From en.wikipedia.org:
[Family of DNA sequences found in prokaryotic organisms]
[name-list-style=vanc] [the prokaryotic antiviral system] {{Infobox nonhuman
protein | Name = Cascade (CRISPR-associated complex for antiviral defense) |
image = 4QYZ.png | caption = CRISPR Cascade protein (cyan) bound to CRISPR RNA
(green) and phage DNA (red)[1] | Organism = _Escherichia coli_ | TaxID =
511145 | Symbol = CRISPR | EntrezGene = 947229 | HomoloGene = | PDB = 4QYZ |
UniProt = P38036 | RefSeqmRNA = | RefSeqProtein = NP_417241.1 }}
[[File:Crispr.png|thumb|262px|Diagram of the CRISPR prokaryotic antiviral
defense mechanism[2]]] CRISPR ([ˈ]; acronym of CLUSTERED REGULARLY INTERSPACED
SHORT PALINDROMIC REPEATS) is a family of DNA sequences found in the genomes
of prokaryotic organisms such as bacteria and archaea.<ref
name="pmid25574773"/> Each sequence within an individual prokaryotic CRISPR is
derived from a DNA fragment of a bacteriophage that had previously infected
the prokaryote or one of its ancestors.<ref name="Hille2018"/>[3] These
sequences are used to detect and destroy DNA from similar bacteriophages
during subsequent infections. Hence these sequences play a key role in the
antiviral (i.e. anti-phage) defense system of prokaryotes and provide a form
of heritable,<ref name="Hille2018"/> acquired immunity.[4][5][6][7] CRISPR is
found in approximately 50% of sequenced bacterial genomes and nearly 90% of
sequenced archaea.[8]
Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR
sequences as a guide to recognize and open up specific strands of DNA that are
complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR
sequences form the basis of a technology known as CRISPR-Cas9 that can be used
to edit genes within living organisms.[9][10] This editing process has a wide
variety of applications including basic biological research, development of
biotechnological products, and treatment of diseases.[11]<ref name="Hsu2014"/>
The development of the CRISPR-Cas9 genome editing technique was recognized by
the Nobel Prize in Chemistry in 2020 awarded to Emmanuelle Charpentier and
Jennifer Doudna.[12][13]
** History
*** Repeated sequences
The discovery of clustered DNA repeats took place independently in three parts
of the world. The first description of what would later be called CRISPR is
from Osaka University researcher Yoshizumi Ishino and his colleagues in 1987.
They accidentally cloned part of a CRISPR sequence together with the "_iap"
gene_ _(isozyme conversion of alkaline phosphatase)_ from their target genome,
that of _Escherichia coli_.[14][15] The organization of the repeats was
unusual. Repeated sequences are typically arranged consecutively, without
interspersing different sequences.<ref name="Hsu2014"/><ref
name="Ishino-1987"/> They did not know the function of the interrupted
clustered repeats.
In 1993, researchers of _Mycobacterium tuberculosis_ in the Netherlands
published two articles about a cluster of interrupted direct repeats (DR) in
that bacterium. They recognized the diversity of the sequences that
intervened in the direct repeats among different strains of _M.
tuberculosis_[16] and used this property to design a typing method called
_spoligotyping_, still in use today.[17][18]
Francisco Mojica at the University of Alicante in Spain studied the function
of repeats in the archaeal species _Haloferax_ and _Haloarcula_. Mojica's
supervisor surmised 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
_Haloferax volcanii_. Transcription of the interrupted repeats was also noted
for the first time; this was the first full characterization of CRISPR.<ref
name="Mojica2016"/>[19] By 2000, Mojica and his students, after an automated
search of published genomes, identified interrupted repeats in 20 species of
microbes as belonging to the same family.[20] Because those sequences were
interspaced, Mojica initially called these sequences "short regularly spaced
repeats" (SRSR).[21] In 2001, Mojica and Ruud Jansen, who were searching for
additional interrupted repeats, proposed the acronym CRISPR (Clustered
Regularly Interspaced Short Palindromic Repeats) to unify the numerous
acronyms used to describe these sequences.<ref name="Mojica2016b"/>[22] In
2002, Tang, et al. showed evidence that CRISPR repeat regions from the genome
of _Archaeoglobus fulgidus_ were transcribed into long RNA molecules
subsequently processed into unit-length small RNAs, plus some longer forms of
2, 3, or more spacer-repeat units.[23][24]
In 2005, yogurt researcher Rodolphe Barrangou discovered that _Streptococcus
thermophilus_, after iterative phage infection challenges, develops increased
phage resistance due to the incorporation of additional CRISPR spacer
sequences.[25] Barrangou's employer, the Danish food company Danisco, then
developed phage-resistant _S. thermophilus_ strains for yogurt production.
Danisco was later bought by DuPont, which owns about 50 percent of the global
dairy culture market, and the technology spread widely.[26]
*** CRISPR-associated systems
A major advance in understanding CRISPR came with Jansen's observation that
the prokaryote repeat cluster was accompanied by four homologous genes that
make up CRISPR-associated systems, _cas_ 1–4. The Cas proteins showed
helicase and nuclease motifs, suggesting a role in the dynamic structure of
the CRISPR loci.[27] In this publication, the acronym CRISPR was used as the
universal name of this pattern, but its function remained enigmatic.
[[File:SimpleCRISPR.jpg|thumb|Simplified diagram of a CRISPR locus. The three
major components of a CRISPR locus are shown: _cas_ genes, a leader sequence,
and a repeat-spacer array. Repeats are shown as gray boxes and spacers are
colored bars. The arrangement of the three components is not always as
shown.[28][29] In addition, several CRISPRs with similar sequences can be
present in a single genome, only one of which is associated with _cas_
genes.[30]]] In 2005, three independent research groups showed that some
CRISPR spacers are derived from phage DNA and extrachromosomal DNA such as
plasmids.[31][32][33] In effect, the spacers are fragments of DNA gathered
from viruses that previously attacked the cell. The source of the spacers was
a sign that the CRISPR-_cas_ system could have a role in adaptive immunity in
bacteria.<ref name="pmid20056882"/>[34] All three studies proposing this idea
were initially rejected by high-profile journals, but eventually appeared in
other journals.[35]
The first publication<ref name="pmid15791728"/> proposing a role of CRISPR-Cas
in microbial immunity, by Mojica and collaborators at the University of
Alicante, predicted a role for the RNA transcript of spacers on target
recognition in a mechanism that could be analogous to the RNA interference
system used by eukaryotic cells. Koonin and colleagues extended this RNA
interference hypothesis by proposing mechanisms of action for the different
CRISPR-Cas subtypes according to the predicted function of their proteins.[36]
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.<ref name="pmid17379808"/><ref
name="Hsu2014"/> A CRISPR region in _Streptococcus thermophilus_ acquired
spacers from the DNA of an infecting bacteriophage. The researchers
manipulated the resistance of _S. thermophilus_ to different types of phages
by adding and deleting spacers whose sequence matched those found in the
tested phages.[37]<ref name="Marraffini2015"/> In 2008, Brouns and Van der
Oost identified a complex of Cas proteins called Cascade, that in _E. coli_
cut the CRISPR RNA precursor within the repeats into mature spacer-containing
RNA molecules called CRISPR RNA (crRNA), which remained bound to the protein
complex.[38] Moreover, it was found that Cascade, crRNA and a
helicase/nuclease (Cas3) were required to provide a bacterial host with
immunity against infection by a DNA virus. By designing an anti-virus CRISPR,
they demonstrated that two orientations of the crRNA (sense/antisense)
provided immunity, indicating that the crRNA guides were targeting dsDNA.
That year Marraffini and Sontheimer confirmed 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 _Pyrococcus furiosus_.<ref name="Hsu2014"/><ref
name="Marraffini2015"/> A 2010 study showed that CRISPR-Cas cuts strands of
both phage and plasmid DNA in _S. thermophilus_.[39]
*** Cas9
[Cas9] A simpler CRISPR system from _Streptococcus pyogenes_ uses the protein
Cas9, an endonuclease functioning with two small RNAs—crRNA and tracrRNA—to
form a four-component complex.[40][41] In 2012, Jennifer Doudna and Emmanuelle
Charpentier simplified this into a two-component system by fusing the RNAs
into a "single-guide RNA", enabling Cas9 to target and cut specific DNA
sequences—a breakthrough that earned them the Nobel Prize in Chemistry in
2020.[42] Parallel work showed the _S. thermophilus_ Cas9 could be similarly
reprogrammed by altering the crRNA sequence.<ref name="Mojica2016"/> These
developments spurred genome editing efforts, including demonstrations by
groups led by Feng Zhang and George Church showing genome editing in human
cells using CRISPR-Cas9.[43][44][45]
*** Cas12a
[Cas12a] Cas12a, a Class II Type V CRISPR-associated nuclease, was
characterized in 2015 and was formerly known as Cpf1.<ref name="Rath_2015" />
This nuclease is found in the CRISPR-Cpf1 system of bacteria such as
_Francisella novicida_.<ref name="Redman_2016"/>[46] The initial designation,
derived from a TIGRFAMs protein family definition established in 2012,
reflected the prevalence of this CRISPR-Cas subtype in the _Prevotella_ and
_Francisella_ lineages. Cas12a exhibits several key distinctions from Cas9:
it generates staggered cuts in double-stranded DNA, in contrast to the blunt
ends produced by Cas9;<ref name="Proteintech"/> it relies on a 'T-rich'
protospacer adjacent motif (PAM) (typically 5'-TTTV-3', where V is A, C, or
G), offering alternative targeting sites compared to the 'G-rich' PAMs
(typically 5'-NGG-3') favored by Cas9;<ref name="Nobel_Foundation"/> and it
requires only a CRISPR RNA (crRNA) for effective targeting, whereas Cas9
necessitates both a crRNA and a _trans_-activating crRNA (tracrRNA).<ref
name="Rath_2015"/>
*** Cas13a
[ Cas13a] In 2016, the nuclease (formerly known as C2c2) from the bacterium
_Leptotrichia shahii_ was characterized by researchers in Feng Zhang's group
at MIT and the Broad Institute. Cas13 is an RNA-guided RNA endonuclease,
which means that it does not cleave DNA, but only single-stranded RNA. Cas13
is guided by its crRNA to a ssRNA target and binds and cleaves the target.
Similar to Cas12a, the Cas13 remains bound to the target and then cleaves
other ssRNA molecules non-discriminately.[47] This collateral cleavage
property has been exploited for the development of various diagnostic
technologies.[48][49][50]
** Locus structure
*** Repeats and spacers
The CRISPR array is made up of an AT-rich leader sequence followed by short
repeats that are separated by unique spacers.[51] 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.[52] Some show dyad symmetry, implying the formation
of a secondary structure such as a stem-loop ('hairpin') in the RNA, while
others are designed to be unstructured. The size of spacers in different
CRISPR arrays is typically 32 to 38 bp (range 21 to 72 bp).<ref
name="Barrangou2014"/> New spacers can appear rapidly as part of the immune
response to phage infection.<ref name="pmid17894817"/> There are usually fewer
than 50 units of the repeat-spacer sequence in a CRISPR array.<ref
name="Barrangou2014"/>
*** CRISPR RNA structures
<gallery title="Gallery of secondary structure images" perrow="5">
Image:RF01315.png| CRISPR-DR2: Secondary structure taken from the Rfam (see
http://rfam.xfam.org) database. Family RF01315 (see
http://rfam.xfam.org/family/RF01315) . Image:RF01318.png| CRISPR-DR5:
Secondary structure taken from the Rfam (see http://rfam.xfam.org) database.
Family RF011318 (see http://rfam.xfam.org/family/RF01318) .
Image:RF01319.png| CRISPR-DR6: Secondary structure taken from the Rfam (see
http://rfam.xfam.org) database. Family RF01319 (see
http://rfam.xfam.org/family/RF01319) . Image:RF01321.png| CRISPR-DR8:
Secondary structure taken from the Rfam (see http://rfam.xfam.org) database.
Family RF01321 (see http://rfam.xfam.org/family/RF01321) . Image:RF01322.png|
CRISPR-DR9: Secondary structure taken from the Rfam (see http://rfam.xfam.org)
database. Family RF01322 (see http://rfam.xfam.org/family/RF01322) .
Image:RF01332.png| CRISPR-DR19: Secondary structure taken from the Rfam (see
http://rfam.xfam.org) database. Family RF01332 (see
http://rfam.xfam.org/family/RF01332) . Image:RF01350.png| CRISPR-DR41:
Secondary structure taken from the Rfam (see http://rfam.xfam.org) database.
Family RF01350 (see http://rfam.xfam.org/family/RF01350) . Image:RF01365.png|
CRISPR-DR52: Secondary structure taken from the Rfam (see
http://rfam.xfam.org) database. Family RF01365 (see
http://rfam.xfam.org/family/RF01365) . Image:RF01370.png| CRISPR-DR57:
Secondary structure taken from the Rfam (see http://rfam.xfam.org) database.
Family RF01370 (see http://rfam.xfam.org/family/RF01370) . Image:RF01378.png|
CRISPR-DR65: Secondary structure taken from the Rfam (see
http://rfam.xfam.org) database. Family RF01378 (see
http://rfam.xfam.org/family/RF01378) . </gallery>
*** Cas genes and CRISPR subtypes
Small clusters of _cas_ genes are often located next to CRISPR repeat-spacer
arrays. Collectively the 93 _cas_ genes 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.<!-- The
numbers are from Makarova2015, and the Makarova2018 source has expanded them a
bit. Its fig1 mentions 13 core gene families (instead of 11), but there's no
mention of total gene families nor total gene count. -->[53]
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.[54] The 6 system
types are divided into 33 subtypes.[55] 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,[56] but exceptions
exist due to module shuffling.<ref name="Makarova2018"/> Many organisms
contain multiple CRISPR-Cas systems suggesting that they are compatible and
may share components.[57][58] The sporadic distribution of the CRISPR-Cas
subtypes suggests that the CRISPR-Cas system is subject to horizontal gene
transfer during microbial evolution.
[table] {| class="wikitable center" style="width:100%" |+ Signature genes and
their putative functions for the major and minor CRISPR-cas types |- ! Class
!! Cas type !Cas subtype!! Signature protein !! Function !! [Refh] |- |
rowspan="19" | 1 || rowspan="8" | I | [sdash] ||Cas3 || Single-stranded DNA
nuclease (HD domain) and ATP-dependent helicase ||[59][60] |- |[I-A]|| Cas8a,
Cas5 || rowspan="3" | Cas8 is a Subunit of the interference module that is
important in targeting of invading DNA by recognizing the PAM sequence. Cas5
is required for processing and stability of crRNAs. || rowspan="3" |<ref
name="Makarova2015"/>[61] |- |[I-B]|| Cas8b |- |[I-C]|| Cas8c |- |[I-D]||
Cas10d || rowspan="2" | contains a domain homologous to the palm domain of
nucleic acid polymerases and nucleotide cyclases || rowspan="2" |[62][63] |-
|[I-E]|| Cse1, Cse2 |- |[I-F]|| Csy1, Csy2, Csy3 || Type IF-3 have been
implicated in CRISPR-associated transposons||<ref name="Makarova2015"/> |-
|[I-G][ Subtype [I-G] was previously known as subtype [I-U] . <ref
name="Makarova2015"/> ]|| GSU0054 || ||[64] |- | rowspan="7" | III | [sdash]
|| Cas10 || Homolog of Cas10d and Cse1. Binds CRISPR target RNA and promotes
stability of the interference complex ||<ref name="pmid21756346"/>[65] |-
|[III-A]|| Csm2 || Not determined ||<ref name="Makarova2015"/> |- |[III-B]||
Cmr5 || Not determined ||<ref name="Makarova2015"/> |- |[III-C]|| Cas10 or
Csx11 || ||<ref name="Makarova2015"/><ref name="pmid30840895"/> |- |[III-D]||
Csx10 || ||<ref name="Makarova2015"/> |- |[III-E]|| || ||<ref
name="Makarova2019"/> |- |[III-F]|| || ||<ref name="Makarova2019"/> |- |
rowspan="4" | IV | [sdash] || Csf1 || ||<ref name="Makarova2019"/> |-
|[IV-A]|| || ||<ref name="Makarova2019"/> |- |[IV-B]|| || ||<ref
name="Makarova2019"/> |- |[IV-C]|| || ||<ref name="Makarova2019"/> |- |
rowspan="23" | 2 || rowspan="4" | II | [sdash] ||Cas9 || Nucleases RuvC and
HNH together produce DSBs, and separately can produce single-strand breaks.
Ensures the acquisition of functional spacers during adaptation. ||[66][67]
|- |[II-A]|| Csn2 || Ring-shaped DNA-binding protein. Involved in primed
adaptation in Type II CRISPR system. ||[68] |- |[II-B]||Cas4 || Endonuclease
that works with cas1 and cas2 to generate spacer sequences || [69] |-
|[II-C]|| || Characterized by the absence of either Csn2 or Cas4 ||[70] |- |
rowspan="12" | V | [sdash] || Cas12 || Nuclease RuvC. Lacks HNH. ||<ref
name="Wright2016"/>[71] |- |[V-A]|| Cas12a (Cpf1) || Auto-processing pre-crRNA
activity for multiplex gene regulation||<ref name="Makarova2019"/>[72] |-
|[V-B]|| Cas12b (C2c1) || ||<ref name="Makarova2019"/> |- |[V-C]|| Cas12c
(C2c3) || ||<ref name="Makarova2019"/> |- |[V-D]|| Cas12d (CasY) || ||<ref
name="Makarova2019"/> |- |[V-E]|| Cas12e (CasX) || ||<ref
name="Makarova2019"/> |- |[V-F]|| Cas12f (Cas14, C2c10) || ||<ref
name="Makarova2019"/> |- |[V-G]|| Cas12g || ||<ref name="Makarova2019"/> |-
|[V-H]|| Cas12h || ||<ref name="Makarova2019"/> |- |[V-I]|| Cas12i || ||<ref
name="Makarova2019"/> |- |[V-K][ Subtype [V-K] was previously known as subtype
[V-U] 5. <ref name="Makarova2019"/> ]|| Cas12k (C2c5) || Type V-K have been
implicated in CRISPR-associated transposons.||<ref name="Makarova2019"/> |-
|[V-U]|| C2c4, C2c8, C2c9 || ||<ref name="Makarova2019"/> |- | rowspan="7" |
VI | [sdash] || Cas13 || RNA-guided RNase || <ref name="Wright2016"/>[73] |-
|[VI-A]|| Cas13a (C2c2) || ||<ref name="Makarova2019"/> |- |[VI-B]|| Cas13b ||
||<ref name="Makarova2019"/> |- |[VI-C]|| Cas13c || ||<ref
name="Makarova2019"/> |- |[VI-D]|| Cas13d || ||<ref name="Makarova2019"/> |-
|VI-X |Cas13x.1 |RNA dependent RNA polymerase, Prophylactic RNA-virus
inhibition |[74] |- |VI-Y | | |<ref name="Xu-2021"/> |} [Clear]
** Mechanism
CRISPR-Cas immunity is a natural process of bacteria and archaea.[75]
CRISPR-Cas prevents bacteriophage infection, conjugation and natural
transformation by degrading foreign nucleic acids that enter the cell.[76]
*** Spacer acquisition
When a microbe is invaded by a bacteriophage, the first stage of the immune
response is to capture phage DNA and insert it into a CRISPR locus in the form
of a spacer. Cas1 and Cas2 are found in both 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.[77][78][79][80][81]
Multiple Cas1 proteins have been characterised and their structures
resolved.[82][83][84] 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.<ref name="pmid22337052"/> Representative Cas2
proteins have been characterised and possess either (single strand) ssRNA-[85]
or (double strand) dsDNA-[86][87] 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.[88] In this complex, Cas2 performs a
non-enzymatic scaffolding role,<ref name="pmid24793649"/> binding
double-stranded fragments of invading DNA, while Cas1 binds the
single-stranded flanks of the DNA and catalyses their integration into CRISPR
arrays.[89][90][91] New spacers are usually added at the beginning of the
CRISPR next to the leader sequence creating a chronological record of viral
infections.[92] 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.[93] IHF also enhances integration efficiency in
the type I-F system of _Pectobacterium atrosepticum ,_[94] but in other
systems, different host factors may be required[95]
**** Protospacer adjacent motifs (PAM)
[Protospacer adjacent motif] 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 type I and type II, but not
type III systems during acquisition.<ref
name="pmid16079334"/>[96][97][98][99][100] 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.[101][102] The conservation
of the PAM sequence differs between CRISPR-Cas systems and appears to be
evolutionarily linked to Cas1 and the leader sequence.<ref
name="pmid19143596"/>[103]
New spacers are added to a CRISPR array in a directional manner,<ref
name="pmid15758212"/> occurring preferentially,[104]<ref
name="pmid18065539"/><ref name="pmid18065545"/>[105][106] but not exclusively,
adjacent<ref name="pmid19239620"/><ref name="pmid22834906"/> 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
newly acquired spacer inserted between the first and second direct
repeats.<ref name="pmid22402487"/><ref name="pmid23445770"/>
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.<ref name="pmid22558257"/>[107][108] 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.<ref name="pmid23403393"/> 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 _Sulfolobus solfataricus_ 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.<ref name="pmid22834906"/>
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.<ref
name="pmid22558257"/><ref name="pmid22771574"/><ref name="pmid22781758"/> This
observation led to the hypothesis that the acquisition machinery slides along
the foreign DNA after priming to find a new protospacer.<ref
name="pmid22781758"/>
*** 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.<ref name="pmid20125085"/> 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[109][110][111] created by the pairing
of identical repeats that flank the crRNA.[112] 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.[113][114][115]
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).<ref
name="Deltcheva2011"/> 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.<ref name="pmid22949671"/>
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.<ref name="pmid19095942"/><ref name="Garneau2010"/><ref
name="pmid19120484"/><ref name="pmid22558257"/>[116][117][118] The type I-E
complex (commonly referred to as Cascade) requires five Cas proteins bound to
a single crRNA.[119][120]
*** 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.<ref name="pmid22949671"/> Cas9 requires both the crRNA and
the tracrRNA to function and cleave 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.[121][122] The type III systems analysed from _S. solfataricus_ and
_P. furiosus_ both target the mRNA of phages rather than phage DNA
genome,<ref name="pmid23320564"/><ref name="pmid19945378"/> which may make
these systems uniquely capable of targeting RNA-based phage genomes.<ref
name="pmid22337052"/> Type III systems were also found to target DNA in
addition to RNA using a different Cas protein in the complex, Cas10.[123] The
DNA cleavage was shown to be transcription dependent.[124]
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.[125] RNA-guided CRISPR enzymes are classified
as type V restriction enzymes.
** Evolution
{{Infobox protein family | Symbol = CRISPR_Cas2 | Name = CRISPR associated
protein Cas2 (adaptation RNase) | image = PDB 1zpw EBI.jpg | width = | caption
= Crystal structure of a hypothetical protein tt1823 from Thermus thermophilus
| Pfam = PF09827 | Pfam_clan = | InterPro = IPR019199 | SMART = | PROSITE = |
MEROPS = | SCOP = | TCDB = | OPM family = | OPM protein = | CAZy = | CDD =
cd09638 }}
{{Infobox protein family | Symbol = CRISPR_Cse1 | Name = CRISPR-associated
protein CasA/Cse1 (Type I effector DNase) | image = | width = | caption = |
Pfam = PF09481 | Pfam_clan = | InterPro = IPR013381 | SMART = | PROSITE = |
MEROPS = | SCOP = | TCDB = | OPM family = | OPM protein = | CAZy = | CDD =
cd09729 }} {{Infobox protein family | Symbol = CRISPR_assoc | Name = CRISPR
associated protein CasC/Cse3/Cas6 (Type I effector RNase) | image = PDB 1wj9
EBI.jpg | width = | caption = Crystal structure of a crispr-associated protein
from Thermus thermophilus | Pfam = PF08798 | Pfam_clan = CL0362 | InterPro =
IPR010179 | SMART = | PROSITE = | MEROPS = | SCOP = | TCDB = | OPM family = |
OPM protein = | CAZy = | CDD = cd09727 }}
The cas genes in the adaptor and effector modules of the CRISPR-Cas system are
believed to have evolved from two different ancestral modules. A
transposon-like element called casposon encoding the Cas1-like integrase and
potentially other components of the adaptation module was inserted next to the
ancestral effector module, which likely functioned as an independent innate
immune system.[126] The highly conserved cas1 and cas2 genes of the adaptor
module evolved from the ancestral module while a variety of class 1 effector
cas genes evolved from the ancestral effector module.[127] The evolution of
these various class 1 effector module cas genes was guided by various
mechanisms, such as duplication events.[128] On the other hand, each type of
class 2 effector module arose from subsequent independent insertions of mobile
genetic elements.[129] These mobile genetic elements took the place of the
multiple gene effector modules to create single gene effector modules that
produce large proteins which perform all the necessary tasks of the effector
module.<ref name="Shmakov-2017"/> The spacer regions of CRISPR-Cas systems are
taken directly from foreign mobile genetic elements and thus their long-term
evolution is hard to trace.[130] The non-random evolution of these spacer
regions has been found to be highly dependent on the environment and the
particular foreign mobile genetic elements it contains.[131]
CRISPR-Cas can immunize bacteria against certain phages and thus halt
transmission. For this reason, Koonin described CRISPR-Cas as a Lamarckian
inheritance mechanism.[132] However, this was disputed by a critic who noted,
"We should remember [Lamarck] for the good he contributed to science, not for
things that resemble his theory only superficially. Indeed, thinking of
CRISPR and other phenomena as Lamarckian only obscures the simple and elegant
way evolution really works".[133] But as more recent studies have been
conducted, it has become apparent that the acquired spacer regions of
CRISPR-Cas systems are indeed a form of Lamarckian evolution because they are
genetic mutations that are acquired and then passed on.[134] On the other
hand, the evolution of the Cas gene machinery that facilitates the system
evolves through classic Darwinian evolution.<ref name="Koonin-2016"/>
*** Coevolution
Analysis of CRISPR sequences revealed coevolution of host and viral
genomes.[135]
The basic model of CRISPR evolution is newly incorporated spacers driving
phages to mutate their genomes to avoid the bacterial immune response,
creating diversity in both the phage and host populations. To resist a phage
infection, the sequence of the CRISPR spacer must correspond perfectly to the
sequence of the target phage gene. Phages can continue to infect their hosts'
given point mutations in the spacer.<ref name="pmid20072129"/> Similar
stringency is required in PAM or the bacterial strain remains phage
sensitive.<ref name="pmid18065545"/><ref name="pmid20072129"/>
*** Rates
A study of 124 _S. thermophilus_ strains showed that 26% of all spacers were
unique and that different CRISPR loci showed different rates of spacer
acquisition.<ref name="pmid18065539"/> Some CRISPR loci evolve more rapidly
than others, which allowed the strains' phylogenetic relationships to be
determined. A comparative genomic analysis showed that _E. coli_ and _S.
enterica_ evolve much more slowly than _S. thermophilus_. The latter's
strains that diverged 250,000 years ago still contained the same spacer
complement.[136]
Metagenomic analysis of two acid-mine-drainage biofilms showed that one of the
analyzed CRISPRs contained extensive deletions and spacer additions versus the
other biofilm, suggesting a higher phage activity/prevalence in one community
than the other.<ref name="pmid17894817"/> In the oral cavity, a temporal study
determined that 7–22% of spacers were shared over 17 months within an
individual while less than 2% were shared across individuals.<ref
name="pmid21149389"/>
From the same environment, a single strain was tracked using PCR primers
specific to its CRISPR system. Broad-level results of spacer presence/absence
showed significant diversity. However, this CRISPR added three spacers over
17 months,<ref name="pmid21149389"/> suggesting that even in an environment
with significant CRISPR diversity some loci evolve slowly.
CRISPRs were analysed from the metagenomes produced for the Human Microbiome
Project.[137] Although most were body-site specific, some within a body site
are widely shared among individuals. One of these loci originated from
streptococcal species and contained ≈15,000 spacers, 50% of which were unique.
Similar to the targeted studies of the oral cavity, some showed little
evolution over time.<ref name="pmid22719260"/>
CRISPR evolution was studied in chemostats using _S. thermophilus_ to
directly examine spacer acquisition rates. In one week, _S. thermophilus_
strains acquired up to three spacers when challenged with a single phage.[138]
During the same interval, the phage developed single-nucleotide polymorphisms
that became fixed in the population, suggesting that targeting had prevented
phage replication absent these mutations.<ref name="pmid23057534"/>
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.[139]
The chemostat and observational studies suggest many nuances to CRISPR and
phage (co)evolution.
** Identification
CRISPRs are widely distributed among bacteria and archaea<ref
name="pmid24728998"/> and show some sequence similarities.<ref
name="pmid17442114"/> 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.[140]
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.<ref name="pmid18065539"/><ref
name="pmid21149389"/>[141][142][143][144] However, this approach yields
information only for specifically targeted CRISPRs and for organisms with
sufficient representation in public databases to design reliable polymerase
PCR primers. Degenerate repeat-specific primers can be used to amplify CRISPR
spacers directly from environmental samples; amplicons containing two or three
spacers can be then computationally assembled to reconstruct long CRISPR
arrays.<ref name="pmid31729390"/>
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[145] or by using direct repeat sequences in partially
assembled CRISPR arrays from contigs (overlapping DNA segments that together
represent a consensus region of DNA)<ref name="pmid22719260"/> and direct
repeat sequences from published genomes[146] as a hook for identifying direct
repeats in individual reads.
** Use by phages
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.[147] PICIs are induced, excised,
replicated, and finally packaged into small capsids by certain staphylococcal
temperate phages. PICIs use several mechanisms to block phage reproduction.
In the first mechanism, PICI-encoded Ppi differentially blocks phage
maturation by binding or interacting specifically with phage TerS, hence
blocking phage TerS/TerL complex formation responsible for phage DNA
packaging. In the second mechanism PICI CpmAB redirects the phage capsid
morphogenetic protein to make 95% of SaPI-sized capsid and phage DNA can
package only 1/3rd of their genome in these small capsids and hence become
nonviable phage.[148] The third mechanism involves two proteins, PtiA and
PtiB, that target the LtrC, which is responsible for the production of virion
and lysis proteins. This interference mechanism is modulated by a modulatory
protein, PtiM, binds to one of the interference-mediating proteins, PtiA, and
hence achieves the required level of interference.[149]
One study showed that lytic ICP1 phage, which specifically targets _Vibrio
cholerae_ 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 I-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.[150][151]
Certain archaeal viruses were shown to carry mini-CRISPR arrays containing one
or two spacers. It has been shown that spacers within the virus-borne CRISPR
arrays target other viruses and plasmids, suggesting that mini-CRISPR arrays
represent a mechanism of heterotypic superinfection exclusion and participate
in interviral conflicts.<ref name="pmid31729390"/>
** Applications
[CRISPR gene editing]
[name-list-style=vanc] CRISPR gene editing is a revolutionary technology that
allows for precise, targeted modifications to the DNA of living organisms.
Developed from a natural defense mechanism found in bacteria, CRISPR-Cas9 is
the most commonly used system. Gene editing with CRISPR-Cas9 involves a Cas9
nuclease and an engineered guide RNA, which come together to allow for the
precise "cutting" of one or both strands of DNA at specific locations within
the genome.[152] It makes use of the cell's natural DNA repair systems,
including non-homologous end joining, homology-directed repair, or mismatch
repair, to modify, insert, or delete genetic material at these specific cut
sites. <ref name="Koblan_2020" />[153] This technology has transformed fields
such as genetics, medicine,[154][155][156] and agriculture,[157] offering
potential treatments for genetic disorders, advancements in crop engineering,
and research into the fundamental workings of life. However, its ethical
implications and potential unintended consequences have sparked significant
debate.[158][159]
** See also
[colwidth=18em]
- CRISPR activation
- Anti-CRISPR
- CRISPR/Cas Tools
- CRISPR gene editing
- The CRISPR Journal
- "Designer baby "
- DRACO
- Gene knockout
- Genome-wide CRISPR-Cas9 knockout screens
- Glossary of genetics
- Human germline engineering
- _Human Nature_ (2019 documentary film)
- MAGESTIC
- _New_ eugenics
- Prime editing
- RNAi
- SiRNA
- Surveyor nuclease assay
- Synthetic biology
- Zinc finger
[div col end]
** Notes
[group=Note]
[clear]
** References
[reflist]
** Further reading
[30em]
- [ vauthors = Doudna J, Mali P ]
- [ vauthors = Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV, van der Oost J ]
- [ vauthors = Sander JD, Joung JK ]
- [ vauthors = Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F ]
- [ vauthors = Terns RM, Terns MP ]
- [ vauthors = Westra ER, Buckling A, Fineran PC ]
- [ vauthors = Andersson AF, Banfield JF ]
- [ vauthors = Hale C, Kleppe K, Terns RM, Terns MP ]
- [ vauthors = van der Ploeg JR ]
- [ vauthors = van der Oost J, Brouns SJ ]
- [ vauthors = Karginov FV, Hannon GJ ]
- [ vauthors = Pul U, Wurm R, Arslan Z, Geissen R, Hofmann N, Wagner R ]
- [ vauthors = Díez-Villaseñor C, Almendros C, García-Martínez J, Mojica FJ ]
- [ vauthors = Deveau H, Garneau JE, Moineau S ]
- [ vauthors = Koonin EV, Makarova KS ]
- [title = The age of the red pen]
- [ vauthors = Ran AF, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F ]
[refend]
** External links
[Commons category] [topic]
- [url=https://fas.org/sgp/crs/misc/R44824.pdf ]
- [url=https://www.ibiology.org/ibiomagazine/jennifer-doudna-genome-engineering-with-crispr-cas9-birth-of-a-breakthrough-technology.html ]
- [title=Human Nature]
*** Protein Data Bank
- [Q46901]
- [P76632]
- [Q46899]
- [Q46898]
- [Q46897]
[Glossaries of science and engineering] [Repeated sequence] [Biology]
[authority control]
[DEFAULTSORT:Crispr] Category:1987 in biotechnology Category:2015 in
biotechnology Category:Biological engineering Category:Biotechnology
Category:Genetic engineering Category:Genome editing Category:Jennifer Doudna
Category:Molecular biology Category:Non-coding RNA Category:Repetitive DNA
sequences Category:Immune system Category:Prokaryote genes