISPR
arrays.[87][88][89] New spacers are always added at the beginning of the CRISPR
next to the leader sequence creating a chronological record of viral
infections.[90] In E. Coli a histone like protein called integration host factor
(IHF), which binds to the leader sequence, is responsible for the accuracy of
this integration.[91]
Protospacer adjacent motifs
Bioinformatic analysis of regions of

phage genomes that were excised
as spacers (termed protospacers)
revealed that they were not
randomly selected but instead were
found adjacent to short (3 5 bp)
DNA sequences termed
protospacer adjacent motifs (PAM).
Analysis of CRISPR-Cas systems
showed PAMs to be important for
CRISPR-Cas9 as a Molecular Tool
type I and type II, but not type III Introduces Targeted Double Strand
systems during DNA Breaks.
acquisition.[29][92][93][94][95][96] In
type I and type II systems,
protospacers are excised at
positions adjacent to a PAM
sequence, with the other end of the
spacer cut using a ruler
mechanism, thus maintaining the
regularity of the spacer size in the
CRISPR array.[97][98] The
conservation of the PAM sequence
differs between CRISPR-Cas
systems and appears to be
evolutionarily linked to Cas1 and
the leader sequence.[96][99] Double Strand DNA Breaks
Introduced by CRISPR-Cas9 Allows
New spacers are added to a Further Genetic Manipulation By
CRISPR array in a directional Exploiting Endogenous DNA Repair
manner,[27] occurring Mechanisms.
preferentially,[61][92][93][100][101] but
not exclusively, adjacent[95][98] to
the leader sequence. Analysis of the type I-E system from E.coli demonstrated
that the first direct repeat adjacent to the leader sequence, is copied, with the
ages of CRISPR immunity for each of the three major types of adaptive
ity. (1) Acquisition begins by recognition of invading DNA by Cas1 and Cas2
eavage of a protospacer. (2) The protospacer is ligated to the direct repeat
nt to the leader sequence and (3) single strand extension repairs the CRISPR
plicates the direct repeat. The crRNA processing and interference stages occur
ntly in each of the three major CRISPR systems. (4) The primary CRISPR
ipt is cleaved by cas genes to produce crRNAs. (5) In type I systems
/Cas6f cleave at the junction of ssRNA and dsRNA formed by hairpin loops in
ect repeat. Type II systems use a trans-activating (tracr) RNA to form dsRNA,
s cleaved by Cas9 and RNaseIII. Type III systems use a Cas6 homolog that
ot require hairpin loops in the direct repeat for cleavage. (6) In type II and type
ems secondary trimming is performed at either the 5 or 3 end to produce
crRNAs. (7) Mature crRNAs associate with Cas proteins to form interference
exes. (8) In type I and type II systems, interactions between the protein and
equence are required for degradation of invading DNA. Type III systems do not
a PAM for successful degradation and in type III-A systems basepairing
between the crRNA and mRNA rather than the DNA, targeted by type III-B
ms.
newly acquired spacer inserted between the first and second direct
repeats.[78][97]
The PAM sequence appears to be important during spacer insertion in type I-E
systems. That sequence contains a strongly conserved final nucleotide (nt)
adjacent to the first nt of the protospacer. This nt becomes the final base in the
first direct repeat.[79][102][103] This suggests that the spacer acquisition
machinery generates single-stranded overhangs in the second-to-last position
of the direct repeat and in the PAM during spacer insertion. However, not all
CRISPR-Cas systems appear to share this mechanism as PAMs in other
organisms do not show the same level of conservation in the final position.[99]
It is likely that in those systems, a blunt end is generated at the very end of the
direct repeat and the protospacer during acquisition.
Insertion variants
Analysis of Sulfolobussolfataricus CRISPRs revealed further complexities to
the canonical model of spacer insertion, as one of its six CRISPR loci inserted
new spacers randomly throughout its CRISPR array, as opposed to inserting
closest to the leader sequence.[98]
Multiple CRISPRs contain many spacers to the same phage. The mechanism
that causes this phenomenon was discovered in the type I-E system of E.coli.
A significant enhancement in spacer acquisition was detected where spacers
already target the phage, even mismatches to the protospacer. This priming
requires the Cas proteins involved in both acquisition and interference to
interact with each other. Newly acquired spacers that result from the priming
mechanism are always found on the same strand as the priming
spacer.[79][102][103] This observation led to the hypothesis that the acquisition
machinery slides along the foreign DNA after priming to find a new
protospacer.[103]
Biogenesis
CRISPR-RNA (crRNA), which later guides the Cas nuclease to the target
during the interference step, must be generated from the CRISPR sequence.
The crRNA is initially transcribed as part of a single long transcript
encompassing much of the CRISPR array.[10] This transcript is then cleaved by
Cas proteins to form crRNAs. The mechanism to produce crRNAs differs
among CRISPR/Cas systems. In type I-E and type I-F systems, the proteins
Cas6e and Cas6f respectively, recognise stem-loops[104][105][106] created by the
pairing of identical repeats that flank the crRNA.[107] These Cas proteins cleave
the longer transcript at the edge of the paired region, leaving a single crRNA
along with a small remnant of the paired repeat region.
Type III systems also use Cas6, however their repeats do not produce stem-
loops. Cleavage instead occurs by the longer transcript wrapping around the
Cas6 to allow cleavage just upstream of the repeat sequence.[108][109][110]
Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage.
Functional type II systems encode an extra small RNA that is complementary
to the repeat sequence, known as a trans-activating crRNA (tracrRNA).[76]
Transcription of the tracrRNA and the primary CRISPR transcript results in
base pairing and the formation of dsRNA at the repeat sequence, which is
subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two

systems the crRNA does not contain the full spacer, which is instead truncated
at one end.[71]
CrRNAs associate with Cas proteins to form ribonucleotide complexes that

recognize foreign nucleic acids. CrRNAs show no preference between the
coding and non-coding strands, which is indicative of an RNA-guided DNA-
targeting system.[6][37][75][79][111][112][113] The type I-E complex (commonly
referred to as Cascade) requires five Cas proteins bound to a single
crRNA.[114][115]
Interference
During the interference stage in type I systems the PAM sequence is
recognized on the crRNA-complementary strand and is required along with
crRNA annealing. In type I systems correct base pairing between the crRNA
and the protospacer signals a conformational change in Cascade that recruits
Cas3 for DNA degradation.
Type II systems rely on a single multifunctional protein, Cas9, for the

interference step.[71] Cas9 requires both the crRNA and the tracrRNA to
function and cleaves DNA using its dual HNH and RuvC/RNaseH-like
endonuclease domains. Basepairing between the PAM and the phage genome
is required in type II systems. However, the PAM is recognized on the same
strand as the crRNA (the opposite strand to type I systems).
Type III systems, like type I require six or seven Cas proteins binding to
crRNAs.[116][117] The type III systems analysed from S. solfataricus and P.
furiosus both target the mRNA of phages rather than phage DNA
genome,[66][117] which may make these systems uniquely capable of targeting
RNA-based phage genomes.[65]
The mechanism for distinguishing self from foreign DNA during interference is
built into the crRNAs and is therefore likely common to all three systems.
Throughout the distinctive maturation process of each major type, all crRNAs
contain a spacer sequence and some portion of the repeat at one or both ends.
It is the partial repeat sequence that prevents the CRISPR-Cas system from
targeting the chromosome as base pairing beyond the spacer sequence signals
self and prevents DNA cleavage.[118] RNA-guided CRISPR enzymes are
classified as type V restriction enzymes.
Evolution CRISPR associated protein

A bioinformatic study has
suggested that CRISPRs are
evolutionarily conserved and
cluster into related types. Many
show signs of a conserved
secondary structure.[107]
CRISPR/Cas can immunize

bacteria against certain phages crystal structure of a crispr-associated
and thus halt transmission. For protein from Thermus thermophilus
this reason, Koonin described Identiers
CRISPR/Cas as a Lamarckian Symbol CRISPR_assoc
inheritance mechanism.[119]
Pfam PF08798 (http://pfam.xfam.
However, this was disputed by a
org/family?acc=PF08798)
critic who noted, "We should
remember [Lamarck] for the
Pfam CL0362 (http://pfam.xfam.or
good he contributed to science,
clan g/clan/CL0362)
not for things that resemble his InterPro IPR010179 (https://www.eb
theory only superficially. Indeed, i.ac.uk/interpro/entry/IPR01
thinking of CRISPR and other 0179)
phenomena as Lamarckian only CDD cd09727 (https://www.ncbi.
obscures the simple and elegant nlm.nih.gov/Structure/cdd/c
way evolution really works".[120] ddsrv.cgi?uid=cd09727)
Available protein structures:
Coevolution Pfam structures (http://pfam.xfam.or

g/family/PF08798?tab=pdbBl
Analysis of CRISPR sequences ock)

revealed coevolution of host and PDB RCSB PDB (http://www.rcsb.o
viral genomes.[121] Cas9 proteins rg/pdb/search/smartSubquery.
are highly enriched in pathogenic do?smartSearchSubtype=Pfa
mIdQuery&pfamID=PF08798);
and commensal bacteria.
PDBe (https://www.ebi.ac.uk/
CRISPR/Cas-mediated gene
pdbe/entry/search/index?pfa
regulation may contribute to the m_accession:PF08798); PDBj
regulation of endogenous (https://pdbj.org/searchFor?qu
bacterial genes, particularly ery=PF08798)
during interaction with PDBsum structure summary (https://ww
eukaryotic hosts. For example, w.ebi.ac.uk/thornton-srv/datab
Francisella novicida uses a ases/cgi-bin/pdbsum/GetPfam
unique, small, CRISPR/Cas- Str.pl?pfam_id=PF08798)
associated RNA (scaRNA) to

repress an endogenous transcript CRISPR associated protein
encoding a bacterial lipoprotein Cas2
that is critical for F. novicida to
dampen host response and
promote virulence.[122]
The basic model of CRISPR

evolution is newly incorporated
spacers driving phages to mutate
their genomes to avoid the
bacterial immune response, crystal structure of a hypothetical
creating diversity in both the protein tt1823 from Thermus
phage and host populations. To thermophilus
fight off a phage infection, the
Identiers
sequence of the CRISPR spacer
must correspond perfectly to the
Symbol CRISPR_Cas2
sequence of the target phage Pfam PF09827 (http://pfam.xfam.
gene. Phages can continue to org/family?acc=PF09827)
infect their hosts given point InterPro IPR019199 (https://www.eb
mutations in the spacer.[118] i.ac.uk/interpro/entry/IPR01
Similar stringency is required in 9199)

PAM or the bacterial strain CDD cd09638 (https://www.ncbi.
remains phage sensitive.[93][118] nlm.nih.gov/Structure/cdd/c
ddsrv.cgi?uid=cd09638)
Rates Available protein structures:
A study of 124 S. thermophilus Pfam structures (http://pfam.xfam.or

g/family/PF09827?tab=pdbBl
strains showed that 26% of all
ock)
spacers were unique and that
PDB RCSB PDB (http://www.rcsb.o
different CRISPR loci showed
rg/pdb/search/smartSubquery.
different rates of spacer
do?smartSearchSubtype=Pfa
acquisition.[92] Some CRISPR loci mIdQuery&pfamID=PF09827);
evolve more rapidly than others, PDBe (https://www.ebi.ac.uk/
which allowed the strains' pdbe/entry/search/index?pfa
phylogenetic relationships to be m_accession:PF09827); PDBj
(https://pdbj.org/searchFor?qu
determined. A comparative
ery=PF09827)
genomic analysis showed that E.
coli and S. enterica evolve much PDBsum structure summary (https://ww
w.ebi.ac.uk/thornton-srv/datab
more slowly than S.
ases/cgi-bin/pdbsum/GetPfam
thermophilus. The latter's strains Str.pl?pfam_id=PF09827)
that diverged 250 thousand years
ago still contained the same
CRISPR-associated protein
spacer complement.[123]
Cse1
Metagenomic analysis of two acid Identiers
mine drainage biofilms showed Symbol CRISPR_Cse1
that one of the analyzed CRISPRs
contained extensive deletions and
spacer additions versus the other
InterPro IPR013381 (https://www.eb
biofilm, suggesting a higher
i.ac.uk/interpro/entry/IPR01
phage activity/prevalence in one
3381)
community than the other.[61] In
the oral cavity, a temporal study CDD cd09729 (https://www.ncbi.
determined that 7-22% of spacers nlm.nih.gov/Structure/cdd/c
were shared over 17 months ddsrv.cgi?uid=cd09729)

within an individual while less Available protein structures:
than 2% were shared across
Pfam structures (http://pfam.xfam.or
individuals.[101] g/family/PF09481?tab=pdbBl
ock)
From the same environment a
single strain was tracked using PDB RCSB PDB (http://www.rcsb.o
rg/pdb/search/smartSubquery.
PCR primers specific to its
CRISPR system. Broad-level mIdQuery&pfamID=PF09481);
results of spacer PDBe (https://www.ebi.ac.uk/
presence/absence showed pdbe/entry/search/index?pfa
significant diversity. However, m_accession:PF09481); PDBj
this CRISPR added 3 spacers (https://pdbj.org/searchFor?qu
ery=PF09481)
over 17 months,[101] suggesting
that even in an environment with PDBsum structure summary (https://ww
w.ebi.ac.uk/thornton-srv/datab
significant CRISPR diversity
ases/cgi-bin/pdbsum/GetPfam
some loci evolve slowly.
Str.pl?pfam_id=PF09481)
CRISPRs were analysed from the

metagenomes produced for the CRISPR-associated protein
human microbiome project.[124] Cse2
Although most were body-site Identiers
specific, some within a body site Symbol CRISPR_Cse2
are widely shared among
individuals. One of these loci
originated from streptococcal
species and contained ~15,000
InterPro IPR013382 (https://www.eb
spacers, 50% of which were
i.ac.uk/interpro/entry/IPR01
unique. Similar to the targeted
3382)
studies of the oral cavity, some CDD cd09670 (https://www.ncbi.
showed little evolution over nlm.nih.gov/Structure/cdd/c
time.[124] ddsrv.cgi?uid=cd09670)
Available protein structures:
Pfam structures (http://pfam.xfam.or
CRISPR evolution was studied in g/family/PF09485?tab=pdbBl

chemostats using S. ock)
thermophilus to directly examine PDB RCSB PDB (http://www.rcsb.o

spacer acquisition rates. In one rg/pdb/search/smartSubquery.
week, S. thermophilus strains
mIdQuery&pfamID=PF09485);
acquired up to three spacers
PDBe (https://www.ebi.ac.uk/
when challenged with a single pdbe/entry/search/index?pfa
phage.[125] During the same m_accession:PF09485); PDBj
interval the phage developed (https://pdbj.org/searchFor?qu
single nucleotide polymorphisms ery=PF09485)
that became fixed in the PDBsum structure summary (https://ww

population, suggesting that w.ebi.ac.uk/thornton-srv/datab
targeting had prevented phage ases/cgi-bin/pdbsum/GetPfam
Str.pl?pfam_id=PF09485)
replication absent these
mutations.[125]
Another S. thermophilus experiment showed that phages can infect and

replicate in hosts that have only one targeting spacer. Yet another showed that
sensitive hosts can exist in environments with high phage titres.[126] The
chemostat and observational studies suggest many nuances to CRISPR and
phage (co)evolution.
Identification
CRISPRs are widely distributed among bacteria and archaea[69] and show
some sequence similarities.[107] Their most notable characteristic is their
repeating spacers and direct repeats. This characteristic makes CRISPRs easily
identifiable in long sequences of DNA, since the number of repeats decreases
the likelihood of a false positive match. Three programs used for CRISPR
repeat identification search for regularly interspaced repeats in long
sequences: CRT,[127] PILER-CR[128] and CRISPRfinder.[129]
Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci

do not typically assemble, due to their repetitive nature or through strain
variation, which confuses assembly algorithms. Where many reference
genomes are available, polymerase chain reaction (PCR) can be used to amplify
CRISPR arrays and analyse spacer content.[92][101][130][131][132] However, this
approach yields information only for specifically targeted CRISPRs and for
organisms with sufficient representation in public databases to design reliable
polymerase chain reaction (PCR) primers.
The alternative is to extract and reconstruct CRISPR arrays from shotgun

metagenomic data. This is computationally more difficult, particularly with
second generation sequencing technologies (e.g. 454, Illumina), as the short
read lengths prevent more than two or three repeat units appearing in a single
read. CRISPR identification in raw reads has been achieved using purely de
novo identification[133] or by using direct repeat sequences in partially
assembled CRISPR arrays from contigs (overlapping DNA segments that
together represent a consensus region of DNA)[124] and direct repeat
sequences from published genomes[134] as a hook for identifying direct repeats
in individual reads.
Usebyphages
Another way for bacteria to defend against phage infection is by having
chromosomal islands. A subtype of chromosomal islands called phage-
inducible chromosomal island (PICI) is excised from a bacterial chromosome
upon phage infection and can inhibit phage replication.[135] The mechanisms
that induce PICI excision and how PICI inhibits phage replication are not well
understood. One study showed that lytic ICP1 phage, which specifically targets
Vibriocholerae serogroup O1, has acquired a CRISPR/Cas system that targets
a V.cholera PICI-like element. The system has 2 CRISPR loci and 9 Cas genes.
It seems to be homologous to the 1-F system found in Yersinia pestis.
Moreover, like the bacterial CRISPR/Cas system, ICP1 CRISPR/Cas can
acquire new sequences, which allows phage and host to co-evolve.[136]
Applications
By the end of 2014 some 1000 research papers had been published that
mentioned CRISPR.[137][138] The technology had been used to functionally
inactivate genes in human cell lines and cells, to study Candida albicans, to
modify yeasts used to make biofuels and to genetically modify crop strains.[138]
CRISPR can also be used to change mosquitos so they cannot transmit diseases
such as malaria.[139]
CRISPR-based re-evaluations of claims for gene-disease relationships have led

to the discovery of potentially important anomalies.[140]
DNA repair after double-strand break
Genome engineering
CRISPR/Cas9 genome editing is carried out with a Type II CRISPR system.

When utilized for genome editing, this system includes Cas9, crRNA, tracrRNA
along with an optional section of DNA repair template that is utilized in either
non-homologous end joining (NHEJ) or homology directed repair (HDR).
view of CRISPR Cas9 plasmid construction[141][142]
Major components
Component Function
Contains the guide RNA that locates the correct section of
crRNA host DNA along with a region that binds to tracrRNA
(generally in a hairpin loop form) forming an active complex.
tracrRNA Binds to crRNA and forms an active complex.
Single guide RNAs are a combined RNA consisting of a
sgRNA
tracrRNA and at least one crRNA
Protein whose active form is able to modify DNA. Many
variants exist with differing functions (i.e. single strand
Cas9
nicking, double strand break, DNA binding) due to Cas9's
DNA site recognition function.
Repair DNA that guides the cellular repair process allowing
template insertion of a specic DNA sequence
CRISPR/Cas9 often employs a plasmid to transfect the target cells.[143] The

main components of this plasmid are displayed in the image and listed in the
table. The crRNA needs to be designed for each application as this is the
sequence that Cas9 uses to identify and directly bind to the cell's DNA. The
crRNA must bind only where editing is desired. The repair template is
designed for each application, as it must overlap with the sequences on either
side of the cut and code for the insertion sequence.
Multiple crRNAs and the tracrRNA can be packaged together to form a single-
guide RNA (sgRNA).[144] This sgRNA can be joined together with the Cas9
gene and made into a plasmid in order to be transfected into cells.
Structure
CRISPR/Cas9 offers a high degree of fidelity and relatively simple
construction. It depends on two factors for its specificity: the target sequence
and the PAM. The target sequence is 20 bases long as part of each CRISPR
locus in the crRNA array.[143] A typical crRNA array has multiple unique target
sequences. Cas9 proteins select the correct location on the host's genome by
utilizing the sequence to bond with base pairs on the host DNA. The sequence
is not part of the Cas9 protein and as a result is customizable and can be
view of the transfection and DNA cleaving by CRISPR Cas9 (crRNA and
RNA are often joined as one strand of RNA when designing a plasmid)[143]
independently synthesized.[145][146]
The PAM sequence on the host genome is recognized by Cas9. Cas9 cannot be
easily modified to recognize a different PAM sequence. However this is not too
limiting as it is a short sequence and nonspecific (e.g. the SpCas9 PAM
sequence is 5'-NGG-3' and in the human genome occurs roughly every 8 to 12
base pairs).[143]
Once these have been assembled into a plasmid and transfected into cells the
Cas9 protein with the help of the crRNA finds the correct sequence in the host
cell's DNA and depending on the Cas9 variant creates a single or double
strand break in the DNA.[147]
Properly spaced single strand breaks in the host DNA can trigger homology
directed repair, which is less error prone than the non-homologous end joining
that typically follows a double strand break. Providing a DNA repair template
allows for the insertion of a specific DNA sequence at an exact location within
the genome. The repair template should extend 40 to 90 base pairs beyond the
Cas9 induced DNA break.[143] The goal is for the cell's HDR process to utilize
the provided repair template and thereby incorporate the new sequence into
the genome. Once incorporated, this new sequence is now part of the cell's
genetic material and passes into its daughter cells.
Many online tools are available to aid in designing effective sgRNA

sequences.[148]
Delivery
Scientists can use viral or non-viral systems for delivery of the Cas9 and
sgRNA into target cells. Electroporation of DNA, RNA or ribonucleocomplexes
is the most common and cheapest system. This technique was used to edit
CXCR4 and PD-1, knocking in new sequences to replace specific genetic
"letters" in these proteins. The group was then able to sort the cells, using cell
surface markers, to help identify successfully edited cells.[149] Deep sequencing
of a target site confirmed that knock-in genome modifications had occurred
with up to 20% efficiency, which accounted for up to approximately one-third
of total editing events.[150] However, hard-to-transfect cells (stem cells,
neurons, hematopoietic cells, etc.) require more efficient delivery systems such
as those based on lentivirus (LVs), adenovirus (AdV) and adeno-associated
virus (AAV).
Editing
CRISPRs have been used to cut five[36] to 62 genes at once: pig cells have been
engineered to inactivate all 62 Porcine Endogenous Retroviruses in the pig
genome, which eliminated transinfection from the pig to human cells in
culture.[151] CRISPR's low cost compared to alternatives is widely seen as
revolutionary.[11][12]
Selective engineered redirection of the CRISPR/Cas system was first

demonstrated in 2012 in:[152][153]
Immunization of industrially important bacteria, including some used in

food production and large-scale fermentation
Cellular or organism RNA-guided genome engineering. Proof of concept
studies demonstrated examples both in vitro[13][39][71] and in
vivo[49][154][155]
Bacterial strain discrimination by comparison of spacer sequences
Controlled genome editing

Several variants of CRISPR/Cas9 allow gene activation or genome editing with
an external trigger such as light or small molecules.[156][157][158] These include
photoactivatable CRISPR systems developed by fusing light-responsive protein
partners with an activator domain and a dCas9 for gene activation,[159][160] or
fusing similar light responsive domains with two constructs of split-
Cas9,[161][162] or by incorporating caged unnatural amino acids into Cas9,[163]
or by modifying the guide RNAs with photocleavable complements for genome
editing.[164]
Methods to control genome editing with small molecules include an allosteric

Cas9, with no detectable background editing, that will activate binding and
cleavage upon the addition of 4-hydroxytamoxifen (4-HT),[156] 4-HT
responsive intein-linked Cas9s[165] or a Cas9 that is 4-HT responsive when
fused to four ERT2 domains.[166] Intein-inducible split-Cas9 allows
dimerization of Cas9 fragments[167] and Rapamycin-inducible split-Cas9
system developed by fusing two constructs of split Cas9 with FRB and FKBP
fragments.[168] Furthermore, other studies have shown to induce transcription
of Cas9 with a small molecule, doxycyline.[169][170] Small molecules can also be
used to improve Homology Directed Repair (HDR),[171] often by inhibiting the

Non-Homologous End Joining (NHEJ) pathway.[172] These systems allow
conditional control of CRISPR activity for improved precision, efficiency and
spatiotemporal control.
Knockdown/activation
Using "dead" versions of Cas9 (dCas9) eliminates CRISPR's DNA-cutting

ability, while preserving its ability to target desirable sequences. Multiple
groups added various regulatory factors to dCas9s, enabling them to turn
almost any gene on or off or adjust its level of activity.[173] Like RNAi, CRISPR
interference (CRISPRi) turns off genes in a reversible fashion by targeting, but
not cutting a site. The targeted site is methylated, epigenetically modifying the
gene. This modification inhibits transcription. Conversely, CRISPR-mediated
activation (CRISPRa) promotes gene transcription.[174] Cas9 is an effective way
of targeting and silencing specific genes at the DNA level.[175] In bacteria, the
presence of Cas9 alone is enough to block transcription. For mammalian
applications, a section of protein is added. Its guide RNA targets regulatory
DNA sequences called promoters that immediately precede the target gene.[36]
Cas9 was used to carry synthetic transcription factors that activated specific
human genes. The technique achieved a strong effect by targeting multiple
CRISPR constructs to slightly different locations on the gene's promoter.[36]
RNA editing
In 2016 researchers demonstrated that CRISPR from an ordinary mouth
bacterium could be used to edit RNA. The researchers searched databases
containing hundreds of millions of genetic sequences for those that resembled
Crispr genes. They considered the fusobacteria Leptotrichia shahii. It had a
group of genes that resembled CRISPR genes, but with important differences.
When the researchers equipped other bacteria with these genes, which they
called C2c2, they found that the organisms gained a novel defense.[176]
Many viruses encode their genetic information in RNA rather than DNA that
they repurpose to make new viruses. HIV and poliovirus are such viruses.
Bacteria with C2c2 make molecules that can dismember RNA, destroying the
virus. Tailoring these genes opened any RNA molecule to editing.[176]
Disease models
CRISPR simplifies creation of animals for research that mimic disease or show
what happens when a gene is knocked down or mutated. CRISPR may be used
at the germline level to create animals where the gene is changed everywhere,
or it may be targeted at non-germline cells.[177][178][179]
CRISPR can be utilized to create human cellular models of disease. For

instance, applied to human pluripotent stem cells CRISPR introduced targeted
mutations in genes relevant to polycystic kidney disease (PKD) and focal
segmental glomerulosclerosis (FSGS).[180] These CRISPR-modified pluripotent
stem cells were subsequently grown into human kidney organoids that
exhibited disease-specific phenotypes. Kidney organoids from stem cells with
PKD mutations formed large, translucent cyst structures from kidney tubules.
The cysts were capable of reaching macroscopic dimensions, up to one
centimeter in diameter.[181] Kidney organoids with mutations in a gene linked
to FSGS developed junctional defects between podocytes, the filtering cells
affected in that disease. This was traced to the inability of podocytes ability to
form microvilli between adjacent cells.[182] Importantly, these disease
phenotypes were absent in control organoids of identical genetic background,
but lacking the CRISPR modifications.[180]
A similar approach was taken to model long QT syndrome in cardiomyocytes

derived from pluripotent stem cells.[183] These CRISPR-generated cellular
models, with isogenic controls, provide a new way to study human disease and
test drugs.
Gene drive
Gene drives may provide a powerful tool to restore balance of ecosystems by
eliminating invasive species. Concerns regarding efficacy, unintended
consequences in the target species as well as non-target species have been
raised particularly in the potential for accidental release from laboratories into
the wild. Scientists have proposed several safeguards for ensuring the
containment of experimental gene drives including molecular, reproductive,
and ecological.[184] Many recommend that immunization and reversal drives
be developed in tandem with gene drives in order to overwrite their effects if
necessary.[185] There remains consensus that long-term effects must be studied
more thoroughly particularly in the potential for ecological disruption that
cannot be corrected with reversal drives.[186]
Biomedicine
CRISPR/Cas-based "RNA-guided nucleases" can be used to target virulence
factors, genes encoding antibiotic resistance and other medically relevant
sequences of interest. This technology thus represents a novel form of
antimicrobial therapy and a strategy by which to manipulate bacterial
populations.[187][188] Recent studies suggested a correlation between the
interfering of the CRISPR/Cas locus and acquisition of antibiotic
resistance[189] This system provides protection of bacteria against invading
foreign DNA, such as transposons, bacteriophages and plasmids. This system
was shown to be a strong selective pressure for the acquisition of antibiotic
resistance and virulence factor in bacterial pathogens.[189] Some of the affected
genes are tied to human diseases, including those involved in muscle
differentiation, cancer, inflammation and fetal hemoglobin.[36]
Research suggests that CRISPR is an effective way to limit replication of

multiple herpesviruses. It was able to eradicate viral DNA in the case of
Epstein-Barr virus (EBV). Anti-herpesvirus CRISPRs have promising
applications such as removing cancer-causing EBV from tumor cells, helping
rid donated organs for immunocompromised patients of viral invaders, or
preventing cold sore outbreaks and recurrent eye infections by blocking HSV-1
reactivation. As of August 2016, these were awaiting testing.[190] CRISPR is
being applied to develop tissue-based treatments for cancer and other
diseases.[173][191]
CRISPR may revive the concept of transplanting animal organs into people.
Retroviruses present in animal genomes could harm transplant recipients. In
2015 a team eliminated 62 copies of a retrovirus's DNA from the pig genome in
a kidney epithelial cell.[173] Researchers recently demonstrated the ability to
birth live pig specimens after removing these retroviruses from their genome
using CRISPR for the first time.[192]
CRISPR may have applications in tissue engineering and regenerative

medicine, such as by creating human blood vessels that lack expression of
MHC class II proteins, which often cause transplant rejection.[193]
CRISPR in Cancer
As of 2016 CRISPR had been studied in animal models and cancer cell lines, to
learn if it can be used to repair or thwart mutated genes that cause cancer.[194]
The first clinical trial involving CRISPR started in 2016. It involved removing
immune cells from people with lung cancer, using CRISPR to edit out the gene
expressed PD-1, then administrating the altered cells back to the same person.
20 other trials were under way or nearly ready, mostly in China, as of 2017.[195]
In 2016 the United States Food and Drug Administration (FDA) approved a
clinical trial in which CRISPR would be used to alter T cells extracted from
people with different kinds of cancer and then administer those engineered T
cells back to the same people.[196]
Gene function
In 2015, multiple studies attempted to systematically disable each individual
human gene, in an attempt to identify which genes were essential to human
biology. Between 1,600 and 1,800 genes passed this testof the 20,000 or so
known human genes. Such genes are more strongly activated, and unlikely to
carry disabling mutations. They are more likely to have indispensable
counterparts in other species. They build proteins that unite to form larger
collaborative complexes. The studies also catalogued the essential genes in four
cancer-cell lines and identified genes that are expendable in healthy cells, but
crucial in specific tumor types and drugs that could target these rogue
genes.[197]
The specific functions of some 18 percent of the essential genes are

unidentified. In one 2015 targeting experiment, disabling individual genes in
groups of cells attempted to identify those involved in resistance to a
melanoma drug. Each such gene manipulation is itself a separate "drug",
potentially opening the entire genome to CRISPR-based regulation.[173]
In 2016-2017, a CRISPR/Cas-based approach to genetically engineering adult

rodent brains invivo was successfully demonstrated.[198][199]
In vitro genetic depletion

Unenriched sequencing libraries often have abundant undesired sequences.
Cas9 can specifically deplete the undesired sequences with double strand
breakage with up to 99% efficiency and without significant off-target effects as
seen with restriction enzymes. Treatment with Cas9 can deplete abundant
rRNA while increasing pathogen sensitivity in RNA-seq libraries.[200]
Patentsandcommercialization
As of December 2014, patent rights to CRISPR were contested. Several
companies formed to develop related drugs and research tools.[201] As
companies ramp up financing, doubts as to whether CRISPR can be quickly
monetized were raised.[202] In February 2017 the US Patent Office ruled on a
patent interference case brought by University of California with respect to
patents issued to the Broad Institute, and found that the Broad patents, with
claims covering the application of CRISPR/cas9 in eukaryotic cells, were
distinct from the inventions claimed by University of California.[203][204][205]
Shortly after, University of California filed an appeal of this ruling.[206][207]
As of November 2013, SAGE Labs (part of Horizon Discovery group) had

exclusive rights from one of those companies to produce and sell genetically
engineered rats and non-exclusive rights for mouse and rabbit models.[208] By
2015, Thermo Fisher Scientific had licensed intellectual property from
ToolGen to develop CRISPR reagent kits.[209]
In March 2017, the European Patent Office (EPO) announced its intention to
allow claims to Max-Planck Institute in Berlin, University of California, and
University of Vienna,[210][211] and in August 2017, the EPO announced its
intention to allow CRISPR claims in a patent application that MilliporeSigma
had filed.[210] As of August 2017 the patent situation in Europe was complex,
with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for
claims, along with University of California and Broad.[212]
Societyandculture
Human germline modication

At least four labs in the US, labs in China and the UK, and a US biotechnology
company called Ovascience announced plans or ongoing research to apply
CRISPR to human embryos.[213] Scientists, including a CRISPR co-inventor,
urged a worldwide moratorium on applying CRISPR to the human germline,
especially for clinical use. They said "scientists should avoid even attempting,
in lax jurisdictions, germline genome modification for clinical application in
humans" until the full implications "are discussed among scientific and
governmental organizations".[51][214] These scientists support basic research on
CRISPR and do not see CRISPR as developed enough for any clinical use in
making heritable changes to humans.[215]
In April 2015, Chinese scientists reported results of an attempt to alter the

DNA of non-viable human embryos using CRISPR to correct a mutation that
causes beta thalassemia, a lethal heritable disorder.[216][217] The study had
previously been rejected by both Nature and Science in part because of ethical
concerns.[218] The experiments resulted in changing only some genes, and had
off-target effects on other genes. The researchers stated that CRISPR is not
ready for clinical application in reproductive medicine.[218] In April 2016
Chinese scientists were reported to have made a second unsuccessful attempt
to alter the DNA of non-viable human embryos using CRISPR - this time to
alter the CCR5 gene to make the embryo HIV resistant.[219]
In December 2015, an International Summit on Human Gene Editing took

place in Washington under the guidance of David Baltimore. Members of
national scientific academies of America, Britain and China discussed the
ethics of germline modification. They agreed to support basic and clinical
research under appropriate legal and ethical guidelines. A specific distinction
was made between somatic cells, where the effects of edits are limited to a
single individual, versus germline cells, where genome changes could be
inherited by future generations. Heritable modifications could have
unintended and far-reaching consequences for human evolution, genetically
(e.g. gene/environment interactions) and culturally (e.g. Social Darwinism).
Altering of gametocytes and embryos to generate inheritable changes in
humans was defined to be irresponsible. The group agreed to initiate an

international forum to address such concerns and harmonize regulations
across countries.[220]
Policy barriers to genetic engineering

Policy regulations for the CRISPR/cas9 system vary around the globe. In
February 2016, British scientists were given permission by regulators to
genetically modify human embryos by using CRISPR-Cas9 and related
techniques. However, researchers were forbidden from implanting the
embryos and the embryos were to be destroyed after seven days.[221]
The US has an elaborate, interdepartmental regulatory system to evaluate new

genetically modified foods and crops. For example, the Agriculture Risk
Protection Act of 2000 gives the USDA the authority to oversee the detection,
control, eradication, suppression, prevention, or retardation of the spread of
plant pests or noxious weeds to protect the agriculture, environment and
economy of the US. The act regulates any genetically modified organism that
utilizes the genome of a predefined 'plant pest' or any plant not previously
categorized.[222] In 2015, Yang successfully deactivated 16 specific genes in the
white button mushroom. Since he had not added any foreign DNA to his
organism, the mushroom could not be regulated under by the USDA under
Section 340.2.[223] Yang's white button mushroom was the first organism
genetically modified with the Crispr/cas9 protein system to pass US
regulation.[224] In 2016, the USDA sponsored a committee to consider future
regulatory policy for upcoming genetic modification techniques. With the help
of the US National Academies of Sciences, Engineering and Medicine, special
interests groups met on April 15 to contemplate the possible advancements in
genetic engineering within the next 5 years and potential policy regulations
that would need to come into play.[225] With the emergence of rogue genetic
engineers employing the technology, the FDA has begun issuing new
regulations.[226]
In China, where social conditions sharply contrast both the USA and England,
genetic diseases carry a heavy stigma, individuals with mental and physical
disabilities do not get much federal or public support and religiously there are
no barriers against the use of genetic modifications to change the genotypes of
their people. [227] This leaves China with far fewer policy barriers and an
advantage over the use of the technology. Time will tell what direction they
choose to take, one thing is for certain, China has many policies to consider.
[228]
Recognition
In 2012 and 2013, CRISPR was a
runner-up in Science Magazine's
Breakthrough of the Year award. In
2015, it was the winner of that
award.[173] CRISPR was named as
one of MIT Technology Review's
10 breakthrough technologies in
2014 and 2016.[229][230] In 2016,
Jennifer Doudna, Emmanuel
Charpentier, along with Rudolph
Barrangou, Philippe Horvath, and
Feng Zhang won the Gairdner
International award. In 2017,
Jennifer Doudna and Emmanuel
Charpentier were awarded the
Japan Prize for their revolutionary
invention of CRISPR-Cas9 in Jennifer Doudna
Tokyo, Japan.
Alternative cutters
CRISPR-DR2: CRISPR-DR3:
Secondary structure Secondary structure
taken from the Rfam (htt taken from the Rfam (htt
p://rfam.xfam.org) p://rfam.xfam.org)
database. Family database. Family U1
RF01315 (http://rfam.xfa snRNA (http://rfam.xfam.
m.org/family/RF01315). org/family/RF00003).
database. Family database. Family
RF011318 (http://rfam.xf RF01319 (http://rfam.xfa
am.org/family/RF01318). m.org/family/RF01319).
RF01321 (http://rfam.xfa RF01322 (http://rfam.xfa
m.org/family/RF01321). m.org/family/RF01322).
CRISPR-DR65:
Secondary structure
taken from the Rfam (htt
p://rfam.xfam.org)
database. Family
RF01378 (http://rfam.xfa
m.org/family/RF01378).
Seealso
CRISPR/Cas Tools Synthetic biology

Gene knockout Surveyor nuclease assay
RNAi DRACO
SiRNA
Notes
1. 71/79 Archaea, 463/1008 Bacteria CRISPRdb (http://crispr.u-psud.fr/crisp
r/CRISPRdatabase.php), Date: 19.6.2010 Archived (https://web.archive.or
g/web/20150516061838/http://crispr.u-psud.fr/crispr/CRISPRdatabase.ph
p) May 16, 2015, at the Wayback Machine.
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&action=full&--eqskudatarq=1073) Edited by Jennifer Doudna, University
of California, Berkeley; Prashant Mali, University of California, San Diego
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Externallinks
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Jennifer Doudna talk: Genome Engineering with CRISPR-Cas9: Birth of a
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nology.html)
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12/20/2017 CRISPR - Wikipedia

CRISPR (/krspr/) is a family of DNA sequences in bacteria. The sequences

The CRISPR/Cas system is a prokaryotic immune system that confers

CRISPR is an abbreviation of Clustered Regularly Interspaced Short

Diagram of the CRISPR prokaryotic antiviral defense mechanism.[1]

A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified

location, allowing existing genes to be removed and/or new ones

CRISPR/Cas genome editing techniques have many potential applications,

Structure of crRNA-guided E. coli Cascade

The discovery of clustered DNA repeats began independently in three parts of

In 1993 researchers of Mycobacterium tuberculosis in the Netherlands

At the same time, repeats were observed in the archaeal organisms of

In 2005, three independent research groups showed that some CRISPR

The first publication[28] proposing a

Experimental work by several groups revealed the basic mechanisms of

RNA was later found in Pyrococcusfuriosus.[19][35] A 2010 study showed that

Feng Zhang's and George Church's groups simultaneously described genome

CRISPR has been modified to make programmable transcription factors that

Hemoglobin (HBB) in 28 out of 54 embryos. 4 out of the 28 embryos were

In the early 2000s, researchers developed zinc finger nucleases, synthetic

Repeats and spacers

Cas genes and CRISPR subtypes

III Cas10 Homolog of Cas10d and Cse1 [70]

IIIA Csm2 Not Determined [62]

IIIB Cmr5 Not Determined [62]

IIID Csx10 [62]

IIA Csn2 Ring-shaped DNA-binding [73]

Multiple Cas1 proteins have been

In the I-E system of E. coli Cas1

Protospacer adjacent motifs

Bioinformatic analysis of regions of

closest to the leader sequence.[98]

subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two

CrRNAs associate with Cas proteins to form ribonucleotide complexes that

Type II systems rely on a single multifunctional protein, Cas9, for the

Evolution CRISPR associated protein

CRISPR/Cas can immunize

Coevolution Pfam structures (http://pfam.xfam.or

Analysis of CRISPR sequences ock)

associated RNA (scaRNA) to

The basic model of CRISPR

Similar stringency is required in 9199)

Rates Available protein structures:

A study of 124 S. thermophilus Pfam structures (http://pfam.xfam.or

were shared over 17 months ddsrv.cgi?uid=cd09729)

CRISPRs were analysed from the

Pfam structures (http://pfam.xfam.or

CRISPR evolution was studied in g/family/PF09485?tab=pdbBl

thermophilus to directly examine PDB RCSB PDB (http://www.rcsb.o

that became fixed in the PDBsum structure summary (https://ww

Another S. thermophilus experiment showed that phages can infect and

Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci

The alternative is to extract and reconstruct CRISPR arrays from shotgun

CRISPR-based re-evaluations of claims for gene-disease relationships have led

DNA repair after double-strand break

CRISPR/Cas9 genome editing is carried out with a Type II CRISPR system.

view of CRISPR Cas9 plasmid construction[141][142]

CRISPR/Cas9 often employs a plasmid to transfect the target cells.[143] The

Many online tools are available to aid in designing effective sgRNA

Selective engineered redirection of the CRISPR/Cas system was first

Immunization of industrially important bacteria, including some used in

Controlled genome editing

Methods to control genome editing with small molecules include an allosteric

used to improve Homology Directed Repair (HDR),[171] often by inhibiting the