Figures (5)  Tables (5)

    • Figure 1. 

      Genome editing using MNs. (a) Two MNs are required to generate the DSB at the target genomic site. (b) After DNA cleavage, repair systems based on NHEJ or HR are activated, allowing subsequent modification of the genomic locus for the biotechnological purpose of the experiment. (c) Protein/DNA interactions are established between the MN (through its specific DNA recognition/binding domain and endonuclease domain, for specific DNA cleavage) and the genomic locus to be edited. (d) The endonuclease domain must interact with the genomic spacer region to allow DNA cleavage. (e) Both MNs recognize a long cleavage spacer region, typically 14 to 40 base pairs (bp) long. Created with BioRender.com.

    • Figure 2. 

      Main aspects of genome editing with ZFNs. (a) Functional domains of ZFNs: the FokI cleavage domain and the DNA-specific recognition and binding domain; Zinc-finger domain (containing an alpha-helix and a beta-sheet with two antiparallel beta-strands, in addition to a Zn2+ cation. (b) Two ZFNs are required for the cleavage of the genomic locus, with simultaneous double activation of the FokI domains, which need to interact after touching each other. The provoked DSB activates DNA repair systems (NHEJ or HR). (c) Multiple DNA/protein interactions and between FokI domains allow two compatible ZFNs to catalyze two phosphodiester bond cleavage reactions within the spacer region. (d) Tandem Zinc-finger domains are essential for the specificity of recognition and binding to DNA at the genomic editing locus. (e) The spacer region capable of presenting an editable site for the FokI domains is short, between 5 and 7 bp. Created with BioRender.com.

    • Figure 3. 

      Most important aspects of using TALENS for genome editing. (a) TALENS have components varying in length and structural complexity. Each TALEN has two stabilization domains (N- and C-terminal), a FokI cleavage domain, and an extensive DNA-specific recognition and binding domain, made up of RVD modules with pairs of amino acid residues organized in tandem. Each combination of residues in an RVD pair can recognize and specifically bind to a base along a DNA strand. (b) The RVD DNA recognition code indicates which pair of amino acid residues are most likely to recognize and bind to a given DNA base. The same pair can bind to more than one base but with different probabilities. The figure shows the pairs with their respective most frequent bases. (c) Two TALENs with opposite orientations are needed to produce a DSB. (d) TALEN/DNA interactions are extensive, which allows for a higher level of editing precision of this technology when compared to MNs and ZFNs. (e) The RVD modules present sequential binding cooperation to the genomic editing locus, which increases the specificity of DNA recognition. (f) A spacer of 12 to 21 bp is required for the mutual activation of the FokI domains and the precise cleavage of the DNA. Created with BioRender.com.

    • Figure 4. 

      Genome editing using the classic CRISPR/Cas9 system and Prime editing. (a) Editing elements typically present in the CRISPR/Cas9 system: the DNA cleavage enzyme Cas9, the recognition RNAs of the genomic editing locus: crRNA, tracrRNA, a single guide RNA (sgRNA) and a prime editing guide RNA (pegRNA) – in the case of Prime editing. (b) In typical editing, a sgRNA internal to Cas9, containing a sequence of bases that are complementary and homologous to the bases of a DNA strand at the genomic editing locus, is used to access the editing region, which is doubly cleaved by a single Cas9 with both internal endonuclease domains, resulting in a DSB and activation of DNA repair systems. In the Prime editing system, a modified Cas9 (nCas9), linked to a reverse transcriptase (RT) and carrying only one active DNA cleavage domain, is guided by a peg RNA to the genomic editing locus. After pairing the bases of an extensive region of the pegRNA with one of the DNA strands of the editing locus, nCas9 catalyzes the production of a single-strand break (SSB) by a single nick in the DNA. RT reads the editing sequence present in a region of the pegRNA and synthesizes a DNA strand containing the editing, which is attached to the end of the nicked DNA. (c) Multiple and extensive interactions between Cas9, sgRNA and these with both DNA strands at the genomic editing locus confer a high level of specificity, precision and editing accuracy. (d) The main cause of the high specificity of DNA editing provided by the CRISPR system is the multiple interactions between nucleic acids (sgRNA and DNA), governed by complementarity and homology, which are not observed in MNs, ZFNs, and TALENs. Created with BioRender.com.

    • Figure 5. 

      Some recurring biotechnological approaches to genome editing in plants. Diagram shows the different methodological applications of the main plant genome editing tools, as well as their limitations of use. Created with BioRender.com.

    • Parameter ZFNs TALENS CRISPR/Cas9 system
      Design complexity
      		 ++ ++ +
      Versatility in editing + ++ +++
      Simultaneous editing in multiple sites + + +++
      Large-scale library delivery
      		 + + +++
      Specificity + ++ ++++
      Efficiency ++ ++ +++
      Cost + +++ +
      Homologous recombination frequency + + +
      Mutation frequency after non-homologous end joining (NHEJ) + + ++
      Use as epigenetic modifiers ++ +++ ++++
      Use as gene-knockout in model organisms − − +++
      − absent, + low, ++ moderate, +++ high, ++++ very high. Modified from Khan[11].

      Table 1. 

      Technical, biotechnological aspects, and possibilities of using ZFNs, TALENs, and CRISPR/Cas9 system for the genomic manipulation of plants.

    • Standard Cas nucleases
      Class Type Signature
      Cas protein      		 Main features Ref.
      2 II Cas9 Most popular Cas for editing the
      plant genome recognizes PAM NGG
      Requires a crRNA-tracrRNA duplex
      or a single-guide RNA for target recognition      		 [48,49]
      V Cas12a More specific than wild-type SpCas9 Requires a crRNA for target recognition, but the crRNA is
      short (~42 nt) Targets T-rich regions generates a DSB distal from the PAM site, with staggered ends      		 [50−57]
      Cas12j
      (CasΦ)      		 Recognizes PAM at the 3' end of target sequences and initiate staggered cleravages on both target and non-target DNA strands Is smaller than other Cas proteins, with 700 to 800 residues [58,59]
      Based on Zhang et al.[3]

      Table 2. 

      Some examples of classes/types and signature/standard proteins of CRISPR/cas systems for editing the plant genome.

    • Type Name Modification PAM Ref.
      Engineered Cas9 variants Cas9 nickase (nCas9) Point mutations D10A in the RuvC domain or H840A in the HNH domain
      only cleaves the targeting or non-targeting strand, respectively      		 NGG [61]
      Dead Cas9 (dCas9) Simultaneously promotes point mutations D10A in the RuvC domain or
      H840A in the HNH domain nuclease activity is abolished      		 NGG, NGN, NNG, and NNN [62]
      SpCas9 VQR Point mutations 1135V/R1335Q/T1337R NGA [63]
      SpCas9 EQR Point mutations D1135E, R1335Q, and T1337R NGAG [63]
      SpCas9 VRER Point mutations D1135V, G1218R, R1335E, and T1337R NGCG [63]
      xCas9 Point mutations A262T/R324L/S409I/E480K/E543D/M694I/E1219V NG, GAA and GAT [64]
      SpCas9-NG Point mutations R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R NG, GAA and GAT [65]
      SpRY Point mutations A61R/L1111R/N1317R/A1322R/R1333P NGN [66]
      iSpyMacCas9 Point mutations R221K and N394K NAAR [67]
      Cas9 orthologs Staphylococcus aureus
      Cas9 (SaCas9 KKH)      		 Point mutations E782K/N968K/R1015H NNGRRT [68]
      Streptococcus thermophilus Cas9 (St1Cas9) − NNAGAA [69]
      Brevibacillus laterosporus Cas9 (BlatCas9) − NNNNCND [70]
      Streptococcus macacae Cas9 (SmacCas9) − NAA [71]
      Lactobacillus rhamnosus Cas9 (LrCas9) − NGAAA [72]
      Faecalibaculum rodentium Cas9 (FrCas9) Point mutations E796A, H1010A, and D1013A NNTA [73]
      Cas12 orthologs AsCas12a − TTTV [74]
      AsCas12a RR Point mutations S542R/K607R TYCV and CCCC [75]
      AsCas12a RVR Point mutations S542R/K548V/N552R TATV [75]
      enAsCas12a Point mutations E174R/S542R/K548R VTTV, TTTT, TTCN, and TATV [76]
      LbCas12a − TTTV [74]
      LbCas12a RR Point mutations G532R/K595R TYCV and CCCC
      LbCas12a RVR Point mutations G532R/K538V/Y542R TATV [75]
      FnCas12a − TTV, TTTV, and KYTV [77]
      FnCas12a RR Point mutations N607R/K671R TYCV and TCTV [77]
      FnCas12a RVR Point mutations N607R/K613V/N617R TWTV [77]
      N can be any purine or pyrimidine base (A, G, C, or T). Based on Zhang et al.[3]

      Table 3. 

      Some examples of Cas9 engineered/orthologs and other Cas mutated for new possibilities for editing plant genomes.

    • ModificationApproachMain features
      knock-inHDR after SSN
      action      		Gene activation enhanced by
      long ssDNA donors
      knock-outNHEJ or HDR after
      the action of SSNs      		Lower in polyploid than diploids
      plant species
      Point mutationsBase editingDepends on CBEs and ABEs
      Epigenome editingCRISPR-directed methyltransferases
      and demethylases      		Editing epigenetic marks for
      transcription regulation
      Gene replacementNHEJ or HDR
      (preferred) after
      the action of SSNs      		Homology-dependency among
      DSB flanks and donor template
      flanks
      Multiplexed
      genome
      editing      		CRISPR arrayLong transcripts with multiplexed
      sgRNAs for multiple
      simultaneous editings
      Expression or delivery of sgRNAs
      through ribonucleoproteins
      (RNPs) or particle bombardment
      Gene
      transcriptional
      regulation      		CRISPR arrayCas9 fusion with various
      transcription effectors can be
      used to repress or activate
      transcription.
      Chromosomal
      rearrangement      		CRISPR arrayTarget highly endogenous
      retrotransposons and LINEs

      Table 4. 

      Main approaches available for editing the plant genome and the types of alterations brought about by them.

    • Application Main features Purposes Limitations Edited plants Ref.
      Thermal sensitivity of CRISPR/Cas9 and Cas12a Optimal editing efficiency under higher temperatures Increased efficiency of CRISPR/
      Cas9 and Cas12a by thermal
      sensitivity      		 Few plants tested Arabidopsis (29 °C) and maize (28 °C) [131]
      Generation of transgene-free edited plants Elimination of CRISPR transgenes
      by segregation associated with screening with selection marker
      genes/reporter genes
      Editing of plant genomes without
      the integration of exogenous DNA,
      by the transient expression of the CRISPR/Cas machinery      		 Minimize regulatory issues,
      enhance public acceptance
      and improve biosafety      		 Not feasible for vegetatively propagated plants, trees, polyploids and self-incompatible plants Arabidopsis, wild tobacco lettuce. rice, apple, grape, Petunia, hybrid, and soybean [120]
      Editing of polyploid
      plants      		 Multiallelic genome editing in polyploid plants Introduction of valuable
      traits in polyploid plants      		 Low efficiency, labor
      and time-consuming      		 Autopolyploid lines of Arabidopsis, Tragopogon (Asteraceae), potato (Solanum tuberosum) and Rape (Brassica napus) [132]
      Generation of germline-edited plants Genome editing using CRISPR delivered by Agrobacterium before embryogenic cell division To improve downstream genetic
      and trait analysis      		 Low editing efficiency
      in germlines      		 Arabidopsis [133]
      Off-target minimization Use wild-type Cas9 and Cas12a,
      nCas9s and high-fidelity versions of SpCas9 design of low-mismatch sgRNAs to limit the exposure of the genome to CRISPR components      		 Technical improvement of
      editing systems
      Minimize the major concern of
      CRISPR technology      		 Many high-fidelity SpCas9s show intrinsically lower nuclease activities
      in plants      		 rice [134,135]
      Improve plant breeding To express plant growth-stimulating genes use of haploid induction-edit and haploid-inducer mediated genome editing (IMGE) To enhance genome editing by
      CRISPR in recalcitrant plant
      species and varieties
      To increase stable inheritable of desirable traits through many generations      		 Inevitable segregation
      in seed production      		 Sorghum (Sorghum bicolor), sugarcane, and indica rice [136,137]

      Table 5. 

      Recent applications of CRISPR/Cas for genomic manipulation in plants and their main challenges.