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The most recent addition to the SSN family is the CRISPR/Cas system that is normally present within bacteria and archaea, and provides an adaptive immunity against invading plasmids or viruses. CRISPR/Cas system functions to destroy invading nucleic acids by introducing targeted DNA breaks (Garneau et al. 2010).

There are three major types of CRISPR/Cas system: Types I – III (Makarova et al. 2011). The Type II system was adopted for genome engineering a few years ago (Cong et al. 2013; Zhang et al. 2011). In this system, two components enable targeted DNA cleavage: a Cas9 protein and an RNA complex consisting of a CRISPR RNA (crRNA; contains 20 nucleotides of RNA that are homologous to the target site) and a trans‐activating CRISPR RNA (tracrRNA). Cas9 protein causes double‐stranded DNA break at the sequences homologous to the crRNA sequence and upstream of a protospacer‐adjacent motif (PAM) (PAM; e.g. NGG for Streptococcus pyogenes Cas9). For genome engineering purposes, the complexity of the system was reduced by fusing the crRNA and tracrRNA to generate a single‐guide RNA (gRNA). Moreover, off‐target cleavage is a limitation of the CRISPR/Cas system (Cho et al. 2014; Fu et al. 2013).

The target site recognition in CRISPR‐Cas system is facilitated through RNA: DNA interaction (as opposed to a protein: DNA interaction used by meganucleases, zinc‐finger nucleases, and TALENs). Redirecting of Cas9 targets involves modification of 20 nucleotides within the crRNA or gRNA. These 20 nucleotides are used to direct Cas9 binding and cleavage, the system has been shown to tolerate mismatches, with a higher tolerance closer to the 5′ end of the target sequence (Fu et al. 2013). Results from recent studies suggest the first 8–12 nucleotides, in addition to the PAM sequence, are most critical for target site recognition (Sternberg et al. 2014; Wu et al. 2014). To reduce off‐targeting, several methods have been developed, including dual‐nicking of DNA (Mali et al. 2013; Ran et al. 2013), a fusion of catalytically‐dead Cas9 to FokI (Guilinger et al. 2014; Tsai et al. 2014) and shortening of gRNA sequence (Fu et al. 2014). Several softwares and programs have been developed in recent years for the identification of target sequences in the genome and the design of specific gRNA, which are listed in Table 1.1.

The Cas9 is an endonuclease consisting of two discrete nuclease domains: the HNH domain which is responsible for the cleavage of the DNA strand complementary to the guide RNA sequence (target strand) and the RuvC‐like domain that cleaves the DNA strand opposite the complementary strand (Chen et al. 2014; Gasiunas et al. 2012; Jinek et al. 2012). The double‐strand breaks (DSBs) are repaired through Non‐Homologous End Joining or Homology directed Repair in the presence of a template. Mutations in both nuclease domains (Asp10 → Ala, His840 → Ala) result in an RNA‐guided DNA‐binding protein without endonuclease activity that is called dCas9 (Jinek et al. 2012; Qi et al. 2013). This dCas9 is then supplemented with effector domains for the execution of distinct functions in the genome (Figure 1.1B). Fusion of a transcriptional activator VP64 with dCas9 exhibited targeted gene activation by altering the flowering time regulation in Arabidopsis (Xu et al. 2019). Similarly, dCas9‐VP64 regulated transcriptional activation of endogenous genes and dCas9‐SRDX‐regulated transcriptional repression in Arabidopsis and tobacco (Lowder et al. 2015, 2018). These regulatory domains can also perform multiplex gene targeting using multiple sgRNAs. As a new dimension to CRISPR/Cas technology, there are the base editing enzymes, for example, cytidine deaminase fused with the dCas9, which can replace specific bases in the targeted region of DNA and RNA.

Table 1.1 List of available softwares and programs for designing gRNA.

Software	Features	Link	References
Cas‐OFFinder	Identifies gRNA target sequence from an input sequence and checks off‐target binding site	http://www.rgenome.net/cas‐offinder	Bae et al. (2014)
Cas‐Designer	Identifies gRNA target sequence from an input with low probability of off‐target effect	http://www.rgenome.net/cas‐designer/	Park et al. (2015)
Cas9 Design	Designs gRNA	http://cas9.cbi.pku.edu.cn/database.jsp	Ma et al. (2013)
E‐CRISP	Designs gRNA	http://www.e‐crisp.org/E‐CRISP/designcrispr.html	Heigwer et al. (2014)
CRISPR‐P	Designs gRNA	http://cbi.hzau.edu.cn/crispr2/	Lei et al. (2014)
CHOP	Identifies target site	https://chopchop.rc.fas.harvard.edu/	Montague et al. (2014)
CRISPR‐PLANT	Designs gRNA	http://www.genome.arizona.edu/crispr/	Xie et al. (2014)
CCTop	Identifies candidate gRNA target sites with reduced off‐target quality	http://crispr.cos.uni‐heidelberg.de/	Stemmer et al. (2015)
CRISPRdirect	Identifies candidate gRNA target sequences	http://crispr.dbcls.jp/	Naito et al. (2015)
COSMID	Identifies target sites	https://crispr.bme.gatech.edu	Cradick et al. (2014)
CRISPR Finder	Identifies CRISPR	http://crispr.u‐psud.fr/Server	Grissa et al. (2007)
CrisprGE	Identifies target sites	http://crdd.osdd.net/servers/crisprge	Kaur et al. (2015)
CRISPR Multitargeter	Identifies target sites	http://www.multicrispr.net	Prykhozhij et al. (2015)
CRISPRseek	Identifies target specific guide RNAs	http://www.bioconductor.org/packages/release/bioc/html/CRISPRseek.html	Zhu et al. (2014)
flyCRISPR	Identifies target sites and evaluate its specificity	http://flycrispr.molbio.wisc.edu	Gratz et al. (2014)
GT‐SCAN	Identifies target sites and ranking them with their potential off target sites	http://flycrispr.molbio.wisc.edu	O'Brien and Bailey (2014)
sgRNAcas9	Identifies target sites with their potential off target sites	www.biootools.com	Xie et al. (2014)
SSFinder	Identifies target sites	https://code.google.com/p/ssfinder	Upadhyay and Sharma (2014)
ZiFiT	Identifies target sites	http://zifit.partners.org/ZiFiT	Mandell and Barbas (2006)
sgRNA Designer	Guide RNA design based on efficiency score	http://broadinstitute.org/rnai/public/analysis‐tools/sgrna‐design	Doench et al. (2014)

Several plant species have been edited using CRISPR/Cas system, including rice, wheat (Shan et al. 2013b; Upadhyay et al. 2013), sorghum (Jiang et al. 2013), tobacco (Li et al. 2013), Arabidopsis (Fauser et al. 2014; Feng et al. 2014; Li et al. 2013), Brassica napus (Kang et al. 2018), watermelon (Tian et al. 2018), etc. (Table 1.2). Moreover, dCas9 can be fused with various epigenetic regulatory factors which can modulate DNA acetylation/methylation, post‐ translational histone modification, ubiquitination and protein sumoylation and phosphorylation to carry out epigenetic modifications (Shrestha et al. 2018; Yamamuro et al. 2016). This has been more recently explored in Arabidopsis for demethylation (Gallego‐Bartolomé et al. 2018).

Table 1.2 List of examples of genes edited by CRISPR Cas system in various plant species.

Plant system	Gene	Description of Experiment	References
Arabidopsis thaliana	GSS21/2	Host adaptation against P. xylostella	Chen et al. (2020)
Rice	EPFL9	a positive regulator of stomatal development	Yin et al. (2017)
OsDEP1 OsROC5 OsPDS	Carotenoid biosynthesis, leaf morphology	Tang and Tang (2017)
OsDL and OsALS	loss of midrib in the leaf blade	Endo et al. (2016)
OsPDS, OsBEL	Herbicide resistant and Nutritional improvement	Xu et al. (2017)
OsRLK, OsBEL	receptor‐like kinases	Wang et al. (2017)
OsPDS OsDL	Herbicide resistant and loss of midrib in the leaf blade	Tang et al. (2019)
OsERF922	Enhanced resistance to blast disease	Wang et al. (2016)
GW2, GW5, and TGW6	Improvement of grain weight	Xu et al. (2016)
ALS	Enhanced herbicide resistance	Sun et al. (2016)
SBEIIb and SBEI	Generation of high amylose rice	Sun et al. (2017)
Hd 2, Hd 4, and Hd 5	Early maturity of rice varieties	Li et al. (2017)
OsMATL	Induction of haploid plants	Yao et al. (2018)
ALS	Herbicide resistance	Butt et al. (2017)
EPSPS	Herbicide resistance	Li et al. (2016)
ALS	Herbicide resistance	Endo et al. (2016)
Gn1a, GS3, DEP1	Enhanced yield, dense erect panicles	Li et al. (2016)
LAZY1	Tiller‐spreading	Miao et al. (2013)
OsSWEET13	Bacterial blight resistance	Zhou et al. (2015)
OsDEP1 OsROC5	Herbicide resistant	Yao et al. (2018)
Soybean	FAD2‐1A, FAD2‐1B	Biosynthesis of lipids	Kim et al. (2017)
ALS	Herbicide resistance	Li et al. (2015)
GmPDS11&18	Carotenoid Biosynthesis	Du et al. (2016)
Tobacco	FAD2‐1A, FAD2‐1B	Lipid biosynthesis	Kim et al. (2017)
NtPDS and NtPDR6	etiolated leaves for the psd mutant and more branches for the pdr6 mutant	Gao et al. (2015)
Cotton	Cloroplastos alterados (GhCLA)	Photosynthesis	Li et al. (2019)
CABs, replication associated protein (Rep) and non‐coding intergenic regions (IR), a‐Satellite Rep and b‐Sat IR.	CLCuD associated Begomoviruses (CABs) and Helper begomoviruses a and b satellites.	Iqbal et al. (2016), Uniyal et al. (2019)
Ashbya gossypii	HIS3, ADE2, TRP1, LEU2 and URA3	auxotrophic markers	Jiménez et al. (2020)
Maize	Maize glossy2 gene	Cuticular wax deposition	Lee et al. (2019)
ARGOS8	Novel variants of ARGOS8 for drought‐tolerance	Shi et al. (2017)
ALS	Herbicide resistance	Svitashev et al. (2015)
ZmIPK	Reduction of anti‐nutritional compound phytic acid	Liang et al. (2014)
TMS5	Thermosensitive male‐sterile	Li et al. (2017)
Wheat	MLO	Resistance to powdery mildew	Wang et al. (2014)
GW2	Enhanced yield	Zhang et al. (2018)
EDR1	Powdery mildew resistance	Zhang et al. (2017)
Barley	HvPM19	Positive regulation of grain dormancy	Lawrenson et al. (2015)
HvCKX1/3	Cytokinin metabolism and root morphology	Gasparis et al. (2019)
Tomato	SlMlo1	Resistant to powdery mildew	Nekrasov et al. (2017)
SlWUS	Increased fruit size	Rodríguez‐Leal et al. (2017)
SlAGL6	Facultative parthenocarpy	Klap et al. (2017)
SP5G	Day neutrality and early flowering	Soyk et al. (2016)
SP, SP5G, CLV3, WUS, GGP1	Tomato domestication	Li et al. (2018)
SIAN2	Anthocyanin biosynthesis	Zhi et al. (2020)
SlJAZ2	Bacterial speck resistance	Ortigosa et al. (2019)
Potato	VInv	Reduction of sugar accumulation	Clasen et al. (2016)
ALS	Herbicide resistance	Butler et al. (2016)
Wx1	High amylopectin content	Andersson et al. (2017)
StPPO	Reduced Enzymatic Browning in Tubers	González et al. (2020)
Cassava	MePDS	Carotenoid Biosynthesis	Odipio et al. (2017)
Citrus paradise	CsLOB1	Citrus canker resistance	Jia et al. (2017)
Citrus sinensis	CsLOB1 promoter	Citrus canker resistance	Peng et al. (2017)
Citrus paradise	CsLOB1 promoter	Alleviated citrus canker	Jia et al. (2016)
Manihot esculenta	EPSPS	Herbicide resistance	Hummel et al. (2018)
Cucumas sativus	eIF4E	Virus resistance	Chandrasekaran et al. (2016)
Camelina sativa	FAD2	Low polyunsaturated fatty acids	Jiang et al. (2017)
Linumusitatissimum	EPSPS	Herbicide resistance	Sauer et al. (2016)
Carrot	DcPDS and DcMYB113‐like genes	depigmented carrot plants	Xu et al. (2019)
Strawberry	PDS	Carotenoid Biosynthesis	Wilson et al. (2019)
Grapes	VvPDS	Carotenoid Biosynthesis	Nakajima et al. (2017)
CsLOB1	Increased resistance to citrus canker	Jia et al. (2017)
MLO‐1	Negative regulator of resistance to Powdery mildew	Malnoy et al. (2016)
Pear	MdPDS and TFL1.1	Carotenoid Biosynthesis & Floral repression	Charrier et al. (2019)
Apple	PDS	Carotenoid Biosynthesis	Charrier et al. (2019, Nishitani et al. (2016)
DIMP‐1/2/3	Negative regulator of resistance to blight disease	Malnoy et al. (2016)
Banana	eBSV	Resistance against Banana streak virus (eBSV)	Tripathi et al. (2019)
Chicory	phytoene desaturase gene (CiPDS)	Fruit ripening	Jansing et al. (2019)
Rubber	HbFT HbTFL1	Delayed‐flowering and early‐flowering	Fan et al. (2020)
Brassica napus L.)	BnLPAT2 and BnLPAT5	size of the oil bodies increased	Zhang et al. (2019)
Populus	phytoene desaturase gene 8	Carotenoid Biosynthesis	Fan et al. (2015)
Flax	EPSPS	Aromatic amino acid biosynthesis	Sauer et al. (2016)