*5.2. DNA-Free Genome Editing Through Ribonucleoproteins (RNPs)*

Foreign DNA-free GE is another approach in the CRSIPR/Cas9 toolkit to produced transgene-free plants [153]. In conventional approaches to genetic engineering, the foreign DNA with editing components is incorporated into the host organism. Due to the random incorporation, the genetic modifications can be unpredictable. Even if the expression cassettes are abolished, the fragments of foreign DNA can still be integrated into the host genome and cause mutations [154]. Furthermore, the introduction of genetically modified organisms has increased globally [155]. So, there is an increased demand to produce transgene-free plants. To develop DNA-free genome-edited plants, particle bombardment and protoplast transformation techniques have been employed.

Transgene integration may be reduced by using the CRISPR/Cas DNA transient expression system, but it does not entirely eliminate it; additionally, the discarded DNA portion may still get incorporated into different non-targeted locations within the plant genome. To escape the shortcomings of mRNA and plasmid-based expression system of sgRNA/Cas9, a most competent transgene-free editing strategy was established by designing the RNPs sgRNA/Cas9 system in plants [156–158]. Hence, sgRNA/Cas9 RNPs have a greater ability to produce DNA-free edited plants with low off-target frequency and is more efficient than a plasmid-mediated editing system. The RNP-based system does not need transcriptional and translational apparatuses for creating nicks in the target sites and, after cleaving, it is then disintegrates itself. In 2015, Woo and colleagues performed DNA-free GE for the first time in rice, tobacco, lettuce, and *Arabidopsis* using the RNPs system [156]. Potato, apple, and grape explants were subjected to targeted mutations carrying CRISPR/Cas9-mediated RNPs [159–161]. In addition, DNA-free GE in maize and wheat has been developed by particle bombardment-mediated transformation of RNPs and Cas9 proteins into cells [58,148,162]. Recently, a new CRISPR/Cas variant Cpf1 has been added to the RNP-based GE toolkit, carrying AsCpf1/crRNA and LbCpf1/crRNA RNPs into tobacco and soybean [163]. However, in several cereal crops protoplast regeneration is a bigger task, hence, the biolistic-mediated RNP editing system is the most appropriate technique for GE in plants. The delivery of RNP-mediated CRISPR/Cas9 machinery has been demonstrated by two different groups in wheat and maize [145,146]. The discovery of a DNA-free editing system will surely simplify the GE of plants and helps to commercialize the edited plants in the future.

### *5.3. CRISPR*/*Cas9 Toolbox: Ways Toward Precise Editing*

#### 5.3.1. Base Editing

As compared to DSB-governed GE, single-nucleotide modification at a specific site of the genome is called base editing, and is not based on donor DNA or an HDR mechanism and also does not require DSB generation, which provides a simple, highly accurate, and universal mechanism for editing a single base at a target site. Thus, base editing with the CRISPR/Cas9 tool is gaining interest for precise targeted gene editing in plants [164]. Currently, the use of the HDR repair mechanism with donor DNA for DSBs has been found to be less effective in contrast to NHEJ repair with a template-free system, posing a great hurdle in plants for base substitution. Genome-wide association studies (GWAS) have demonstrated that crop plants having a single nucleotide insertion/deletion are more significant for screening the elite germplasm [16]. Therefore, powerful tools are required immediately for generating accurate base editing in crop plants [165]. The CRISPR/Cas9 is an exceptional technique for precise substitution of a single base in target DNA [166]. The CRISPR/Cas9-directed base editing strategy has used the gRNA system, which is homologous to the natural CRISPR system. But in case of a cytosine base-editor (CBE) system, modified Cas9 endonucleases called nickase (nCas9) are used as compared to the natural CRISPR system. These nCas9 proteins, in addition to dead Cas9 proteins fused with an enzyme having base cleaving activity such as cytidine, are converted to uridine by the cytidine deaminase [149,167]. Recently, an effective base-editor 3 (BE3) platform was developed, which involves the merger of APOBECI known as rat cytidine deaminase and which has been extensively employed for GE in many organisms including plants [168]. In addition, several improvements in the BE3 system has allowed modifications in PAM sites to enhance its editing specificity and accuracy [168]. Likewise, three cytidine deaminase orthologs such as human APOBEC3A [150,169], human AID [170], and lamprey PmCDA1 [167] have been fused with nCas9 to attain highly precise C-to-T substitution. For example, a plant CBE system based on APOBEC3A has been widely applied for C-to-T substitution in potato, rice, and wheat [150,171]. In rice and *Arabidopsis*, CBE has been used to create point mutations. In addition, CBE can also be applied to generate non-specific mutations that manipulate the desired gene and disrupt its function. CBE was found to be precise and more accurate than SSN-mediated editors, producing rare if any indels [172].

Similarly, adenine converts to inosine by adenine deaminase [168]. In wheat and watermelon, this strategy has been adopted to develop herbicide-resistance plants [50,173]. Yan and colleagues identified a fluorescence-tracking mechanism in rice which converts the adenine to guanine by a single-base editing system [174]. An adenine base editor (ABE) was developed for multiplex base substitutions in rice [175]. Similarly, ABE was applied to study the germline transmission and preferred phenotypic changes in *Arabidopsis* [176]. Recently, Li et al. (2018) upgraded the ABE for generating base editing in wheat and rice plants. They have also successfully developed an herbicide-resistant rice by producing the point mutation [177]. So, GE has been provided novel dimensions by base-editing tools, widening its prospective applications by manipulating desired nucleotides in the plant genome.

#### 5.3.2. Multiplex Genome Editing

In plants, cellular processes are fine-tuned by several redundant genes. Sometimes, mutating a single gene may not confer a desired phenotype because of the compensation effect produced by other genes in same gene family. Hence, an upgraded editing system with improved efficiency is needed for multiplex gene editing in plants. In CRISPR/Cas9-mediated multiplex GE, many sgRNA cassettes can be designed by using single or multiple promoters into a single-vector system [108,178]. In 2013, Mao and coworkers designed two sgRNAs for two homologous of *magnesium-chelatase subunit I* (*CHLI*) having function in the photosynthesis mechanism, and it successfully transformed the vector in *Arabidopsis thaliana.* The result showed the albino phenotype in plants in which both genes were disrupted [179]. In another study, four subunits of katanin p80 were mutated in *A. thaliana* using multiplex genome editing. For this, three sgRNA expression cassettes were designed for simultaneous gene editing and the results demonstrated the dwarf phenotype in quadruple-mutant plants [180].

The group of Xie reported the editing of eight genes simultaneously by designing multiple sgRNA expression cassettes. An endogenous t-RNA-processing platform was used for the expression of multiple sgRNAs. All the sgRNAs were released after the nick produced by endogenous t-RNA-processing-based RNase [181]. Similarly, this t-RNA-based strategy has also been efficiently demonstrated in *Zea mays* [182]. A multiplexing system was developed by Tang and colleagues in which hammerhead self-cleaving ribozyme was applied. Additionally, the same promoter Po1II was used for the expression of multiple sgRNAs that govern Cas9 activity. Ribozyme cleavages separated sgRNAs and Cas9 after transcription and released functional sgRNAs and Cas9 [183]. Furthermore, the ability of the CRISPR-Cpf1 system was harnessed for multiplex GE in rice. A single promoter was applied to produce a construct composed of numerous repeated units of crRNA attached with a target sequence. A target repeat sequence was recognized by Cpf1 and produced cleavage, which resulted in releasing of crRNAs [184]. Hence, CRISPR/Cas9-mediated multiplex GE is a convenient approach for knocking out multiple genes at once and helping to decipher the function of a desired gene family that regulates multiple biological networks. Moreover, it is also beneficial in finding out the epistatic association among genes in numerous genetic processes.

#### *5.4. Beyond Cas9: New Cas Variants Broadening the CRISPR Toolbox*

The CRISPR/Cas9 system which originated from *Streptococcus pyogenes* has some drawbacks which hinder its editing activity like multiple incompatible off-targets due to the gRNA mismatches. Thus, several changes have been made to enhance the editing efficiency and to minimize the off-target nicking of Cas9 enzymes including SpCas9n (Cas9n) [23], Dead cas9 (dcas9) [185], and *FokI* Cas9 (fCas9) [186,187]. Various bacterial species have been used for the extraction of Cas9 proteins having novel- and stretched-PAM sequences that can help in enhancing the non-target cleavages. *Neisseria meningitides* have unique a CRISPR/Cas machinery named Nmecas9 which is specific for 8-mer (50 -NNNNGATT) PAM sequence targets that can minimize the chance of off-target cleavage and

enhance specificity [188]. Besides, of the other identified orthologs of Cas9, SpCas9 has been most commonly used for GE. SpCas9, derived from *Staphylcoccus aureus*, detects the 6-mer PAM sequence (50 -NNGRRT) [189]. The modification of SpCas9 has been carried out which targets the PAM sequence (50 NGA) and edits the target gene efficiently [190]. Besides SpCas9, the shorter length of SaCas9 permits it to overcome the delivery challenges faced by SpCas9 in utilizing the multi-dimensional adeno-virus cargo vectors [189]. The CRISPR/SaCas9 system has been efficiently used to edit many plant genomes such as citrus, rice, tobacco, and *A. thaliana* [191]. Furthermore, *Streptococcus thermophilus*-derived St1Cas9 and St3Cas9 have also been employed for CRISPR-mediated GE [191]. Different types of tracrRNA and crRNA are used by these orthologs to identify different PAM sites [192].

### CRISPR/Cpf1 System

Recently, *Francisella novicida* was studied to discover the Class II type CRISPR-Cpfl system [193], recently named Cas13 [194]. In comparison to Cas9 for cleavage and production of cohesive ends, Cpfl needs a single RNA-guided complex having 4–5 nucleotides 50 -overhangs. The CRISPR-Cpfl system has been used successfully with none or fewer off-targets in both animals and plants. In 2016, the CRISPR/Cpf1 tool was effectively applied for GE in plants [195]. Due to the exceptional properties of Cpf1, type V CRISPR/Cpf1 has been considered as another powerful technique for plant GE [196]. The CRISPR/Cpf1 machinery like the conventional CRISPR/Cas9 system is formed by the two major elements: one Cpfl nuclease for target specificity and the other one for target sequence identification called crRNA. Although, in contrast to the Cas9 network, which recognizes PAM sequences with G-rich contents (50 -NGG-30 ), the Cpf1 recognizes a PAM sequence (50 -TTN-30 ) having T-rich contents [194]. Furthermore, CrRNA and tracrRNA interaction is not needed in the Cpfl system, although it is necessary for the Cas9 technique. A size of about 42 to 44 crRNA is required in the CpfI system, which is smaller than that of gRNA [194]. In rice and tobacco, targeted mutagenesis has been carried out through the CRISPR/Cpf1 mechanism derived from *Francisella novicida* (FnCpf1) [195]. In rice, *Lachnospiraceae*-derived Cpf1 (LbCpf1)-mediated targeted mutations have been reported [197,198]. Similarly, LbCpf1 and FnCpf1 nucleases have great potential for precise GE for specific gene addition via HR mechanism [199].

#### **6. Applications of CRISPR**/**Cas9 in Plant Breeding**

Climate change and rapid increases in the world's population are two major concerns that threaten agriculture production and food security globally [200]. Several biotic stressors (bacteria, viruses, fungi, insects, nematodes, etc.) and abiotic stresses (drought, salinity, heat, cold, waterlogging, etc.) hamper crop production and compromise food security around the world. Crop breeders are striving hard to develop climate-resilient, stress-tolerant crops with better quality and increased production [201]. Thus, the CRISPR/Cas9 system has numerous applications for the functional genomic research of plant genes that play a crucial role in genetic improvement of many significant agronomic traits. Especially, the knockout of some genes can encourage superior traits including disease resistance, adaptation to various abiotic stressors, nutrient usage, and yield improvements. Thus, CRISPR/Cas9-mediated GE has great potential in plant breeding for crop improvement.

#### *6.1. CRISPR*/*Cas9 System for Plant Disease Resistance*

Virus, bacteria, fungi, nematodes, and insects are the major causal agents inducing biotic stressors and crop yield reduction. Moreover, the persistent upsurge in several new strains of lethal pests make the battle very challenging against these pathogens [202]. Thus, to protect agriculture from the devastating impact of biotic stressors, it is very crucial to understand the plant–pathogen interaction [203]. GE strategies have been successfully applied to explore plant–pathogen interactions and mechanisms underlying plant responses against pathogen attack.

CRISPR/Cas9-mediated GE can be employed directly to disrupt disease-causing genes, known as "S-genes" and develop disease-resistant crops. For example, targeted knockout plants for the ethylene-responsive gene *OsERF922* were generated via the CRISPR/Cas9 tool, which showed reduced blast lesions and increased resistance against rice blast caused by *Magnaporthe oryzae* [204]. Likewise, bacterial blight-resistant plants were produced by targeted mutagenesis of the *SWEET13* gene [205]. CRISPR/Cas9-based mutagenesis was applied to the promoter region, and transcription factor (TF) of canker *CsLOB1* in *Citrus paradise* was identified. Due to the presence of such mutations, two mutant lines *DLOB10* and *DLOB9* with high mutation rates have been produced. The frame-shift mutation and disruption of the *CsLOB1* gene improved resistance against *Xanthomonas citri* [206]. Peng and colleagues reported the editing of effector binding elements (EBEs) by the CRISPR/Cas9 system in the *CsLOB1* gene promoter region to increase disease resistance in *Citrus sinensis* against *Xanthomonas citri* [207].

In wheat protoplasts, the CRISPR/Cas9 technique was applied by Shan et al. to edit the *TaMLO* gene [141] and produce wheat lines resistant to powdery mildew caused by *Blumeria graminis* f. sp. *Tritici* [62]. In another study, CRISPR/Cas9-mediated multiplex GE was performed to mutate three homologs of the *EDR1* gene to develop resistance against powdery mildew in wheat [208]. Similarly, CRISPR/Cas9-based mutants of *MLO* were produced in tomatoes which conferred resistance against powdery mildew [209].

It has been estimated that about half of plant diseases are caused by virulent viruses, which result in heavy crop losses globally [201]. Gene-targeting efficiencies were improved many folds by DNA virus amplicons. Geminiviral-based DNA replicons of wheat was utilized for transient expression of the CRISPR/Cas9 system against *wheat dwarf virus* (*WDV*), in hexaploid wheat and 12 fold upregulation was observed in ubiquitin gene expression [210]. Stable over-expression of sgRNAs and Cas9 that particularly target the genome of the Gemini-virus to prevent its growth has been applied for virus-resistant crop breeding programs [211–213]. Furthermore, the CRISPR/Cas9 system can also be used to mutate viral genomes in addition to tackling diseases caused by them [201]. The efficiency of CRISPR/Cas9-mediated viral GE can be increased by using virus promoters to govern sgRNA/Cas9 expression cassettes [211]. Recently, a new ortholog of Cas9 has been discovered in *Francisella novicida* (FnCas9) to edit RNA virus genomes. FnCas9 has successfully inhibited the replication of the tobacco mosaic virus as well as the cucumber mosaic virus and provides immunity against them [214]. Therefore, CRISPR/Cas9-mediated GE is an exceptional tool to improve genetic make-up and enables them to combat various pathogens. A list of recent studies indicating the significant success of the CRISPR/Cas9 system against various plant diseases is compiled in Table 3.



translation initiation factor 4E-1 (*elF4E*), Bidirectional sugar transporter SWEET13-like (*SWEET13*).
