Molecular Biotechnology (2023) 65:311–325

https://doi.org/10.1007/s12033-022-00567-0

REVIEW PAPER

A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/

Cas13/Cas14 Proteins Utilized for Genome Engineering
V. Edwin Hillary1 · S. Antony Ceasar1

Received: 16 May 2022 / Accepted: 15 September 2022 / Published online: 27 September 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas)
system has altered life science research offering enormous options in manipulating, detecting, imaging, and annotating
specific DNA or RNA sequences of diverse organisms. This system incorporates fragments of foreign DNA (spacers) into
CRISPR cassettes, which are further transcribed into the CRISPR arrays and then processed to make guide RNA (gRNA).
The CRISPR arrays are genes that encode Cas proteins. Cas proteins provide the enzymatic machinery required for acquiring
new spacers targeting invading elements. Due to programmable sequence specificity, numerous Cas proteins such as Cas9,
Cas12, Cas13, and Cas14 have been exploited to develop new tools for genome engineering. Cas variants stimulated genetic
research and propelled the CRISPR/Cas tool for manipulating and editing nucleic acid sequences of living cells of diverse
organisms. This review aims to provide detail on two classes (class 1 and 2) of the CRISPR/Cas system, and the mechanisms
of all Cas proteins, including Cas12, Cas13, and Cas14 discovered so far. In addition, we also discuss the pros and cons and
recent applications of various Cas proteins in diverse fields, including those used to detect viruses like severe acute respira-
tory syndrome coronavirus-2 (SARS-CoV-2). This review enables the researcher to gain knowledge on various Cas proteins
and their applications, which have the potential to be used in next-generation precise genome engineering.

Keywords CRISPR · Cas system · Genome engineering · DETECTR​· SHERLOCK

Introduction which faced unpredictable off-target effects and delivered

only temporary or partial inhibition of gene function [2].
Genome editing or gene editing is a popular technology The genome-editing technique needs programmable
used in medicines, therapeutic drugs, infectious studies, and sequence-specific endonucleases to produce the site-specific
agricultural biotechnology. The genome-editing tools have single-stranded breaks (SSBs) or double-stranded breaks
been employed to study the precise function of a gene by (DSBs) at the targeted site that allow the endogenous repair
cutting and altering at a programmed locus through inser- mechanisms to fill the breaks [3]. These breaks are fixed by
tion, deletion, or replacement of targeted bases. In the begin- either of the two major repair mechanisms, (1) homology-
ning, conventional gene-editing techniques like homologous directed repair (HDR) and (2) non-homologous end-joining
recombination (HR) were utilized for gene inactivation, but repair (NHEJ) [4]. To facilitate specific DNA breaks, vari-
the effectiveness of HR was extremely low with the labor- ous genome-editing tools are developed previously, such as
intensive process [1]. Later, targeted gene knock-down uti- meganucleases or homing endonucleases [5], zinc-finger
lizing RNA interference (RNAi) technique has provided nucleases (ZFNs) [6], and transcription activator-like effec-
researchers with rapid and low-cost technology to silence tor nucleases (TALENs) [7]. But these tools demand labo-
the gene of interest to study its functions. However, it also rious efforts for cloning and protein construction to make
could not completely knock-down the targeted sequence, DSBs, which hinders these tools from routine applications
of genome editing. In 2012, the researchers developed
* S. Antony Ceasar clustered regularly interspaced short palindromic repeats
[email protected] (CRISPR) and CRISPR-associated proteins (Cas) system-
based genome editing tools [8]. CRISPR/Cas system medi-
1
Department of Biosciences, Rajagiri College of Social ates diverse adaptive immune systems against phages or
Sciences, 683 104, Cochin, Kerala, India

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312 Molecular Biotechnology (2023) 65:311–325

plasmids. Due to unique features of simplicity in design, Following this, several CRISPR/Cas genes were identified.
cost-effectiveness, and labor intensity, the research com- In 2005, spacer sequence was identified in many genomes,
munity has immediately adopted the CRISPR/Cas system and unique spacer regions were found within the CRISPR
as a user-friendly and robust RNA-guided DNA targeting array [19]. These results showed that CRISPR is an adaptive
tool for genome editing in various species [8–11]. Based immune system to defend prokaryotic cells against phage
on the mechanism, the CRISPR system has been divided infection through the RNA-guided process.
into two main classes (1 and 2) and six types (I-VI) [12]. In
these, types I to III were extensively studied, whereas types
IV and VI were recently discovered. Types I, II, and V cut Classification of CRISPR/Cas System
DNA, type VI cleaves RNA, type III cleaves both DNA and
RNA and the cleavage activity of type IV has not yet been The CRISPR system has been classified into two major
identified [13]. classes. In the Class 1 system, the RNA-guided target cleav-
Cas9 protein has been utilized for diverse applications age needs several effector proteins, but the Class 2 system
such as fluorescent imaging, base-editing, and transcrip- requires only one RNA-guided endonuclease to cleave the
tional activation, apart from targeted cleavage of dsDNA. DNA sequences [12, 20]. The class 1 system of CRISPR
Like Cas9, the Cas10 protein is also involved in various is divided into three types I, III, and IV, and the Class 2
applications such as fluorescent imaging, base-editing, and system is divided into types II, V, and VI [21, 22]. In the
RNA tracking. As an alternative to Cas9 and Cas10, the type I system, the CRISPR/Cas locus contains the Cas3 sig-
Cas12 protein enhanced genome-editing efficiency by target- nature gene that encodes a large protein with a helicase to
ing only T-rich motifs without utilizing tracrRNA. So Cas12 unwind DNA-DNA and RNA–DNA duplexes [23]. The type
system has expanded editing applications such as base-edit- II locus encodes multidomain protein to target and cleave the
ing and detecting transcriptional variations. Cas13 protein dsDNA [8]. The type III CRISPR/Cas possesses the Cas10
is also used for diverse applications such as imaging, base- signature gene, encodes a multidomain protein with palm
editing, and detection of transcriptional variations. Recently, domain to target, and cleaves ssDNA [24]. Type IV sys-
the Cas14 protein has advanced genome-editing efficiency tem contains CRISPR-associated splicing factor 1 (Csf1),
without needing an adjacent protospacer motif (PAM) and which encodes a ribonucleic protein, but the detailed func-
performs transcriptional regression and base-editing. This tion of this system is yet to be identified [21]. Type V locus
review describes the Cas variants of two classes of CRISPR possesses Cas12 signature gene (known as CRISPR from
systems used for genome editing. Prevotella and Francisella 1 (Cpf1), C2c1 or C2c3 protein)
encodes RuvC (an E. coli protein involved in DNA repair)
domain, which cleaves both dsDNA or ssDNA [25]. Type
History of CRISPR/Cas System VI contains Cas13 (C2c2) that encodes higher eukaryotes
and prokaryotes' nucleotide-binding domain (HEPN), which
A group of researchers detected CRISPRs by analyzing the cleaves ssRNA [26] (Table 1).
alkaline phosphatase gene, which is liable for the isozyme The CRISPR/Cas system uses three stages to defend
conversion of alkaline phosphatase (iap) in the E. coli K-12 against viruses or foreign genetic materials [27] (Fig. 1). In
strain [14]. They identified a genomic region that contains a the first stage, protospacers are incorporated into the host
series of 32 nucleotides of distinctive sequences flanked by CRISPR locus as spacers between crRNA repeats [28]; next,
invariable palindromic repeats on the 3′ end of the iap gene Cas proteins are expressed, a spacer is transcribed into pre-
[14]. Later, distinctive parallel sequences were found in the crRNA, and the pre-crRNA is cleaved by Cas proteins and
other E. coli strains and Enterobacteria (Shigella dysenteriae becomes functional as a mature crRNA [28]. In the third
and Salmonella enterica) [15]. Similarly, during the study of stage, Cas protein recognizes the target with the help of
Mycobacterium tuberculosis strains, researchers identified crRNA and generates cleavage of the genome [28]. Many
36 bp repeats interspaced with unique spacers of 35–41 bp CRISPR systems act based on the presence of a sequence-
[16]. specific PAM, which is adjacent to the crRNA-specific site
In subsequent works, the CRISPR array was found in within the target genome.
archaea (Haloferax mediterranei and Streptococcus thermo-
pile), and the same was identified in 90% bacterial and 40%
archaeal genomes [17]. However, this odd genomic sequence CRISPR/Cas System
first turned out to be the outline of the CRISPR array. Still,
due to the absence of necessary genome sequence data, the Scientists have discovered a novel microbial defense system
biological function of CRISPR remained elusive. Jansen that defends itself from viral and mobile genetic elements.
et al. in 2002 described the CRISPR-associated genes [18]. One of the defense mechanisms found in bacterium and

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Table 1  Classification of CRISPR/Cas system with their protein, target molecule, mechanism of spacer acquisition, processing of Pre-CRISPR, self-vs non-self-discrimination and their effectors

References

[23]

[24]

[21]

[25]

[26]
[8]
Host organism

S. thermophi-
S. epidermics

lus and S.

F. novicida
pyogenes
E. coli

–
CRISPR repeat Cmr/Csm, crRNA, and
Effectors of CRISPR

Cas9, tracrRNA, and

Cas3, Cascade, and

C2c1, and crRNA

Cpf1, crRNA and
tracrRNA
crRNA

crRNA
Cas10
system

–
nonself-dis-
crimination
Self vs

PAM

PAM

PAM
–

–
Pre-CRISPR process-

RNase III, and tracr-

Fig. 1  CRISPR/Cas adaptive immunity system. The three stages such

RNA
Cas6

Cas6

Cpf1

as CRISPR adaptation (stage 1), CRISPR RNA biogenesis (stage 2),

ing

and CRISPR interference (stage 3) are schematically illustrated. Dur-

–

ing the adaption stage, the injection of genetic material (virus) into
Cas7, Cas5, Cas8, and
Name of CRISPR/Cas

Cas7, Cas5, and Cas1

Cas7, Cas5, and Csf1

bacterial cells triggers the Cas1 and Cas2 adaption module proteins,
which cleaves the invading sequences (spacers) and are then incorpo-
rated into the CRISPR array. During CRISPR/RNA biogenesis stage,
the CRISPR array is transcribed into a precursor to crRNA molecules
(pre-crRNA), which are then cleaved into mature crRNAs. These
system

Cas3

mature crRNAs form effector complexes with Cas proteins. When

Cas12

Cas13
Cas9

foreign genetic material sequences match a CRISPR spacer, the

matching crRNA binds to the invading strand and cleaves the invad-
ing strand with the help of Cas nuclease (CRISPR interference stage)
Spacer acquisition

Cas1/Cas2/ Cas4

Cas1/Cas2/ Cas4

ssDNA and dsDNA Cas1/Cas2/ Cas4

archaea is referred to as CRISPR/Cas system. By integrat-

Cas1/Cas2

Cas1/Cas2

ing DNA sequences into their genome identical to previous

strategy

invaders, bacteria and archaea generate a cellular memory

with the name of isolated organism with references

NA

of them [29]. These acquired sequences permit them to spot

viruses or mobile genetic invaders resulting in the degra-
dation of the invading sequences and work as an adaptive
immune system [29, 30].
CRISPR immunity is denoted by distinct phases. In
dsDNA
ssDNA

ssDNA

ssRNA
Type Protein Target

the adaptation phase, bacterium and archaea gain cellular

–

memory of the invading phages [30]. The phage genome

sequences are integrated into the CRISPR locus of the
Cas10

C2c2
Cas3

Cas9

Cpf1
Csf1

bacterial or archaeal genomes, composed of 24 to 47 base

pair (bp) repeats and separated by spacers. The unique loci
IV

VI
III

of the CRISPR system were first discovered in 1987 [14].

V
Class2 II
Class1 I

Then Bolotin et al. in 2005 identified CRISPR’s function

Class

(enzymes encoding Cas genes generate DNA fragmentation)

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involved in protecting the bacteria and archaea from foreign acquisition in vivo; however, the molecular mechanism
invaders [31]. Later, scientists revealed that the CRISPR/Cas behind these proteins throughout immunity is unclear [43].
system works as an adaptive immune system [32] that was Cas1 and Cas2 proteins formed an integrase complex con-
eventually adopted as a versatile system for RNA program- sisting of two distal Cas1 dimers bridged by a Cas2 dimer
mable genome editing [8]. [44]. The Cas1 and Cas2 proteins bind to the pre-spacer
like twin-folded DNA. The pre-spacer integrated the proxi-
Cas Proteins of the CRISPR System mal leader region of the CRISPR array guided by the leader
sequence with a pair of inverted repeats inside the CRISPR
Cas proteins have gained popularity among the research repeat [45].
community for broader genome engineering applications and
are currently used in diverse fields, including biotechnol- Applications of Cas1 and Cas2 Proteins
ogy, agriculture, and medical research. Recently discovered
programmable Cas proteins like Cas 12, Cas 13, and Cas 14 Cas1 and Cas2 are the conserved proteins among all
have improved the precision of the CRISPR/Cas-mediated CRISPR/Cas systems. The molecular mechanism behind
genome editing (Table 2). The mechanism of these different the Cas1 and Cas2 proteins is still unclear. Hence, research-
Cas proteins, pros and cons, and their applications are sum- ers have not applied these proteins for genome-editing in
marized in detail below. various fields.

Cas1 and Cas2 Proteins Pros and Cons of Cas1 and Cas2 Proteins

Cas1 and Cas2 are generally conserved proteins in the So far, no genome editing studies have been demonstrated
prokaryotic adaptive immune system [28]. CRISPR/Cas via Cas1 and Cas2 proteins; therefore, significant merits
system consists of a CRISPR array containing the repeats and demerits of Cas1 and Cas2 proteins should be evalu-
(∼30 to 40 bp) separated by spacers, an adjacent module ated before applying these in diverse fields.
comprising Cas1 and Cas2, and the distinctive signature
proteins [41].
Cas9 Protein
Mechanism of Cas1 and Cas2 Proteins
Cas9 is a protein associated with the CRISPR adaptive
Cas1 and Cas2 belong to the Type II CRISPR system found immune systems of S. pyogenes and is referred to as SpyCas9
in E. coli [42]. E. coli Cas1-Cas2 complex mediates spacer protein. The SpyCas9 protein had a large multifunctional

Table 2  Details on various CRISPR/Cas proteins with their host organism, sgRNA size, PAM sequence with their target molecule and cut site
Protein name Host organism sgRNA PAM sequence Target Cut site References
sequence
size

Cas9 S. pyogenes 20 5ʹ-NGG-3ʹ dsDNA 5ʹ of PAM [33]

Cas9 S. pyogenes – 5ʹ-NAC, NTG, NTT, and NCG-3ʹ DNA 5ʹ of PAM [34]
Cas9 F. novicida 20 5ʹ-NGG-3ʹ DNA 5ʹ of PAM –
Cas9 S. aureus 21 5ʹ-NNGRRT-3ʹ DNA 5ʹ of PAM [35]
Cas9 Neisseria meningitidis 24 5ʹ-NNNNGATT-3ʹ DNA 5ʹ of PAM [35]
Cas9 S. thermophilus 20 5ʹ-NNAGAAW 5ʹ DNA 5ʹ of PAM –
Cas9 S. thermophilus 20 5ʹ-NGGNG-3 DNA 5ʹ of PAM [36]
Cas9 Campylobacter jejuna 22 NNNNACAC and NNNRYAC​ DNA 5ʹ of PAM [37]
C2c1 Alicyclobacillus acidoterrestris 20 T-rich PAM DNA 5ʹ of PAM [38]
Cpf1 Prevotella and Francisella 20 TTTV DNA 5ʹ of PAM [25]
Cpf1 Acidaminococcus sp. 24 5ʹ-TTTN-3ʹ DNA 3ʹ of PAM [39]
Cas12a Acidaminococcus sp. – Thymine-rich PAM DNA 5ʹ of PAM [37]
Cas13 Lb 28 Non-G nucleotide at the 3ʹ proto- ssRNA – [26]
spacer flanking site (PFS)
Cas14 Uncultivated archaea – – ssDNA – [40]

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domain of 1368 amino acids and acts as a DNA endonucle- responsible for binding with the target sequence. The HNH
ase in natural and artificial CRISPR/Cas systems. and RuvC are nuclease domains to chop the target sequence
[46, 47] (Fig. 2).
Mechanism of Cas9 Protein The Cas9 protein remains inactive due to the absence of
gRNA. The engineered gRNA forms a T-shape involved in
The mechanism of Cas9 protein has been studied extensively. 1 tetra-loop and 3 stem-loops [8, 46]. The gRNA is pro-
Cas9 protein has six domains (1) Recognition lobe (REC I), grammed to own a 5′ end, which is complementary to the
(2) REC II, (3) Arginine-rich bridge helix, (4) PAM Inter- target sequence. The programmed gRNA binds to the Cas9
acting, (5) HNH, and (6) RuvC. REC I is the major domain and generates changes in the protein, which leads the inac-
responsible for binding with the gRNA; the REC II function tive Cas9 protein into its active form. Once triggered, it
is not studied [46]. The arginine-rich bridge helix initiates searches the target sequence by binding with a sequence
cleavage activity upon binding to targeted sequences. The that matches the PAM sequence (5′-NGG-3′). Then Cas9
interaction with PAM confers PAM specificity, which is cuts dsDNA at 3 bp upstream of the PAM using its HNH

Fig. 2  Schematic illustration of CRISPR/Cas9 mechanism. A The nuclease domains to chop the target sequence. The Cas9 protein
Cas9 protein complex contains six domains (Recognition lobe (REC remains inactive due to the absence of gRNA. B The programmed
I), REC II, Arginine-rich bridge helix, PAM Interacting, HNH, and gRNA binds to the Cas9 and generates changes in the protein, which
RuvC). REC I is the major domain responsible for binding with the leads the inactive Cas9 protein into its active form. Once triggered, it
gRNA, the REC II function is not studied. The arginine-rich bridge searches the target sequence by binding with a sequence that matches
helix initiates cleavage activity upon binding to targeted sequences. the PAM sequence (5′-NGG-3′). Then Cas9 generates DSBs at 3 bp
The interaction with PAM confers PAM specificity, which is respon- upstream of the PAM using its HNH and RuvC domains
sible for binding with the target sequence. The HNH and RuvC are

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and RuvC domains. The HNH domain cleaves the DNA nuclease domains to slow down the transcription. Using
strand that is complementary to the 20-nucleotide sequence effector fusion (Cas9 proteins or sgRNA) to alter transcrip-
(gRNA) of crRNA (target strand)) and the RuvC domain tion states of specific genomic loci, or rearrange the genome,
cleaves the DNA strand opposite to the complementary can significantly expand the genome engineering modifi-
strand (non-target DNA strand). The spyCas9 system for cation utilizing the CRISPR/Cas9 system. CRISPR/Cas9
cleaving target DNA recognizes a short ‘seed’ sequence system has been widely adopted by researchers and applied
with the 5'-NGG-3' di-nucleotide containing PAM [8]. The in diverse fields, including microbes [48], plants [49–51],
twin tracrRNA: crRNA was fused into a single guide RNA animals [52], insects [53, 54], and human cell lines [55]
(sgRNA), making the CRISPR/Cas9 system to cut the tar- (briefly explained in Fig. 3).
geted dsDNA or ssDNA sequences [8].
Pros and Cons of Cas9 Protein
Application of Cas9 Protein
The advantage of the CRISPR/Cas9 system is the design
Cas9 protein holds great promise for efficient and targeted simplicity with more efficiency than existing ZFN and
genome engineering applications in research, medicine, and TALEN systems [8]. Multiplexed genome editing is another
biotechnology. The ease of genome modification in many significant advantage of Cas9, which can be achieved by
species by simply designing a gRNA sequence enables large- designing multiple sequence-specific gRNAs simultaneously
scale genome editing experiments to probe genome function [8].
more than traditional gene editing techniques like TALEN Despite numerous advances, CRISPR/Cas9 system faced
and ZFN. Further, the Cas9 protein is also converted into several disadvantages and also opened numerous queries
an RNA-guided homing device (dCas9) by inactivating the about risks associated with editing. One example is the

Fig. 3  Applications of CRISPR/Cas9 system. CRISPR/Cas9 system eases, CRISPR/Cas9 is also being utilized for studying the model and
has revolutionized genome engineering: its accuracy, rapidity, and non-model insects’ biology, somatic genome editing, manufacturing
affordability permit its use in a nearly limitless range of applica- biofuels, and engineering better crops (rice, wheat, etc.), etc. These
tions. Since its discovery, researchers have been using the CRISPR/ advances made possible by the invention of the CRISPR/Cas9 system
Cas9 system to cure diseases, discover new treatments, and for pre- will change the lives of people globally
cision medicine. It does not stop there; beyond treating human dis-

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gRNA, which guides the Cas9 to cleave dsDNA or ssDNA Mechanism of Cas12 Protein
showed higher on- and off-target mutations in the targeted
organisms [9, 55]. Even the finest accessible CRISPR/Cas The Cas12 protein requires only the crRNAs to create an
system that uses HDR also induces undesirable mutations. efficient cut at ssDNA and dsDNA. Cas12 protein contains
However, these off-target effects can be reduced by utilizing the RuvC and nuclease lobe (NUC) domains for cleavage
the modified Cas9 version called null Cas9 (nCas9), which activity [56]. Like Cas9, Cas12 encounters a potential target
generates a nick in only one strand than the DSBs [55]. How- site beside a PAM sequence. Once Cas12 starts encounter-
ever, 100% off-targets cannot be reduced using nCas9, which ing, it initiates R-loop, which forms base-pair hybridization
requires upgraded Cas9 versions in the future. between the crRNA and the target DNA strand. During this
step, Cas12 matches the < 17 bp of the target sequence and
leads to an R-loop formation [47]. Once R-loop is formed,
Cas12 Protein the Cas12 protein uses its active RuvC domain to cleave
the non-target strand with the help of the PAM sequence
Cas12 is a versatile protein with more dynamic applications, [56] (Fig. 4). However, the function of the RuvC domain
including epigenome editing. The Cas12 protein belongs to of Cas12 protein in cleaving the targeted DNA strand is not
the type V CRISPR system [56]. The Cas12 protein had yet clearly studied.
recently emerged as an effector RNA-guided DNA endo-
nuclease that becomes an alternative to the Cas9 protein Application of Cas12 Protein
for genome editing [25]. The Cas12 protein was isolated
from the Acidaminococcus species (AsCas12a) and Lachno- A more concise system, CRISPR/Cas12 cleaves, dsDNA or
spiraceae bacterium (LbCas12a) that fight against invading ssDNA via the RuvC domain and does not require tracrRNA.
viruses. Cas9 protein discriminates more accurately against Recently, Doudna’s group introduced a novel CRISPR/
mismatches within the initial ~ 10 bp of the RNA–DNA helix Cas diagnostic tool termed DNA endonuclease-targeted
proximal to the PAM sequence [25]. Moreover, Cas12 isn’t a CRISPR trans-reporter (DETECTR) based on Cas12 pro-
bit like the Cas9 protein; as a result, this protein can process tein (Fig. 5b) [57]. This DETECTR technique uses type V
the precursor crRNA by itself, which doesn’t require tracr- enzyme to cleave ssDNA sequence in a three-stage process:
RNA or RNase III. This process enhanced researchers to use (1) Cas12a protein and target crRNA are complemented
Cas12 protein for multiplex genome editing [25]. to the DNA reporter probes; once crRNA recognizes its

Fig. 4  Schematic illustration

of CRISPR/Cas12 mechanism.
The Cas12 protein requires only
the crRNAs to generate DSBs.
Cas12 protein cleaves the target
region beside a PAM sequence
(CTA, TTN, TTTN) with the
help of the RuvC and nuclease
lobe (NUC) domains. Once
Cas12 starts encountering, it
initiates R-loop, which forms
base-pair hybridization between
the crRNA and the target DNA
strand. During this step, Cas12
matches the < 17 bp of the target
sequence and leads to an R-loop
formation. Once R-loop is
formed, the Cas12 protein uses
its active RuvC domain and
generates a staggering cut in the
non-target strand with the help
of the PAM sequence

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Fig. 5  a and b Mechanism of SHERLOCK and DETECTR systems. targeted sequence, (B) In DNA endonuclease-targeted CRISPR trans
(A) Targeted double-stranded DNA (dsDNA) or RNA is amplified reporter (DETECTR), DNA is amplified with RPA. The Cas12 sys-
with recombinase polymerase amplification (RPA) or reverse tran- tem pairs with the single-stranded DNA (ssDNA) of interest, and the
scription (RT)-RPA. The RPA is coupled with T7 transcription to DNase activity of Cas12 system is initiated. This amplification step,
covert targeted RNA for detection by Cas13 system. This amplifica- combined with the reporter probe, enables DETECR to detect the tar-
tion step with the combination of reporter probe, enable specific high- geted sequence
sensitivity enzymatic reporter unlocking (SHERLOCK) to detect the

target sequence through Cas12a protein, the Cas12a protein detecting novel severe acute respiratory syndrome coronavi-
turns on collateral action and cleaves the target ssDNA or rus-2 (SARS-Cov-2). Mammoth Biosciences, Inc. targeted
dsDNA. (2) Target DNA probes bind with fluorophores and two Nucleocapsid (N) and Envelope (E) genes of SARS-
a quencher molecule. (3) Degradation of the DNA probes Cov-2 and observed faster virus detection within 1 h [60].
releases fluorophore and quencher, producing a robust In brief, they generated Cas12-gRNAs to specifically target
fluorescent signal for detecting targeted ssDNA or dsDNA the SARS-CoV-2 and optimized the DETECTR assay for E
cleavage [58]. In addition, this system detected even a sin- and N genes. They utilized this upgraded DETECTR assay
gle molecule of viral particles (Fig. 5b). For instance, Chen and detected the SARS-CoV-2 on a lateral flow strip within
et al. (2018) used the DETECTR technique and detected 1 h. Similar to this, Argentina and CASPR Biotech used a
Human Papilloma Viruses (HPV) [57]. In brief, they com- quick and portable SARS-CoV-2 diagnostic method based
bined the non-specific ssDNA of Cas12 with DETECTR on CRISPR/Cas12 system [61]. They gathered saliva sam-
and differentiated several HPV16 and HPV18 strains from ples from COVID-19 patients and reported that the naturally
crude DNA extracts of clinical samples within 1 h. Fur- occurring proteins in saliva had no inhibitory effects on the
thermore, researchers detected African Swine Fever Virus CRISPR/Cas12-based paper strip experiment [61]. Addition-
(ASFV) by employing the CRISPR/Cas12 system [59]. They ally, a Chinese institution used the CRISPR/Cas12-based
combined a fluorescent-based point of care (POC) system DETECTR system to confirm a clear detection of SARS-
with CRISPR/Cas12 and detected ASFV within 2 h. From CoV-2 [62]. They engineered gRNA that targeted the orf1a,
this result, they reported that the CRISPR/Cas12 is very orf1b, N, and E genes of the SARS (Wuhan-Hu-1 strain) and
specific and can detect even up to a single nucleotide of a related viruses, in which they detected single nucleotide pol-
targeted virus [59]. Recently, DETECTR is also utilized for ymorphisms (SNPs). From these results, researchers stated

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that CRISPR/Cas12-based detection could be employed for of a template [68]. Although this system has been success-
the efficient and rapid diagnosis of COVID-19. Jiang et al. fully used to acquire accurate DNA insertion into the desired
(2021) recently developed a magnetic-pull-down-assisted genomic loci, its effectiveness varies depending on the cell
colorimetric (M-CDC) technique coupled to the CRISPR/ type. DNA repair through HDR is also associated with
Cas12 system to detect SARS-CoV-2. They used gold nano- active cell division, making these tools ineffective in cell
particle (AuNP) probes for this technique to detect SARS- division (for example, neurons) [69]. However, significant
CoV-2. Additionally, they screened 41 viral samples and continuing research aims to tailor the Cas12 system further
reported that M-CDC is a useful technique for screening to ensure accurate DNA insertion into the targeted genome.
SARS-CoV-2 variants without requiring advanced instru- Apart from this drawback, this system has a wide range of
ments [63]. These applications of Cas12 nuclease enhanced applicability, and ongoing endeavors are striving to generate
scientists to increase the scope of genome-editing in various enhanced CRISPR/Cas12 for robust genome engineering.
fields, including in diagnosing novel viruses [64–66].

Pros and Cons of Cas12 Protein Cas13 Protein

Like Cas9, Cas12 protein was also considered as a sole The most recently discovered Cas protein is Cas13. CRISPR/
member of the CRISPR family for genome editing. But in Cas13 system functions as an ‘adaptive’ immune system in
most circumstances, Cas12 was deemed superior to Cas9 archaea and bacteria to defend against the invading RNA
protein because Cas12 protein generates staggered DSBs elements [70]. The Cas13 protein family contains two sub-
and promotes HDR repair mechanism instead of both NHEJ types: (1) Cas13a protein from Leptotrichia shahii bacterium
& HDR [67]. Cas12 system also overcomes disadvantages (LshCas13a), which is formally known as C2c2 and belongs
associated with diagnostic strategies. For example, diagnos- to type VI, and (2) Cas13b from Prevotella sp. (PspCas13b)
ing SARS-COV-2 utilizing quantitative qRT-PCR demands belongs to the type III CRISPR/Cas system. This system tar-
a longer time to get the results [66]. But the CRISPR/Cas12- gets and cleaves only the ssRNA, not the ssDNA or dsDNA
based DETECTR detects the SARS-CoV-2 within 1 h [66]. [71].
These results proved the advantage of the CRISPR/Cas12
system, which can also be utilized in detecting newly emerg- Mechanism of Cas13a Protein
ing viruses in the future.
The rapid advancement of the CRISPR-Cas12 system for Cas13a protein is activated through a single crRNA, like
genome editing has proved revolutionary for the life sci- Cas12 protein from pre-crRNA processing. Cas13a protein
ences. Despite this technology's wide application areas, the comprises crRNA, NUC lobes, and two nucleotide-binding
CRISPR/Cas12 system faces several significant flaws. For (HEPN) RNase domains for targeting RNA (Fig. 6). The
example, the CRISPR/Cas12 system is dependent on host LshCas13a cleaves ssRNA, upon recognizing the target
cell DNA repair machinery with or without the presence sequence (22–28 nt) complementary to the crRNA spacer

Fig. 6  Schematic illustration of

CRISPR/Cas13a mechanism.
Cas13a protein is activated
through a single crRNA. Cas13a
protein comprises crRNA,
NUC lobes, and two nucleo-
tide-binding (HEPN) RNase
domains for targeting RNA. The
Cas13a cleaves ssRNA, upon
recognizing the target sequence
(22–28 nt) complementary
to the crRNA spacer. The
target sequence is flanked by a
protospacer-flanking site (PFS)
at the 3′-end and crRNA binds
together and cleaves the target
region of ssRNA without the
tracrRNA

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320 Molecular Biotechnology (2023) 65:311–325

[72, 73]. The target sequence is flanked by a protospacer- via a fluorescent signal [73]. In addition, while SHERLOCK
flanking site (PFS) at the 3′-end, which has a bias to adeno- combined with reverse transcription-recombinase polymer-
sine (A), uracil (U), and cytosine(C). LshCas13a and crRNA ase amplification (RT-RPA) or isothermal-RPA, the crRNA-
bind together and cleave the target region of ssRNA without Cas13a complex binds and cleaves a specific target sequence
the tracrRNA. with high specificity [72]. SHERLOCK is used for various
functions, including RNA detection, sensitive detection of
Mechanism of Cas13b Protein nucleic acid contamination, and general RNA/DNA quanti-
tation of specific qPCR assays [77].
The Cas13b protein is more precise than the Cas13a protein Additionally, Cas13 nuclease could potentially track the
since a PFS flanks RNA targeting with A, U, or G at the 5′ allele-specific expression of transcripts or disease-associ-
end and PAM (NAN/NNA) at the 3′ end [74]. Cas13b pro- ated mutations in cells. These attractive features opened
tein is associated with the mature crRNA. ÇRISPR/Cas13b new avenues to discriminate single nucleotide changes
complex searches for the target ssRNA and induces confor- with high sensitivity in many organisms, including viruses
mational changes at the ssRNA target, resulting in nonspe- (Fig. 5a). For instance, Gootenberg et al. (2018) diagnosed
cific RNA cleavage [75]. However, the mechanism behind E. coli and Pseudomonas aeruginosa with the DNA iso-
the Cas13b protein is not fully revealed, but scientists tested lated from these strains using the SHERLOCK system. They
the ability of the cas13b protein for RNA editing (Fig. 7). have also distinguished the isolates of Klebsiella pneumonia
with two types of resistance genes, such as K. pneumoniae
Application of Cas13 Protein carbapenemase (KPC) and New Delhi metallo-β-lactamase
1 [70]. Furthermore, SHERLOCK differentiated African
Cas13 proteins had wider application in various genome- and American strains of the zika virus, the dengue virus
editing and diagnostic fields. Beyond other applications like (DENV) serotype, and cancer-related DNA targets. In addi-
base editing, the Cas13a is also utilized for single-nucleotide tion, SHERLOCK detected Zika virus and DENV with
detection at any site on the target sequence [25]. Like the higher sensitivity within 30 min. Researchers also developed
DETECTR system, which depends on the activity of Cas12 a unique technique for detecting viral infection by combining
protein, the specific high-sensitivity enzymatic reporter SHERLOCK with heating unextracted diagnostic samples
unlocking (SHERLOCK) technique depends on Cas13 to obliterate nucleases (HUDSON) [75]. Within 2 hours,
protein (Fig. 5a) [76, 77]. The SHERLOCK system works updated SHERLOCK identified DENV in a patient's saliva,
by integrating the targeted RNA fragments with Cas13 blood, and serum. From these existing results, SHERLOCK
crRNA and fluorescent RNA probes. If the corresponding Biosciences, Inc. and Mammoth Biosciences, Inc. used the
target sequence exists in the sample, Cas13 recognizes it SHERLOCK technique for the detection of SARS-CoV-2.
via crRNA and cleaves fluorescent DNA probes, influencing They targeted S and Orf1ab genes and detected the SARS-
fluorophore and quencher. This results in detecting the target Cov-2 RNA sequence within an hour. Metsky et al. (2020)

Fig. 7  Schematic illustration of

CRISPR/Cas13b mechanism.
Cas13b protein is associated
with the mature crRNA. This
ÇRISPR/Cas13b complex
searches for the target ssRNA
and induces precise conforma-
tional changes at the ssRNA
target with the help of the
Protospacer flanking site (PFS),
which flanks RNA targeting at
the 5′ end and PAM sequence
(NAN/NNA) at the 3′ end,
resulting in nonspecific RNA
cleavage

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Molecular Biotechnology (2023) 65:311–325 321

also detected the SARS-CoV-2 RNA using the Cas13a pro- in developing novel RNA knockdown approaches with more
teins. They combined synthetic RNA targets with fluorescent specificity and efficiency.
visualizers and detected SARS-CoV-2-RNA [76]. To test
the effectiveness of detecting SARS-CoV-2, Rauch et al. Cas14 Protein
(2020) recently utilized the Cas13 based-Ragged-Equitable-
Scalable Testing (CREST) method [77]. This technology Doudna’s group investigated other and similar types (Cas9,
uses portable and lightweight LED visualizers built with Cas12, and Cas13) of Cas systems that were available in
plastic filters. Researchers also used lateral flow immu- nature [84]. They explored by creating a metagenomic data-
nochromatography strips to detect target RNAs, but these base of the bacterial genome to search for uncharacterized
strips are expensive. Therefore, they created a P51 cardboard Cas genes [84]. Surprisingly, they found Cas14 protein,
fluorescent visualizer, which was less expensive and could which codes for smaller Cas protein with MW 40–70 kd.
detect 10 copies of target RNA per millilitre. Additionally, This Cas14 protein is extremely smaller (400–700 amino
they utilised the smartphone camera to capture data, which acids) than the other characterized Cas proteins [84]. Due
they then uploaded to the cloud to enable POC testing [77]. to their small size, Doudna’s lab reported that Cas14 protein
It improves nucleic acid detection based on CRISPR and can target ssDNA without a PAM [84].
demonstrates its power to change diagnostic and surveil-
lance efforts by direct screening even on a vast population. Mechanism of Cas14 Protein
Later, the CRISPR/Cas13a test based on single-molecule
RNA diagnosis was developed by the Tian et al. (2020) This Cas14 cleaves ssDNA and confers immunity against
group, which eliminated reverse transcriptase and nucleic viruses with ssDNA genomes or mobile genetic elements
acid extension procedures [78]. They stated that the SARS- (MGEs) [84]. Cas14 protein recognizes the ssDNA, medi-
CoV-2 would be easily detectable using the CRISPR/Cas13a ates seed sequence interaction with the target ssDNA, and
test [78, 79]. These advantages of Cas13 protein enabled cleaves the ssDNA, not dsDNA or ssRNA. Like Cas9, the
researchers to target any non-targetable sequences in diverse Cas14 protein requires both tracrRNA and crRNA to target
organisms [80–82]. the ssDNA (Fig. 8). The cleavage efficiency of the Cas14
protein is more specific than Cas9, Cas12, and Cas 13 pro-
Pros and Cons of Cas13 Protein teins without the presence of the PAM region [40]. Thus,
this system meets all the criteria for high-fidelity genome
Like Cas9 and Cas12, CRISPR/Cas13 is also a robust, editing.
precise, and versatile RNA-targeting system, which opens
novel research horizons in diverse fields. Compared with Application of Cas14 Protein
earlier RNA manipulation systems, CRISPR/Cas13 offers
numerous advantages. For example, its modular construc- CRISPR/Cas14 system is now harnessed as superior to the
tion, which consists of a single protein effector module Cas13 system. Researchers combined the CRISPR/Cas14
and an RNA guide module, allows for significant scalabil- system with the DETECTR as a diagnostic approach for
ity by enabling the production of whole libraries of vari- high-fidelity detection of ssDNA. Harrington et al. (2018)
ous guide RNAs in addition to easy and quick design [83]. first used CRISPR/Cas14 system with the DETECTR tech-
The recently discovered Cas13 mutant versions (dCas13, nique. They designed gRNA targeting the HECT and RLD
Cas13x), which function as programmable RNA-binding Domain Containing E3 Ubiquitin Protein Ligase 2 (HERC2)
proteins, efficiently target different effectors to specific gene of human saliva samples from blue-eyed single nucle-
RNAs to induce specific mutations [83]. Due to the inher- otide polymorphisms (SNP) individuals. They exhibited
ent crRNA biogenesis, multiple RNAs can be targeted using strong activation of recognition of the blue-eyed SNP by
Cas13 precisely. Compared with RNAi, CRISPR/Cas13 Cas14, while the Cas12 system failed to detect the blue-eyed
mediated genome modifications are not limited to targeting SNP [40]. This result represents a cost-effective method for
cytoplasmic transcripts. In addition, Cas13 enables faster screening pathogenic mutations and provides a great oppor-
downregulation of gene expression by directly knocking out tunity for mapping the candidate genes associated with dif-
the cytoplasmic mRNA transcripts [83]. Recently, CRISPR/ ferent pathogens. Another group proposes the Cas14 for
Cas13-based SHERLOCK and SHERLOCKv2 also played viral diagnostic in combination with the simplified nucleic
a vital role in developing novel molecular diagnostic tools acid extraction, which does not involve complicated sample
to detect viruses, including SARS-CoV-2. Apart from these extraction like heating unextracted diagnostic samples to
major advantages, CRISPR/Cas13 faced off-target mutations obliterate nucleases (HUDSON). The results reported that
[55], which is the major drawback of this system. However, Cas14 could provide an advanced platform in the future for
future research would help overcome this obstacle and aid viral screening [72, 79]. This novel CRISPR/Cas14 system

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322 Molecular Biotechnology (2023) 65:311–325

Fig. 8  Schematic illustration

of CRISPR/Cas14 mechanism.
The Cas14 protein comprises
both tracrRNA and crRNA to
target ssDNA. Cas14 protein
recognizes the ssDNA with the
help of tracrRNA and crRNA,
mediates seed sequence interac-
tion with the target ssDNA, and
cleaves the ssDNA, not dsDNA
or ssRNA. The cleavage effi-
ciency of the Cas14 protein is
more specific than Cas9, Cas12,
and Cas 13 proteins without the
presence of the PAM region

crosses criteria with user-friendly, sensitive, and more spe- equipment, and the low cost have made many laboratories
cific applications for future genome engineering. It can be utilize these CRISPR/Cas-based systems to study the func-
harnessed for other purposes, including diagnostic appli- tion of genes in various organisms. The initial discovery of
cations like phylogenetic association (new viruses), epide- the Cas9 protein, which shows its spotlight in the CRISPR
miology association with other pathogens, and taxonomic system, has enhanced researchers to find different Cas vari-
analysis [85]. ants like Cas12, Cas13, and Cas14 proteins. This research
improvement in Cas proteins has provided exciting platforms
Pros and Cons of Cas14 Protein in genome engineering, including detecting RNA viruses
from plants, animals, and humans and treating infections.
The Cas14 protein, which edits dsDNA, ssDNA, and RNA, Several studies are currently underway to find novel Cas
holds various advantages over traditional Cas9 protein. For variants from nature. However, numerous aspects of the
example, the Cas14 protein that has around 500 amino acid mechanism of Cas proteins still lack sufficient understand-
is very small, so this protein could be easily delivered to ing. Therefore, understanding the molecular mechanism
target any tissue than Cas9 protein [86]. Additionally, the behind the Cas proteins, identification of PAM-less Cas
Cas14 protein's selectivity improves the fidelity of single proteins, and accurate targeting specificity to minimize
nucleotide polymorphism (SNP) [83]. Finally, the Cas14 off-target effects will increase the potential of utilizing Cas
protein has less limiting PAM requirements (uses only T-rich proteins for precise applications of genome engineering. In
sequences) [83], allowing it to edit more targeted genomic addition, identifying the uncharacterized advance Cas pro-
sequences than the Cas9 protein. However, only a few stud- teins that may still exist in many bacteria or archaea will
ies have been demonstrated, which requires extensive studies revolutionize multiple fields, including diagnosing novel
to assist Cas14 in several fields of research in the future. viruses, therapeutics, agriculture, breeding, etc. If these
proteins are fully addressed with improved genome-editing
abilities, it might kick-start new CRISPR “fever” offering
Conclusion and Future Perspectives influential and groundbreaking CRISPR technologies into
mainstream use in the near future.
The exploitation of CRISPR/Cas tools made a breakthrough
in genome engineering in the last few years. The discovery
of Cas proteins has revolutionized natural science research Author Contributions All authors contributed to the study's concep-
tion and design. EHV and SAC wrote the article. EHV and SAC drew
and enabled advances in basic research, therapeutics, and the images.
diagnostics. The ease to use, the requirement of very little

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Molecular Biotechnology (2023) 65:311–325 323

Funding No funding was received for this article. 15. Nakata, A., Amemura, M., & Makino, K. (1989). Unusual nucleo-
tide arrangement with repeated sequences in the Escherichia coli
K-12 chromosome. Journal of Bacteriology, 171(6), 3553–3556.
Declarations 16. Hermans, P. W., Van Soolingen, D., Bik, E. M., De Haas, P. E.,
Dale, J. W., & Van Embden, J. D. (1991). Insertion element IS987
Competing interest The authors declare that there are no competing from Mycobacterium bovis BCG is located in a hot-spot integra-
interests associated with the manuscript. tion region for insertion elements in Mycobacterium tuberculosis
complex strains. Infection and Immunity, 59(8), 2695–2705.
Ethical Approval No ethics approval is needed for this article. 17. Mojica, F. J. M., Díez-Villaseñor, C., Soria, E., & Juez, G. (2000).
Biological significance of a family of regularly spaced repeats in
the genomes of archaea, bacteria and mitochondria. Molecular
Microbiology, 36(1), 244–246.
18. Jansen, R., van Embden, J. D. A., Gaastra, W., & Schouls, L.
References M. (2002). Identification of genes that are associated with
DNA repeats in prokaryotes. Molecular Microbiology, 43(6),
1565–1575.
1. Capecchi, M. R. (2005). Gene targeting in mice: Functional
19. Haft, D. H., Selengut, J., Mongodin, E. F., & Nelson, K. E. (2005).
analysis of the mammalian genome for the twenty-first century.
A guild of 45 CRISPR-associated (Cas) protein families and mul-
Nature Reviews Genetics, 6(6), 507–512.
tiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS
2. McManus, M. T., & Sharp, P. A. (2002). Gene silencing in
Computational Biology, 1(6), e60.
mammals by small interfering RNAs. Nature Reviews Genetics,
20. Koonin, E. V., Makarova, K. S., & Zhang, F. (2017). Diversity,
3(10), 737–747.
classification and evolution of CRISPR-Cas systems. Current
3. Rudin, N., Sugarman, E., & Haber, J. E. (1989). Genetic and
Opinion in Microbiology, 37, 67–78.
physical analysis of double-strand break repair and recombina-
21. Koonin, E. V., & Makarova, K. S. (2017). Mobile genetic elements
tion in Saccharomyces cerevisiae. Genetics, 122(3), 519–534.
and evolution of CRISPR-Cas systems: All the way there and
4. Kass, E. M., & Jasin, M. (2010). Collaboration and competition
back. Genome Biology and Evolution, 9(10), 2812–2825.
between DNA double-strand break repair pathways. FEBS Let-
22. Shabalina, S. A., & Koonin, E. V. (2008). Origins and evolution
ters, 584(17), 3703–3708.
of eukaryotic RNA interference. Trends in Ecology & Evolution,
5. Stoddard, B. L. (2005). Homing endonuclease structure and
23(10), 578–587.
function. Quarterly Reviews of Biophysics, 38(1), 49.
23. Brouns, S. J. J., Jore, M. M., Lundgren, M., Westra, E. R., Sli-
6. Sander, J. D., Dahlborg, E. J., Goodwin, M. J., Cade, L., Zhang,
jkhuis, R. J. H., Snijders, A. P. L., & Van Der Oost, J. (2008).
F., Cifuentes, D., & Qi, Y. (2011). Selection-free zinc-finger-
Small CRISPR RNAs guide antiviral defense in prokaryotes. Sci-
nuclease engineering by context-dependent assembly (CoDA).
ence, 321(5891), 960–964.
Nature Methods, 8(1), 67–69.
24. Marraffini, L. A., & Sontheimer, E. J. (2008). CRISPR interfer-
7. Reyon, D., Tsai, S. Q., Khayter, C., Foden, J. A., Sander, J. D.,
ence limits horizontal gene transfer in staphylococci by targeting
& Joung, J. K. (2012). FLASH assembly of TALENs for high-
DNA. Science, 322(5909), 1843–1845.
throughput genome editing. Nature Biotechnology, 30(5), 460.
25. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I.
8. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A.,
M., Makarova, K. S., Essletzbichler, P., & Regev, A. (2015). Cpf1
& Charpentier, E. (2012). A programmable dual-RNA–guided
is a single RNA-guided endonuclease of a class 2 CRISPR-Cas
DNA endonuclease in adaptive bacterial immunity. Science,
system. Cell, 163(3), 759–771.
337(6096), 816–821.
26. Abudayyeh, O. O., Gootenberg, J. S., Essletzbichler, P., Han, S.,
9. Ceasar, S. A., Rajan, V., Prykhozhij, S. V., Berman, J. N., &
Joung, J., Belanto, J. J., & Regev, A. (2017). RNA targeting with
Ignacimuthu, S. (2016). Insert, remove or replace: A highly
CRISPR–Cas13. Nature, 550(7675), 280–284.
advanced genome editing system using CRISPR/Cas9. Bio-
27. Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao,
chimica et Biophysica Acta - Molecular Cell Research, 1863(9),
Y., Pirzada, Z. A., & Charpentier, E. (2011). CRISPR RNA matu-
2333–2344.
ration by trans-encoded small RNA and host factor RNase III.
10. Hille, F., Richter, H., Wong, S. P., Bratovič, M., Ressel, S., &
Nature, 471(7340), 602–607.
Charpentier, E. (2018). The biology of CRISPR-Cas: Backward
28. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval,
and forward. Cell, 172(6), 1239–1259.
P., Moineau, S., & Horvath, P. (2007). CRISPR provides acquired
11. Xu, Y., & Li, Z. (2020). CRISPR-Cas systems: Overview, inno-
resistance against viruses in prokaryotes. Science, 315(5819),
vations and applications in human disease research and gene
1709–1712.
therapy. Computational and Structural Biotechnology Journal,
29. Horvath, P., & Barrangou, R. (2010). CRISPR/Cas, the immune
18, 2401–2415.
system of bacteria and archaea. Science, 327(5962), 167–170.
12. Makarova, K. S., Haft, D. H., Barrangou, R., Brouns, S. J. J.,
30. Semenova, E., Jore, M. M., Datsenko, K. A., Semenova, A.,
Charpentier, E., Horvath, P., & Yakunin, A. F. (2011). Evolution
Westra, E. R., Wanner, B., & Severinov, K. (2011). Interfer-
and classification of the CRISPR–Cas systems. Nature Reviews
ence by clustered regularly interspaced short palindromic repeat
Microbiology, 9(6), 467–477.
(CRISPR) RNA is governed by a seed sequence. Proceedings of
13. Wang, F., Wang, L., Zou, X., Duan, S., Li, Z., Deng, Z., & Chen,
the National Academy of Sciences, 108(25), 10098–10103.
S. (2019). Advances in CRISPR-Cas systems for RNA targeting,
31. Bolotin, A., Quinquis, B., Sorokin, A., & Ehrlich, S. D. (2005).
tracking and editing. Biotechnology Advances, 37(5), 708–729.
Clustered regularly interspaced short palindrome repeats (CRIS-
14. Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata,
PRs) have spacers of extrachromosomal origin. Microbiology,
A. (1987). Nucleotide sequence of the iap gene, responsible for
151(8), 2551–2561.
alkaline phosphatase isozyme conversion in Escherichia coli,
32. Koonin, E. V., & Wolf, Y. I. (2009). Is evolution Darwinian or/
and identification of the gene product. Journal of Bacteriology,
and Lamarckian? Biology Direct, 4(1), 1–14.
169(12), 5429–5433.
33. Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J., &
Soria, E. (2005). Intervening sequences of regularly spaced

13
324 Molecular Biotechnology (2023) 65:311–325

prokaryotic repeats derive from foreign genetic elements. Journal 50. Hillary, V. E., & Ceasar, S. A. (2019). Application of CRISPR/
of Molecular Evolution, 60(2). 174–182. https://​doi.​org/​10.​1007/​ Cas9 genome editing system in cereal crops. The Open Biotech-
s00239-​004-​0046-3 nology Journal, 13(1), 173–179.
34. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., 51. Ceasar, S. A., Maharajan, T., Hillary, E., & Krishna, T. P. A.
Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. (2022). Insights to improve the plant nutrient transport by
(2013). Multiplex genome engineering using CRISPR/Cas sys- CRISPR/Cas system. Biotechnology Advances, 59, 107963.
tems. Science, 339(6121). 819–823. https://​doi.​org/​10.​1126/​scien​ 52. Lee, H., Yoon, D. E., & Kim, K. (2020). Genome editing methods
ce.​12311​43 in animal models. Animal Cells and Systems, 24(1), 8–16.
35. Esvelt, K. M., Mali, P., Braff, J. L., Moosburner, M., Yaung, S. 53. Hillary, V. E., Ceasar, S. A., & Ignacimuthu, S. (2020). Genome
J., & Church, G. M. (2013). Orthogonal Cas9 proteins for RNA- engineering in insects: focus on the CRISPR/Cas9 system.
guided gene regulation and editing. Nature Methods, 10(11), Genome engineering via CRISPR-Cas9 system (pp. 219–249).
1116–1121. https://​doi.​org/​10.​1038/​nmeth.​2681 Elsevier.
36. Horvath, P., Romero, D. A., Coûté-Monvoisin, A. C., Richards, 54. Hillary, V. E., & Ceasar, S. A. (2021). Genome engineering in
M., Deveau, H., Moineau, S., Boyaval, P., Fremaux, C., & Bar- insects for the control of vector borne diseases. Progress in Molec-
rangou, R. (2008). Diversity, activity, and evolution of CRISPR ular Biology and Translational Science, 179, 197–223.
loci in Streptococcus thermophilus. Journal of Bacteriology, 55. Rodríguez-Rodríguez, D. R., Ramírez-Solís, R., Garza-Elizondo,
190(4), 1401–1412. https://​doi.​org/​10.​1128/​JB.​01415-​07 M. A., Garza-Rodríguez, M. D. L., & Barrera-Saldaña, H. A.
37. Kim, E., Koo, T., Park, S. W., Kim, D., Kim, K., Cho, H.-Y., Song, (2019). Genome editing: A perspective on the application of
D. W., Lee, K. J., Jung, M. H., Kim, S., Kim, J. H., Kim, J. H., CRISPR/Cas9 to study human diseases. International Journal of
& Kim, J.-S. (2017). In vivo genome editing with a small Cas9 Molecular Medicine, 43(4), 1559–1574.
orthologue derived from Campylobacter jejuni. Nature Commu- 56. Fonfara, I., Richter, H., Bratovič, M., Le Rhun, A., & Charpen-
nications, 8(1), 14500. https://​doi.​org/​10.​1038/​ncomm​s14500 tier, E. (2016). The CRISPR-associated DNA-cleaving enzyme
38. Yang, H., Gao, P., Rajashankar, K. R., & Patel, D. J. (2016). Cpf1 also processes precursor CRISPR RNA. Nature, 532(7600),
PAM-dependent target DNA recognition and cleavage by C2c1 517–521.
CRISPR-Cas endonuclease. Cell, 167(7), 1814–1828.e12. https://​ 57. Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X.,
doi.​org/​10.​1016/j.​cell.​2016.​11.​053 Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target
39. Yamano, T., Nishimasu, H., Zetsche, B., Hirano, H., Slaymaker, binding unleashes indiscriminate single-stranded DNase activity.
I. M., Li, Y., Fedorova, I., Nakane, T., Makarova, K. S., Koonin, Science, 360(6387), 436–439.
E. V., Ishitani, R., Zhang, F., & Nureki, O. (2016). Crystal struc- 58. Makarova, K. S., Wolf, Y. I., Iranzo, J., Shmakov, S. A., Alkhn-
ture of Cpf1 in complex with guide RNA and target DNA. Cell, bashi, O. S., Brouns, S. J. J., & Horvath, P. (2020). Evolutionary
165(4), 949–962. https://​doi.​org/​10.​1016/j.​cell.​2016.​04.​003 classification of CRISPR–Cas systems: A burst of class 2 and
40. Harrington, L. B., Burstein, D., Chen, J. S., Paez-Espino, D., derived variants. Nature Reviews Microbiology, 18(2), 67–83.
Ma, E., Witte, I. P., & Doudna, J. A. (2018). Programmed DNA 59. He, Q., Yu, D., Bao, M., Korensky, G., Chen, J., Shin, M., & Du,
destruction by miniature CRISPR-Cas14 enzymes. Science, K. (2020). High-throughput and all-solution phase African Swine
362(6416), 839–842. Fever Virus (ASFV) detection using CRISPR-Cas12a and fluores-
41. Wiedenheft, B., Zhou, K., Jinek, M., Coyle, S. M., Ma, W., & cence based point-of-care system. Biosensors and Bioelectronics,
Doudna, J. A. (2009). Structural basis for DNase activity of a con- 154, 112068.
served protein implicated in CRISPR-mediated genome defense. 60. Broughton, J. P., Deng, X., Yu, G., Fasching, C. L., Servellita, V.,
Structure, 17(6), 904–912. Singh, J., & Sotomayor-Gonzalez, A. (2020). CRISPR–Cas12-
42. Datsenko, K. A., Pougach, K., Tikhonov, A., Wanner, B. L., Sev- based detection of SARS-CoV-2. Nature Biotechnology, 38(7),
erinov, K., & Semenova, E. (2012). Molecular memory of prior 870–874.
infections activates the CRISPR/Cas adaptive bacterial immunity 61. Satyanarayana, M. (2020). A COVID-19 diagnostic that uses
system. Nature Communications, 3(1), 1–7. CRISPR gets a nod from the FDA chemical & engineering news.
43. Babu, M., Beloglazova, N., Flick, R., Graham, C., Skarina, 62. Feng, M., & Li, X. (2020). Land cover mapping toward finer
T., Nocek, B., & Binkowski, A. (2011). A dual function of the scales. Science Bulletin, 65(19), 1604–1606.
CRISPR–Cas system in bacterial antivirus immunity and DNA 63. Jiang, Y., Hu, M., Liu, A.-A., Lin, Y., Liu, L., Yu, B., & Pang,
repair. Molecular Microbiology, 79(2), 484–502. D. W. (2021). Detection of SARS-CoV-2 by CRISPR/Cas12a-
44. Yosef, I., Goren, M. G., & Qimron, U. (2012). Proteins and DNA enhanced colorimetry. ACS Sensors, 6(3), 1086–1093.
elements essential for the CRISPR adaptation process in Escheri- 64. Bandyopadhyay, A., Kancharla, N., Javalkote, V. S., Dasgupta, S.,
chia coli. Nucleic Acids Research, 40(12), 5569–5576. & Brutnell, T. P. (2020). CRISPR-Cas12a (Cpf1): A versatile tool
45. Wright, A. V., & Doudna, J. A. (2016). Protecting genome integ- in the plant genome editing tool box for agricultural advancement.
rity during CRISPR immune adaptation. Nature Structural & Frontiers in Plant Science, 11, 1589.
Molecular Biology, 23(10), 876. 65. Tsukamoto, T., Sakai, E., Iizuka, S., Taracena-Gándara, M.,
46. Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. Sakurai, F., & Mizuguchi, H. (2018). Generation of the adenovi-
I., Dohmae, N., & Nureki, O. (2014). Crystal structure of Cas9 in rus vector-mediated CRISPR/Cpf1 system and the application for
complex with guide RNA and target DNA. Cell, 156(5), 935–949. primary human hepatocytes prepared from humanized mice with
47. Anders, C., Niewoehner, O., Duerst, A., & Jinek, M. (2014). chimeric liver. Biological and Pharmaceutical Bulletin, 41(7),
Structural basis of PAM-dependent target DNA recognition by 1089–1095.
the Cas9 endonuclease. Nature, 513(7519), 569–573. 66. Teng, F., Li, J., Cui, T., Xu, K., Guo, L., Gao, Q., & Zhou, Q.
48. Liu, R., Chen, L., Jiang, Y., Zhou, Z., & Zou, G. (2015). Efficient (2019). Enhanced mammalian genome editing by new Cas12a
genome editing in filamentous fungus Trichoderma reesei using orthologs with optimized crRNA scaffolds. Genome Biology,
the CRISPR/Cas9 system. Cell Discovery, 1(1), 1–11. 20(1), 1–6.
49. Zhang, Y., Massel, K., Godwin, I. D., & Gao, C. (2018). Appli- 67. Hillary, V. E., & Ceasar, S. A. (2022). Prime editing in plants
cations and potential of genome editing in crop improvement. and mammalian cells: Mechanism, achievements, limitations, and
Genome Biology, 19(1), 1–11. future prospects. BioEssays, 44, 2200032.

13
Molecular Biotechnology (2023) 65:311–325 325

68. Hillary, V. E., Ignacimuthu, S., & Ceasar, S. A. (2021). Potential Field-deployable viral diagnostics using CRISPR-Cas13. Science,
of CRISPR/Cas system in the diagnosis of COVID-19 infection. 360(6387), 444–448.
Expert Review of Molecular Diagnostics, 21(11), 1179–1189. 80. Metsky, H. C., Freije, C. A., Kosoko-Thoroddsen, T. S. F., Sabeti,
69. Al-Shayeb, B., Sachdeva, R., Chen, L. X., Ward, F., Munk, P., P. C., & Myhrvold, C. (2020). CRISPR-based surveillance for
Devoto, A., & Amano, Y. (2020). Clades of huge phages from COVID-19 using genomically-comprehensive machine learning
across Earth’s ecosystems. Nature, 578(7795), 425–431. design. BioRxiv. https://​doi.​org/​10.​1101/​2020.​02.​26.​967026
70. East-Seletsky, A., O’Connell, M. R., Knight, S. C., Burstein, D., 81. Rauch, S., Roth, N., Schwendt, K., Fotin-Mleczek, M., Mueller,
Cate, J. H. D., Tjian, R., & Doudna, J. A. (2016). Two distinct S. O., & Petsch, B. (2020). mRNA based SARS-CoV-2 vaccine
RNase activities of CRISPR-C2c2 enable guide-RNA processing candidate CVnCoV induces high levels of virus neutralizing anti-
and RNA detection. Nature, 538(7624), 270–273. bodies and mediates protection in rodents. bioRxiv., 11, 12.
71. Liu, L., Li, X., Wang, J., Wang, M., Chen, P., Yin, M., & Wang, Y. 82. Palaz, F., Kalkan, A. K., Can, O., Demir, A. N., Tozluyurt, A.,
(2017). Two distant catalytic sites are responsible for C2c2 RNase Özcan, A., & Ozsoz, M. (2021). CRISPR-Cas13 system as a
activities. Cell, 168(1–2), 121–134. promising and versatile tool for cancer diagnosis, therapy, and
72. Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., research. ACS Synthetic Biology, 10(6), 1245–1267.
Dy, A. J., Joung, J., & Freije, C. A. (2017). Nucleic acid detection 83. Lotfi, M., & Rezaei, N. (2020). CRISPR/Cas13: A potential thera-
with CRISPR-Cas13a/C2c2. Science, 356(6336), 438–442. peutic option of COVID-19. Biomedicine & Pharmacotherapy,
73. Gootenberg, J. S., Abudayyeh, O. O., Kellner, M. J., Joung, J., 131, 110738.
Collins, J. J., & Zhang, F. (2018). Multiplexed and portable 84. Burstein, D., Harrington, L. B., Strutt, S. C., Probst, A. J., Anan-
nucleic acid detection platform with Cas13, Cas12a, and Csm6. tharaman, K., Thomas, B. C., & Banfield, J. F. (2017). New
Science, 360(6387), 439–444. CRISPR–Cas systems from uncultivated microbes. Nature,
74. Konermann, S., Lotfy, P., Brideau, N. J., Oki, J., Shokhirev, M. 542(7640), 237–241.
N., & Hsu, P. D. (2018). Transcriptome engineering with RNA- 85. Nayak, S., Blumenfeld, N. R., Laksanasopin, T., & Sia, S. K.
targeting type VI-D CRISPR effectors. Cell, 173(3), 665–676. (2017). Point-of-care diagnostics: Recent developments in a con-
75. Smargon, A. A., Cox, D. B. T., Pyzocha, N. K., Zheng, K., nected age. Analytical Chemistry, 89(1), 102–123.
Slaymaker, I. M., Gootenberg, J. S., & Makarova, K. S. (2017). 86. Mahas, A., Stewart, C. N., Jr., & Mahfouz, M. M. (2018). Harness-
Cas13b is a type VI-B CRISPR-associated RNA-guided RNase ing CRISPR/Cas systems for programmable transcriptional and
differentially regulated by accessory proteins Csx27 and Csx28. post-transcriptional regulation. Biotechnology Advances, 36(1),
Molecular Cell, 65(4), 618–630. 295–310.
76. Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O.,
& Zhang, F. (2020). SHERLOCK: Nucleic acid detection with Publisher's Note Springer Nature remains neutral with regard to
CRISPR nucleases. Nature Protocols, 15(3), 1311. jurisdictional claims in published maps and institutional affiliations.
77. Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O.,
& Zhang, F. (2019). SHERLOCK: Nucleic acid detection with Springer Nature or its licensor holds exclusive rights to this article under
CRISPR nucleases. Nature Protocols, 14(10), 2986–3012. a publishing agreement with the author(s) or other rightsholder(s);
78. Tian, T., Shu, B., Jiang, Y., Ye, M., Liu, L., Guo, Z., & Zhou, author self-archiving of the accepted manuscript version of this article
X. (2020). An ultralocalized Cas13a assay enables universal and is solely governed by the terms of such publishing agreement and
nucleic acid amplification-free single-molecule RNA diagnostics. applicable law.
ACS Nano., 15, 1167.
79. Myhrvold, C., Freije, C. A., Gootenberg, J. S., Abudayyeh,
O. O., Metsky, H. C., Durbin, A. F., & Parham, L. A. (2018).

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