Figures (1)  Tables (1)

    • Figure 1. 

      Schematic flow of genome editing in cassava. An sgRNA is designed to target the exon or promoter region of specific genes, guided by the target sequence within the sgRNA. This sgRNA associates with Cas9 endonuclease or nickase, forming a Cas9-gRNA complex to enable nuclease editing, base editing or prime editing. The CRISPR/Cas9 binary construct can be delivered directly into cassava FECs through Agrobacterium or particle bombardment, or into protoplasts via PEG-mediated transfection, leading to genome modification. Alternatively, CRISPR/Cas9 components -- whether in the form of DNA (TECCDNA), RNA (TECCRNA) or pre-assembled ribonucleoproteins (RNPs) – are delivered into FECs via particle bombardment or into protoplasts via PEG, respectively. Once transformed, the regenerated protoplasts or FECs develop into plant seedlings under antibiotic selection. These seedlings are then screened for targeted mutations using DNA sequencing techniques such as PacBio SMRT sequencing, Hi-TOM, or Sanger sequencing. Furthermore, cassava plants can be transiently transformed via Agrobacterium tumefaciens infiltration, carrying viral vectors with the Cas9-sgRNA construct to induce gene knockouts. All identified mutants are subsequently evaluated in the field trials based on phenotypic traits and molecular analysis for breeding purposes.

    • Gene names Gene functions Vectors Editing methods Mutant features Ref.
      MeSWEET10a Susceptibility (S) gene for CBB CRISPR-TA-coupled HDR strategy for tagging MeSWEET10a Target the 3' end of MeSWEET10a with HDR repair Susceptible to CBB infection (because GFP insertion is hemizygous) Veley et al.[58]
      MeSWEET10a Susceptibility (S) gene for CBB pCAMBIA1301-Cas9-EBE-sgRNA Target the EBETALE20 of MeSWEET10a promoter Improved resistance to CBB Wang et al.[59]
      MeSWEET10a Susceptibility (S) gene for CBB pEG302-DMS3 (DEFECTIVE IN MERISTEM SILENCING3)-ZF (artificial zinc-fingers)-EBE DNA methylation the EBETALE20 of MeSWEET10a promoter Improved resistance to CBB Veley et al.[60]
      MeSWEET10a Susceptibility (S) gene for CBB pUC18-mini-Tn7T-Gm-dCas9-sgRNA Target the higher conserved sequence of MeSWEET10a in promoters or 5' -UTR regions (ribosome binding site) Improved resistance to CBB Zárate-Chaves et al.[84]
      MeSWEET10a Susceptibility (S) gene for CBB pTRANS_220D-35S-Cas9-multiple gRNA spacer Csy4 array (gRNA1/2, gRNA1/3, gRNA4/5) Target the EBETALE20 of MeSWEET10a promoter and/or coding sequence Improved resistance to CBB Elliott et al.[85]
      nCBP1-nCBP2 Transcripts of eIF4E family comprises to induce CBSD pCAMBIA2300-Cas9-AtU6-26-gRNA (nCBP-1, nCBP-2, nCBP-1/nCBP-2) Target the exons of translation initiation factor 4E (eIF4E) isoforms nCBP-1, nCBP-2, nCBP-1 and nCBP-2 Double mutant lines delayed and attenuated CBSD aerial symptoms,
      but single mutant lines not      		 Gomez et al.[56]
      MePDS Key enzymes in the carotenoid biosynthesis pCAMBIA2300-35S-Cas9-AtU6-26-gMePDS-1, gMePDS-2 Target two target sites in the exon 13 of MePDS Albino or partial albino phenotypes Odipio et al.[69]
      MeEPSPS Catalysing synthesis of aromatic amino acids and secondary metabolites in plant chloroplasts pCAMBIA2300-2x35S-Cas9-AtU6-sgMeEPSPS#7-At7SL-sgMeEPSPS#11 Target sites of second intron and promoter of MeEPSPS Achieving glyphosate tolerance Hummel et al.[61]
      MeGBSS, MePTST1 Synthesizing long-chain glucan amylose and CBM48-containing protein mediate GBSS localization to starch granules 35S-pcoCas9-eGFP-NLS-tHSP-psynU6-gMeGBSS/gMePTST1-AtFT Target the exon 2 of MeGBSS and an exon at the 3′ end of the coding sequence of MePTST1 Diminish or delete amylose Bull et al.[62]
      MeSSIII-1,
      MeSSIII-2      		 Synthesis of long chain in plant amylopectin glucan pCAMBIA1301-Cas9-AtU6-gMeSSIII-1 and gMeSSIII-2 Target the exons of MeSSIII-1 and MeSSIII-2 Resistant calli stage Li et al.[63]
      MeSBE2 Synthesis of short chain during amylopectin biosynthesis pCAMBIA1301-Cas9-AtU6-gMeSBE2 Target the second and fifth exons of MeSBE2 Increase amylose and resistant starch Luo et al.[64]
      MeCYP79D1 Cyanogen biosynthesis 35S-Cas9-AtU6-26-sgMeCYP79D1 Target the exon 3 of MeCYP79D1 Reduce the levels of linamarin and evolved cyanide Juma et al.[65]
      MeCYP79D1, MeCYP79D2 Cyanogen biosynthesis pCAMBIA2300-Cas9-AtU6-sgMeCYP79D1/sgMeCYP79D2/gMeCYP79D1-MeCYP79D2 Target the exons of MeCYP79D1 and MeCYP79D2, respectively, the exons of MeCYP79D1 and MeCYP79D2 simultaneously Reduce the biosynthesis of cyanide with both genes or MeCYP79D2 mutagenesis, but MeCYP79D1 not Gomez et al.[66]
      MeCGTR1 High-affinity transporter of cyanogenic gulcosides 2x35S-Cas9-AtU6-26-sgMeCGTR1 Target the exon 1 of MeCGTR1 Lower level of cyanogenic glucosides in the top leaves and stems, no difference in roots and bottom leaves Lieberman et al.[80]
      MeF6'H1, MeF6'H1- MeF6'H2, MeF6'H3 Conversion of 6′ hydroxyl feruloyl-CoA to scopoletin 35S-Cas9-AtU6-sgMeF6'H1/sgMeF6'H1-MeF6'H2/ /sgMeF6'H3 Target the exon 1 of MeF6'H1, exon 2 of MeF6'H3, and the exons of MeF6'H1 and MeF6'H2 concurrently Delay PPD Mukami et al.[55]
      AC2, AC3 AC2 coding for the multifunctional TrAP protein involved in gene activation, virus pathogenicity, and suppression of gene silencing, and the AC3 gene coding for the REn protein involved in replication enhancement 35S-Cas9-U6-sgAC2-AC3 Target the viral AC2 and AC3 No significant differences in disease incidence, symptom severity, or virus titres Mehta et al.[57]
      MeE3L Hijacking and redirection of ubiquitination by geminiviruses, associated with CMD2 resistance locus pC1380-TMV-Cas9-eGFP-U6-26-gMeE3L-1-gMeE3L-2 Target the exon of MeE3L SACMV DNA accumulation Chatukuta & Rey[92]
      MeRPPL1 Involved in CMD tolerance, recognize pathogen effectors and trigger plant effector-triggered immunity (ETI) pC1380-TMV-Cas9-eGFP-U6-26-gMeRPPL1-1- gMeRPPL1-2 Target the exon of MeRPPL1 SACMV-DNA A accumulation Ramulifho et al.[93]

      Table 1. 

      The applications of gene editing technology in cassava.