The Botanical Review

https://doi.org/10.1007/s12229-021-09250-6

Evolution and Biology of CRISPR System: A New Era

Tool for Genome Editing in Plants

Shilpi Sharma 1 & Jyoti Vakhlu 1,2

1
School of Biotechnology, University of Jammu, Jammu J&K- 180006, India
2
Author for Correspondence; e-mail: jyotimetagenomic@gmail.com

# The New York Botanical Garden 2021

Abstract
It’s an evolution of its own kind that a technology changed the interface of biology in
such a short expanse of time. Merely a decade ago, scientists reported that the CRISPR-
Cas (clustered regularly interspaced short Palindromic repeats-CRISPR associated)
system is the part of bacteria and archea’s adaptive immune system which helps in
withstanding the attack against invading viruses by acquiring genetic records of
invaders to facilitate robust interference upon reinfection. In this Review, we discuss
the evolution of CRISPR along the time and recent advances in understanding the vivid
mechanism by which Cas proteins respond to foreign nucleic acids and how these
systems have been harnessed for precise genome manipulation in plants. With the
advancement in this technology, it will become easier to genetically modify the plants
for crop improvement.

Keywords CRISPR . Cas . Meganucleases . Genome editing . Crop improvement . sgRNA

Introduction

The ground breaking discoveries of Darwin and Mendel laid the scientific foundation
of plant breeding for plant improvement (Priyadarshan 2019). Classical plant breeding
has been used since nineteenth century to enhance the traits of the crops. It is done by
interbreeding closely or distantly related species to produce a hybrid crop with im-
proved traits. Classical breeding relies mostly on homologous recombination events to
generate genetic diversity and to eradicate undesirable traits. Conventional breeding is
an extremely successful method for crop improvement but it is a very slow and
relatively tedious process (Wolter et al. 2019). With the advances in technologies and
knowledge about plant molecular biology, molecular plant breeding came into being.
The era was marked by the landmark reports of production of transgenic and cisgenic
plants using Agrobacterium in 1980s, commonly known as genetically modified plants
(Chan et al. 1993). Transgenesis is referred to the genetic modification of the host plant
by transfer and random insertion of a foreign gene into the genome of the host plant
(Low et al. 2018). The foreign gene can be from non-plant organism or sexually
incompatible plant. The transgene usually includes sequence in antisense orientation,
coding sequence and regulatory sequences (Singh et al. 2018). Whereas, in cisgenic
 S. Sharma, J. Vakhlu

plants, the genetic manipulation of the host plant is done, using a gene from a sexually
compatible plant (Singh et al. 2018). Such genes, in their natural form contain introns
flanked by their native promoters and terminators in sense orientation. The conven-
tional gene delivery methods used for transformation are I) Agrobacterium mediated
transformation, II) gene gun based transformation, III) protoplast fusion. The major
limitation of the process is that transgenesis can extend the gene pool of the host plant
by the addition of foreign gene. It provides the host plant with new traits that are not
native. The traits might affect the fitness of the host plant and spread through the gene
flow between transgenic plants to wild relatives creating a shift in natural vegetation
(Stange et al. 2020). In addition, there are ethical issues related to the production of
such crops. Cisgenesis doesn’t extend the gene pool but it may also lead to new variety
which is indistinguishable from the wild type. Another problem with plants modified
by cisgenesis and transgenesis is the presence of reporters/markers and vector backbone
in the modified plant which are reported to be toxic to the environment (Nap et al.
1992) and are widely controlled by governmental policies (Conko 2003). Many
countries have resisted consumption of the transgenic plants as they have been skeptic
about their long time effects.
To overcome these challenges, nuclease based genome editing techniques have been
developed in the recent years. Genome editing leads to specific modification of a
genome at a precise location and in a predictable manner. Most important feature of the
technique is that it enables specific and efficient manipulation of plant genome without
introduction of any foreign gene. The plants modified using these nucleases are called
as genetic modified, non- transgenic plants (Chen et al. 2018). The major classes of
DNA binding proteins (engineered homing endonucleases or meganucleases) used for
genome wide alteration by site-specific double stranded DNA breaks are transcription
activator-like effectors (TALENs), zinc finger (ZF) nucleases, RNAi and the most
recent of all, RNA-guided DNA endonuclease Cas9 (CRISPR-Cas9 system). TALENs
and ZFNs both are exploited to mutate genomes at specific locus. Zinc Finger
Nucleases (ZFNs): The ZFNs comprise of two independent regions: a recognition
domain and a nuclease. The recognition domain is rich in zinc fingers that play a role
in identification of the target triplet nucleotides in the DNA, while the other region,
which is a non-specific nuclease, FokI creates the double stranded breaks (DSB). In
order to remain active nuclease has be dimerized hence ZFNs are to be used in pairs
(Sovová et al. 2016). Studies indicate that a target region of 9–18 bp can be modified
using ZFNs (Kim et al. 1996; Yang and Chen 2020). Transcription activator-like
effectors nucleases (TALENs): TALENS also comprise of two independent parts.
The first part consists of transcription activator-like factors (TALEs), these bind to
DNA sequences resulting in change in the expression of the plant genes (Puchta and
Fauser 2014). The second part is a FokI nuclease domain, which in turn can create DSB
in the targeted DNA. Another technology used for genome manipulation is RNA
interference (RNAi) that is based on mutation induced by dicer and argonaute protein.
This technique has certain drawbacks. It is generally non-specific, temporary and the
technique is restricted to the knocking down of only transcribed genes (Xu et al. 2019).
Zinc finger nucleases (ZFNs) are the oldest genetic modification tool that was
developed in 1990 by Sangamo BioSciences (Chandrasekharan et al. 2009). Zinc
finger nucleases have been successfully used for genome editing of many plants such
as maize, tobacco, soyabean etc. (Petolino 2015). The technology suffers from certain
Evolution and Biology of CRISPR System: A New Era Tool for Genome...

limitations; such as it is time consuming, has low specificity thus has high off-target
modification frequency and is expensive. These limitations gave way for a new
technology, Transcription activator-like effector nucleases (TALENs) (Zhang et al.
2019). TALENs unlike ZFNs recognize single nucleotide. TALEN DNA binding
domain and its target nucleotide are far simpler to design, than ZFNs and their target
nucleotide triplet. On the contrary, CRISPR system relies on ribonucleotide complex
instead of traditional protein-DNA complex. Specifically designed sgRNA can modify
any sequence in the genome and they are cost effective. Hence, CRISPR Cas based
genome editing systems are efficient and preferred tool of choice for genome editing as
compared to TALEN and ZFN. CRISPR system is known to be 200 times more cost
effective than TALENs and takes lesser time to be designed. A comparison between
CRISPR/Cas9, TALENs, ZFNs, and RNAi is given in Table 1.
Genome editing leads to specific modification of a genome at a precise location and
in a predictable manner. These nucleases ZFNs, TALENs, and meganucleases proves
to be a powerful tool to perform site-specific genome modifications, sequence deletion
activation/inactivation of genes and rearrangement of the chromosomes (Schmidt et al.
2020). But CRISPR Cas system has emerged as a much simple and cost effective
technique for genome engineering. This system provides the option of reprogramming
the CRISPR system specificity by using customizable small non coding RNAs.
The CRISPR sites, first observed in E.coli (Ishino et al. 1987), are present in about 84%
of archaea and 45% of bacteria (Kaushik et al. 2019).The genetic locus of CRISPR is
known as CRISPR array. It contains a 20–50 bp sequence separated by variable short
DNA sequences known as spacer, preceded by AT rich region. The invading microbe
possesses sequence similar to spaces known as Proto-spacers. The process of CRISPR
Cas9 mediated genome editing exploits repair mechanism of double stranded DNA break
after the cleavage by Cas9. The repair mechanism involves the incorporation of homology
coordinated repair (HDR) and non homologous end joining (NHEJ). NHEJ has been
mostly seen to occur during G1,S and G2 stages while homology coordinated repair
(HDR) rules in late S and G2 stage (Heyer et al. 2010). These two pathways have been
exploited by the scientists for the genome wide editing (Cong et al. 2013). CRISPR/Cas9
mediated genome editing is the first of its kind causing change to the genome of the living
organism, its contexts (e.g., epigenetic marks), or its outputs (e.g., transcripts). The
CRISPR loci are the sequences which are present in the coding sequences of the genes.
Cas9 is the CRISPR associated proteins having nuclease activity which mediates the
double stranded breaks in the DNA. The efficiency, with which Cas9 mediates double
stranded cuts, depends upon the sgRNA. The concept of the CRISPR/Cas9 system with
the sgRNA-derived target specificity provides an opportunity to target multiple sites at
once, as is the case in the natural bacterial system. The most common approach used by
some scientists to achieve multiplex sgRNA expression is to simply assemble numerous
sgRNA expression systems, each with its own promoter (Ma et al. 2015).
The emergence of CRISPR technology has over shadowed the use of ZFNs and
TALENs. CRISPR technology was named the “method of the year” in 2011 by nature
methods (Arora and Narula 2017). In 2015, CRISPR technology was considered to be
the “breakthrough of the year” (Travis 2015). Reflecting on the impact made by
CRISPR Cas technology, this revolutionary discovery has been awarded the 2020
Nobel Prize in chemistry to Dr. Jennifer Doudna and Dr.Emmanuelle Charpentier
(Strzyz 2020).
Table 1 Comparison of major tools used in genome editing. Limitations proposed by meganucleases, ZFNs, RNAi and TALENs surmounted by CRISPR Cas system as it is simple,
cost effective and opens up scope for multiplexing

ZFN TALEN CRISPR/Cas9 RNAi Meganucleases

Efficiency High High High High High

Target site 18–36 bp 24–40 bp 19–25 bp Target site should be located 14–40 bp
50–100 nt from ATG
Nuclease FokI FokI Cas9 Dicer and Argonaute protein I-SceI
Recognition molecule Protein-DNA Protein-DNA RNA-DNA Protein-DNA RNA
Target recognition frequency High High High with choice of High Low
multiplexing
Limitations Both expensive and time Tedious, time consuming and Off targets Off targets Limited versatility
consuming to construct takes long to construct works on transcribed genes only in targeting
Cost High Moderate Low Low Low
S. Sharma, J. Vakhlu
Evolution and Biology of CRISPR System: A New Era Tool for Genome...

This mini-review concenters on the contribution of fundamental research to the

discovery and evolution of the CRISPR-Cas system, its function, classification and
mode of action. In addition an overview of the procedure for genome editing in plants,
complimentary bioinformatic tools available and success stories in plant genome
editing have also been elaborated along with the limitation that the technique suffers
from. The review provides an insight into the breakthroughs and latest development of
CRISPR Cas technology and its possible utility in plant biology.

Evolution of CRISPR

CRISPR, for the first time was discovered 30 years ago by Y. Ishino in Escherichia coli
as 29 nucleotide tandem inverted repeat sequences (Ishino et al. 1987). Similarly in
1989, Francisco Mojica, a doctoral student working on an archea Haloferax
mediterranei discovered multiple copies of 36 bp Palindromic repeats separated by
spacers that didn’t match any other family of repeats found in known microbes. He
named them as short regularly spaced repeats (SRSRs) (Mojica et al. 1993). Later, these
sequences were named “clustered regularly interspaced palindromic repeats” (CRISPR)
(Jansen et al. 2002). By 2000, Mojica had discovered CRISPR loci in 20 different
microbes using bioinformatic tools (Mojica et al. 2000). In 2002, scientists validated
the presence of a gene in the vicinity of CRISPR loci and named them CRISPR
associated genes, presumed to be involved in the function of CRISPR (Jansen et al.
2002). These Cas genes were found to have helicases like activity of the superfamily 2,
and the motifs of the RecB family of exonucleases, depicting their involvement in
DNA metabolism or gene expression (Makarova et al. 2002; Jansen et al. 2002).
Initially, various hypothesis were proposed suggesting CRISPR to be involved in
DNA repair, gene regulation etc., which were later proved to be wrong (Mojica and
Garrett 2013). In 2003, Mojica concluded that CRISPR loci must be regulating the
adaptive immune system against the specific pathogen. At the same time, another group
proposed the role of CRISPR in the defence mechanism, as they put it more figura-
tively, “CRISPRs may represent a memory of ‘past genetic aggressions” (Pourcel et al.
2005). In 2007, Barrangou and co workers found CRISPR to be involved in the
resistance to bacteriophage. The specificity was controlled by spacer-phage sequence
similarity which could confer adaptive immunity to its host (Barrangou et al. 2007). In
2008, it was found that CRISPR specific sequence (NNAGAAW) located downstream
of the proto-spacer region called as the Proto-spacer adjacent motif (PAM). PAM is
important for the phage resistance phenotype and that the Cas9 nuclease activity cuts
DNA at precise location encoded by the particular sequence of the crRNA (CRISPR
RNA) (Deveau et al. 2008). In 2009, it was established to be acting as RNA mediated
DNA break down by a CRISPR RNA-Cas protein complex (Hale et al. 2009).
In 2010, Garneau and group showed in Streptococcus thermophilus that the CRISPR-
Cas system can specifically cleave invading double-stranded DNA within the proto-spacer
region, at specific sites (Garneau et al. 2010). In 2011, a small RNA transcribed from a
sequence adjacent to the CRISPR locus with near-perfect 25 base complimentary to
CRISPR repeats was discovered. The complementarily revealed that this tracrRNA and
crRNA hybridize together and are essential for crRNA processing and thus CRISPR
function (Deltcheva et al. 2011). Later studies showed that tracrRNA is crucial for Cas9
nuclease complex to cut DNA (Jinek et al. 2012). In 2011 only, Sapranauskas and co-
 S. Sharma, J. Vakhlu

workers proved that S. thermophilus CRISPR/Cas system can be transferred into E.coli and
provide protection against phage infection. They also concluded that cas9 with its McrA/
HNH and RuvC/RNase H- motifs is the sole Cas gene required for CRISPR-encoded
interference (Sapranauskas et al. 2011). Soon after, in 2012 first report of CRISPR cas9
mediated genome editing in eukyrotic cells was demonstrated by Cong and group (Cong
et al. 2013). In 2013, first report of successful genome editing in plants was reported by
Feng et al. 2013 in Arabdopsis. In 2014, CRISPR/ Cas system was efficiently demonstrated
as a versatile tool for genome editing of animal system using cynomolgus monkey
(Macaca fascicularis), an efficient animal model (Niu et al. 2014). By 2019, CRISPR
Cas system was efficiently used to edit human embryo for disease resistance (Chen et al.
2020a, 2020b). Timeline of the evolution of CRISPR system is indicated in Fig. 1.

Classification and Diversity of CRISPR-Cas System

Haft et al. in 2005, made the first attempt to classify CRISPR/Cas system. His group
identified 45 Cas protein families and categorized them into core proteins (Cas1–6), 8
CRISPR/Cas subtypes and RAMP (repair associated mysterious protein) module in
prokaryotic genome (Haft et al. 2005). Makarova et al. 2011, later classified CRISPER
system into type I, II and III betting on signature Cas proteins. It was further divided
into 10 subtypes based on the presence of additional signature proteins. This

Fig. 1 The timeline of the evolution of CRISPR Cas system

Evolution and Biology of CRISPR System: A New Era Tool for Genome...

classification system was further modified into two class, six type system based on
signature proteins and CRISPR loci as shown in Fig. 2 (Makarova et al. 2015).
Class I CRISPR has multiple subunit effector molecules and has different nucleases
for pre-crRNA processing, targeted cleavage and spacer sequence loading. Class II has
a single effector molecule performing all the functions. CRISPR-Cpf1 is Class II, Type
V; is an advanced class that utilizes a single Cpf1 protein for all the functions. Cpf1 was
first observed in Francisella novicida. Cpf1 stands for (CRISPR from Prevotella and
Francisella) and is functionally conserved to Cas9 protein but differs in recognition of
a thymine rich PAM site (Zetsche et al. 2015). Cpf1 System doesn’t require a tracrRNA
but most importantly it generates a 4 bp overhangs in contrast to cas9, which produces
blunt ends that are more efficient for genome insertion. Type VI system is characterized
by C2c2 (Class 2, candidate 2), now called as cas13a that cleaves ssRNA of the phage
genome (Aman et al. 2018).
Recently, more light has been shed on different type of CRISPR systems. Class I
comprising type I and Type III systems has known to include diverse variants. They are
present in large number of archea but aren’t that common in bacteria. The rare class IV
consisting of elementary CRISPR-Cas loci lacks effector nuclease and adaptation
module as well. Very little is known about type IV system. It has a very small effector
molecule and it doesn’t contain any domain for cleavage. The type I system targets
DNA only whereas the type III can acknowledge both DNA and RNA for cleavage.
The effector molecules of type I & III are complexes consisting of Cas protein subunits
which doesn’t have detectable conserve sequences. However they possess many
analogous as well as homologous protein subunits as revealed by cryo-electron mi-
croscopy. The anchor of all these complexes consist of paralogous repeat associated
mysterious proteins known as RAMPs such as Cas7 & Cas5 that contains a RNA
recognition motif (RRM) and C-terminal glycine-rich loop. Class I system has repeat
specific RNase responsible for pre-crRNA processing. Type I and type III differ in the

Fig. 2 CRISPR/Cas systems classification is based on different signature nucleases. CRISPR/Cas has six
types and is divided into two classes. The class I contains type I, III and IV, whereas, the class II contains type
II, V, and VI
 S. Sharma, J. Vakhlu

Cas protein complex required in pre-cRNA processing and the nuclease. In type I
system, effector enzyme is a homology directed nuclease with a Cas3 helicase domain
whereas, in type III system, the catalytic domain is the RAMPs of the Cas7 that causes
target cleavage (Faure et al. 2019).
On the other hand, class II CRISPR system is very much simpler in organization. It
has an effector module comprising of a large multi-functional and multi-domain
protein. Class II harbours the type II system with effector protein Cas9. This has
become the principal tool in genome editing methods and requires a tracrRNA and
then there are much rarer type V and VI. The type V system mostly uses cas12 as the
endonuclease. Just like Cas9, Cas12 also targets DNA for editing and has a requirement
for tracrRNA. Type VI is a variant in function of Class II system as it targets RNA for
editing. Type VI includes RNA guided RNase. Type VI uses Cas13 as the endonucle-
ase for the editing of RNA. The emerging knowledge of the system, the Cas13 has been
used to search and detects target sequences in the genome, using a system called as
SHERLOCK (Specific High sensitivity Enzymatic Reporter unlocking). SHERLOCK
technology when coupled with SPRINT (SHERLOCK-based Profiling of in vitro
Transcription) can be used to detect metabolites. This system has been successfully
used to detect Zika virus and dengue. (Kellner et al. 2019; Khambhati et al. 2019).

Mechanism of CRISPR Mediated Defence Process

The mechanism of action of CRISPR/Cas9 system imparted adaptive immunity is

divided into three stages: I) adaptation, II) expression and maturation and III) interfer-
ence (Chen et al. 2019; Tong et al. 2019) (Fig. 3).
In the Adaptation stage, Cas protein identifies the invading foreign DNA from
plasmids or bacteriophages and cleaves them into smaller fragments and then integrates
them into the CRISPR locus of the bacteria. Cas1 and Cas2 are found in most CRISPR

Fig. 3 Overview of mechanism of action of CRISPR-Cas system

Evolution and Biology of CRISPR System: A New Era Tool for Genome...

mediated immunity suggesting its role in spacer acquisition. This has been further
confirmed by mutation studies. It has been reported that mutation of one of the two
genes results in no spacer acquisition (Aliyari and Ding 2009; Dugar et al. 2013;
Hatoum-Aslan et al. 2011). Multiple Cas1 proteins have been reported (Nuñez et al.
2014). Most of the Cas1 proteins are metal dependant nucleases and integrases, which
acquire DNA and are sequence independent (Deng et al. 2013). Whereas, Cas2 are
either ssRNA or dsDNA specific endoribonucleases (Nam et al. 2012). In Sulfolibus
solfataricus, a further complex model of spacer acquisition was reported. Here,
CRISPR loci were seen to randomly insert new spacers throughout its CRISPR array
(Deng et al. 2013). This lead to the hypothesis that insertion machinery scans along the
foreign DNA, after priming to acquire a new proto-spacer (Budhathoki et al. 2020).
During maturation/ Biogenesis phase, CRISPR loci are transcribed to generate a
single long transcript containing much part of CRISPR array (Nussenzweig and
Marraffini 2020) which are then cleaved by Cas protein to form small RNA (crRNA)
containing a single spacer flanked by repeat sequence (Barrangou et al. 2007; Yosef
et al. 2012). These crRNA complexes with cas9 forming the active cas-crRNA
structure. These spacer regions get incorporated in the CRISPR locus and form the
base for immunological memory (Jackson et al. 2019). The mechanism of processing of
crRNA differs in different types of CRISPR immune system. In Type 1-V and 1-VI,
Cas6 recognizes a stem loop (Jia et al. 2020) flanking crRNA and cleaves to leave a
crRNA. Type 2 systems lacks cas6 and uses tracrRNA which binds to the primary
CRISPR transcript and forms dsRNA which is then cleaved by RNase III to produce
crRNA (Wiedenheft et al. 2012).
The final stage is interference phase where, Cas9-crRNA structure run down the cell
for foreign nucleic acid target with complementary crRNA sequences and recognize it
(Murugan 2020), leading to cleavage and thus degradation of the target near PAM
sequence (Mulepati et al. 2014). In type 1, binding of crRNA and proto-spacer signals a
conformational change that causes the recruitment of Cas3 and DNA degradation
occurs. In type 2, single Cas9 protein in used. Cas9 requires both tracrRNA and crRNA
to degrade DNA. Type 3 requires six to seven different types of Cas proteins. It is
unique in targeting both RNA and DNA of the phage using different sets of Cas
proteins (Millen et al. 2019).

Mechanism of Genome Editing in Plants

Once, the basic concepts about working of CRISPR/Cas system in bacteria was
established, researchers were quick to develop this into the tool for genome editing
(site specific mutations). The basic tool kit is same for all the organisms be it animals or
plants. For achieving an efficient CRISPR-Cas mediated genome editing, a suitable
vector system with codon optimized Cas9 gene and powerful promoters for Cas9 and
sgRNA are required. The target locus to be edited needs to be identified and sgRNA is
to be designed complementary to that so as to carry Cas endonuclease to the target site
for cleavage. A host specific vector and transformation system is needed and finally,
the methods to detect and confirm editing. In planta, the target gene to be modified
forms the CRISPR locus and the sgRNA is then synthesized complementary to the
selected CRISPR site sequence. The CRISPR site sequence is selected in a gene on the
basis of availability of the protospacer-associated motif (PAM) motif, NGG sequence
 S. Sharma, J. Vakhlu

downstream of the target site (Das et al. 2020). In addition a successful delivery
mechanism and transformation of this cascade into host is a prerequisite. sgRNA is
mostly regulated by tissue specific RNA polymerase III promoters such as AtU6
(Arabidopsis thaliana U6 promoter), TAU6 (T. aestivum U6 promoter) (Xu et al.
2017). Cas9 is integrated downstream of RNA polymerase II promoter such as
ubiquitin promoters. Cas9 in plant system is generally tagged with NLS (nuclear
localization sequence) to target nuclear DNA. sgRNA and Cas9 both can be expressed
in a single plasmid such as pFGC-pcoCas9, pRGEB32, pHSE401 (Qin et al. 2020).
Fig. 4, gives a brief account of the footprints of CRISPR/Cas9 system by which, it
works in plants.
A web portal called as CRISPR-P has also been designed by Penn State and Arizona
Genomics Institute (AGI) to help researchers to efficiently use the CRISPR Cas
technology for genome engineering in plant system (http://crispr.hzau.edu.cn/
CRISPR2/) (Lei et al. 2014). It uses the bioinformatics to hit highly specific set of
sgRNA for the target genes so as to avoid off -target editing (Liu et al. 2017).

Fig. 4 Comprehensive experimental layout for the CRISPR Cas mediated genome editing in plant systems
Evolution and Biology of CRISPR System: A New Era Tool for Genome...

Resources to Optimize CRISPR System

There are various online tools available for optimizing CRISPR/Cas9 gene editing
system. A vast number of online applications have been developed which are utilized
for application in genome manipulations, for identification of CRISPR target site and
for excerption of sgRNA. Few of such resources are mentioned in Tables 2 and 3.

Applications of CRISPR Cas9 System in Plants

The use of CRISPR Cas technology, for genetic modification has revolutionized the
plant science, over the past few years. The first discovered as a part of adaptive immune
system in bacteria, has now been used for selective genome editing, transcriptome
regulation and epigenetic modifications in plants (Zhang et al. 2019). Since 2014,
nearly 20 crop species have been edited using CRISPR Cas technology (Ricroch et al.
2017). Various plant traits have been modified using this technology such as yield
improvement, abiotic and biotic stress mechanisms and traits enhancement (Zafar et al.
2020). Biotic stresses posed by pathogenic microbes and abiotic stresses such as
drought and salinity, downgrades the yield. The major focus in the field of plant
biology is on the generation of plants which can tolerate worst climatic conditions
and fulfil the need of growing human population. CRISPR system can produce stable
and heritable modifications without hindering with the existing valuable traits. This
leads to the production of homozygous transgene free plants in only one generation
(Zhang et al. 2016).Various works done on crop plants since the advance in CRISPR
Cas9 technology is mentioned in Table 4.
In addition CRISPR system is now routinely used in plant biology for understanding
the function of the gene and turning the crop into an ideo-agro-type. CRISPR system
has also been used to understand the underlying function of many important genes in
agronomically important crops by conventional loss of function mutations. CRISPR-
TSKO (clustered regularly interspaced short palindromic repeats (CRISPR)-based
tissue-specific knockout) system has been generated to specially turn off a gene in
particular tissue, organ or cell type. In Arabidopsis thaliana, CRISPR-TSKO mutations
in important genes lead to the understanding of gene function in both spatial and
temporal setting of plant, while bypassing the pleiotropic effect of whole genome loss
of gene function (Decaestecker et al. 2019). In another experiment, complete shutdown
of PDS3 (phytoene-desaturase3) gene in Arabidopsis thaliana resulted in albino plants
with severe growth and survival difficulties on soil. On knockdown of PDS3 gene in a
specific subset (stomatal lineage) ruled out the pleotropic effect on the whole plant and
allowed the characterization of the gene (Ali et al. 2020).
Genome editing is one of the major and widely adapted applications of
CRISPR system but it is not the only application. In addition to genome wide
manipulation, CRISPR system has been used to regulate gene expression (Yeo
et al. 2018). CRISPRi has been developed for genome silencing to understand
the regulation of gene expression in a sequence specific manner over the
genome wide scale (Larson et al. 2013; Liu et al. 2017; Bassaganyas et al.
2019). The gene down regulation by CRISPRi is highly specific, reversible in
nature (Shariati et al. 2018) and can target multiple genes simultaneously (Yao
et al. 2015).
 S. Sharma, J. Vakhlu

Table 2 Commercially available application for successful genome editing using CRISPR

S.No Trade name Link

Thermo Fisher Scientific www.thermofisher.com/pk/en/home/life-science/genome-editing/

geneart-crispr/crispr-libraries/lentiarray-crispr-libraries/lentiarray-
cas9-lentivirus.html
2 Addgene www.addgene.org/crispr/guide/
3 Synthego www.synthego.com/products/synthetic-sgrna/
4 ATUM www.atum.bio/products/expression-vectors/mammalian?exp=5
5 Clonetech http://www.clontech.com/US/Products/Genome_
Editing/CRISPR_Cas9/Resources/About_Guide-it_Kits
6 CHOPCHOP https://chopchop.rc.fas.harvard.edu/
7 Origene http://www.origene.com/CRISPR-CAS9/
8 GeneCopoeia www.genecopoeia.com/product/transgenic-mouse/

Recently, inactive Cas9 was fused with GFP and was co-expressed with sgRNA to
analyze and visualize the genetic system dynamics in vivo (Kamiyama et al. 2016).
This technology has also been exploited for epigenome editing via installing the
proteins responsible for histone modification/ DNA methylation to understand the
cellular functions (Hilton et al. 2015; Klann et al. 2018). CRISPR-Cas9 is a dynamic
system that is getting modified with each passing day. Recently enough ribonucleo-
protein (RNP) editing using CRISPR system has been introduced, which eliminates
some basis drawbacks with CRISPR system like appropriate promoter for Cas9,
potency of cell to transfection, off-targeting etc. (Svitashev et al. 2016; Liang et al.
2017). Here, a RNP complexed with Cas9 and target gRNA is delivered directly to the
cell of interest (Mout et al. 2017). This approach is less off target effect, as the RNPs
get degraded soon after delivery (Liang et al. 2018). RNP based editing has wide range
of applications and this technology is seen to work in both plants and mammalian cells.
Cas9-RNP delivery system holds great potential for therapeutics as well (Nasri et al.
2019; Lattanzi et al. 2019). Some other applications of CRISPR Cas technology are
reported in the field of metabolic engineering in which plant cells are directed to the
production of specific metabolite. Next generation metabolic engineering in
P. somniferum was done by knocking out 3-OMT2 gene via NHEJ DNA repair,
CRISPR/Cas9 mechanism to edit the biosynthesis pathway of benzylisoquinoline
alkaloids (BIAs) gene involved in the production of codeine, noscapine, papaverine,

Table 3 Tools for optimizing sgRNA for CRISPR system

S.No Trade name Link

1 Atum https://www.atum.bio/eCommerce/cas9/input
2 CRISPR design http://crispr.mit.edu/
3 Casoff finder http://www.rgenome.net/cas-offinder/
4 sgRNA scorer 2.0 https://crispr.med.harvard.edu/
5 CRISPOR http://crispor.tefor.net/
Evolution and Biology of CRISPR System: A New Era Tool for Genome...

Table 4 List of few of the targeted genes via CRISPR mediated genome editing in different plants

S.No. Plant Gene of interest Method Reference

1 Cucumber eIF4E (transcription factor) NHEJ Chandrasekaran

et al. 2016
2 Bread Wheat TaMLO-A1, TaMLO-B1, and NHEJ Wang et al. 2014
TaMLOD1
3 Rice OsERF922 (ethylene responsive factor) NHEJ Wang et al. 2016
4 Arabidopsis thaliana AP1,SVP and TFL1 (floral NHEJ Liu et al. 2019
development genes)
5 Maize ARGOS8 (increase grain in HDR Shi et al. 2017
drought conditions)
6 Tomato SlMAPK3 (drought tolerance) NHEJ Wang et al. 2017
7 Rice OsADH2 (fragrance gene) NHEJ Ashokkumar
et al. 2020
8 Rice OsPRX2 (potassium deficiency tolerance) NHEJ Mao et al. 2018
9 Potato ALS1 (herbicide resistance) HDR Butler et al. 2016
10 Cassava MePDS (Carotenoid biosynthesis) NHEJ Odipio et al. 2017
11 Wheat TaVIT2 (Fe content) HDR Connorton et al. 2017
12 Soybean GmPDS11 and GmPDS18 NHEJ Du et al. 2016
(carotenoid biosynthesis)
13 Citrus CsLOB1 (canker susceptibility gene) NHEJ Jia et al. 2019
14 Gossypium hirsutum GhCLA1 (endogenous gene) NHEJ Wang et al. 2018
15 Tomato and Potato ALS (Acetolactate synthase) NHEJ Veillet et al. 2019

and morphine via different BIA pathways (Alagoz et al. 2016). Using such strategies,
valuable medicinal plants can be converted to bio-factories for mass production of
specific metabolite (Li et al. 2017). A graphical abstract of application of CRISPR
system is given in Fig. 5.
These findings suggest that the application of the CRISPR–Cas9 technology is a
possible molecular tool of future with wide range of utility.

Off- Targeting Effect of CRISPR/ Cas9 System

The limitation of the CRISPR Cas9 system is its low specificity. The low specificity is
accounted by 20 nucleotide short recognition sequence followed by 3 nucleotide PAM
sequence thus leading to off target modifications. Though, the programmable sgRNA
and the Cas9 endonuclease have made genome engineering in plants easy, it can also
cause non specific editing due to the capacity of gRNA to tolerate mismatch upto five
bp at its 5′ end (Haeussler 2020). These off targets can have “hard-to-calculate” effect
in the genome. Off-targets have been categorized into three major types. First type
includes the off-target regions at other PAM regions than the specified PAM (5′-NGG-
3′) with substitutions or mismatches. Second type includes the off target regions at
other PAM regions than specified PAM (5′-NGG-3′) with insertions or deletions
(indels) and the third type, where the cleavage occurs at the sequences other than the
specified PAM sites such as 5′-NAG-3′. (Anders et al. 2014).
 S. Sharma, J. Vakhlu

Fig. 5 Application of CRISPR- Cas9 technology

A relatively higher frequency of off-target modification has been seen in animals

and human genome editing experiments as compared to that of plants (Graham et al.
2020). The low rate of off-target modification in plants can be compiled due to
limitations in detecting off-target modifications in plant system. Majorly, the off-
target sorting is done on small data sets of the genome which contain homologous
regions to the target sites. This is done by using high fidelity sequencing techniques
such as whole genome sequencing (WGS). In plant system this process is hindered by
the procedural limitation like low sequence information of reference genomes (Ahmad
et al. 2020).
Off-target modifications can not only cause non-specificity in scientific discoveries
about gene functions but can also limit genome editing applications for therapeutic
purposes and crop improvement. The off target genetic modifications related to
CRISPR/Cas9 system may cause chromosomal instability, loss of function gene
activity leading to physiological and signaling anomalies (Manghwar et al. 2020).
Thus, it is important to design an optimum gRNA for high targeted modifications with
little scope for off-target effects. The strategies used to overcome off-targeting in
genome editing can be classified as such: Bioinformatic predictions, experimental
validations, Cas9 engineering and gRNA modifications. A lot of different assay and
approaches are used to unravel the mechanism of selection and cleavage of targets by
gRNA-Cas9 complex. Various methods used to quantify off target modifications
caused by CRISPR/Cas system are described in Table 5.
Table 5 Various methods used for the detection of off-target mutation sites
S.No Method
1 T7E1 assay
2 WGS (whole genome
sequencing)
3 WES (Whole exome
sequencing
4 ChIP-seq
5 GUIDE-seq
6 BLESS
7 LAM-HTGTS
8 Digenome-seq
9 CIRCLE-seq
10 IDLV
Description
Endonuclease based cleavage of
hetero-duplex formed by hy-
bridization of mutant and wild
type DNA
sequencing the whole genome
for mutations
Sequencing to detects off-target
mutations in the exome
It identifies genome-wide
sgRNA:dCas9 binding sites
Detects DSBs caused by nuclease
activity
Direct in situ breaks labeling,
enrichment with
high-throughput sequencing
and streptavidin
Detects genomic translocations
caused by end-joining be-
tween genomic DSBs.
Used to detect genome-wide
profiling of off-target effects
Used to identify genome-wide
CRISPR/Cas9 off-target mu-
tations
Detects off-target mutations of
the CRISPR/Cas9 system
Advantage Limitation Reference
Simple Expensive and poor sensitivity Cho et al. 2013
Unbiased and accurate Very expensive Li et al. 2019
Unbiased and cheaper than WGS Ignores mutations in non-coding sites Chen et al. 2020a, 2020b
Unbiased, genome-wide detection of Shows false positives Naeem et al. 2020
sgRNA/cas9 binding loci
Unbiased, detects translocations and Limited co-transfection efficiency and ZAMBONINI 2020
breakpoint hotspots shows false negatives
Unbiased, genome-wide detection of Very expensive and requires a reference Manghwar et al. 2020
DSBs genome
Evolution and Biology of CRISPR System: A New Era Tool for Genome...
Unbiased, genome-wide detection of Difficult to detect all loci because of low Yin et al. 2019
translocations frequency of translocations
Unbiased, sensitive, cost-effective When testing several sgRNAs, sequencing Kim et al. 2019
depth can be challenging, requires a
reference genome
Unbiased, higher sensitivity than Limited sensitivity Lin et al. 2020
Digenome-seq
Unbiased, applicable for primary cells False positives caused by randomly Wang et al. 2015
insertion
 S. Sharma, J. Vakhlu

Conclusion

CRISPR-Cas9 is a simple technology with a large range of applications which has

made this technology the revolution of the era and bagged Jennifer Doudna and
Emmanuelle Charpentier Nobel prize in chemistry for the year 2020. The desirability
of the system is attributed to its simplicity, proficiency, specificity, low off-target effect
as compared to other tools and availability of multiplexing option. This technology is
promising for plant systems have already been proved beyond doubt. The targeted
mutagenesis of genes would surely provide an overlook about the functions of the
genes and can lead to pathway level studies in plant and also help manipulate the
complex agronomical traits in crop plants. The application of CRISPR-Cas system for
genome-wide knockout screening can be preferred over all other genome editing
techniques discussed before because of its simplicity, precision and low cost. Another
promising application of CRISPR system would be to study the function of lethal genes
by maintaining spatial and temporal control over expression of such genes. This
technology can be optimized to express in specific tissue/ developmental stage/
environmental conditions by using tissue specific/ inducible promoters for the expres-
sion of Cas9. This technology also provides the visualization of the expression of genes
in vivo by tagging endogenous genes with GFP and their localization within the cell.
The CRISPR system could be used for the production of novel allelic variants for
breeding in crops. The introduction of precise genetic/epigenetic modifications by
CRISPR Cas technology can provide a successful platform to manipulate important
agronomical traits, such as increase in yield and stress tolerant/resistant crop plants.
Despite the progress of this plant editing system over the past 3–4 years, there are
certain lags which still needs to be optimized such as, influence over local chromatin,
the length of sgRNA for maximum efficiency, the off targeting, efficient delivery
system in plants etc. More studies are needed for the understanding of the mechanism
of germline transmission and heritability of modifications caused by CRISPR-Cas9
system. Thus, it can be concluded that the CRISPR-Cas9 technology can cater human
control over agronomic traits in crop plants leading to the production of better variety
of crop plants with less disease incidence and enhanced nutrition traits.

Abbreviations BIAs, Benzylisoquinoline alkaloids; C2c2, Class 2, candidate 2; Cas, CRISPR associ-
ated; Cpf1, CRISPR from Prevotella and Francisella1; CRISPR, Clustered regularly interspaced short
Palindromic repeats; crRNA, CRISPR RNA; DNA, Deoxyribonucleic acid; DSB, Double stranded
break; GFP, Green fluorescent protein; GMO, Genetically modified organism; NHEJ, Non homolo-
gous end joining; PAM, Proto spacer adjacent motif; RAMP, Repair associated mysterious protein;
RNA, Ribonucleic acid; RNAi, RNA interference; RNP, Ribonucleoprotein; sgRNA, Single guide RNA;
SRSRs, Short regularly spaced repeats; ssRNA, Single stranded RNA; TALEN, Transcription activator-
like effectors; tracrRNA, Trans-activating crRNA; ZFN, Zinc finger nucleases

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