Development of in planta genome editing by transient expression of genome-editing tools in tomato.

RNA-Guided Genome Editing in Plants Using a CRISPR–Cas System

Critical considerations and computational tools in plant genome editing.

Secondary metabolites, including alkaloids, flavonoids, and tannins, are crucial for human health, agriculture, and ecosystem functioning. Their synthesis is often species-specific, influenced by both genetic and environmental factors. The increasing demand for these compounds across various industries highlights the need for advancements in plant breeding and biotechnological approaches. Transcription activator-like effector nucleases (TALENs) have emerged as a powerful tool for precise genome editing, offering significant potential for enhancing the synthesis of secondary metabolites in plants. However, while plant genome editing technologies have advanced significantly, the application of TALENs in improving secondary metabolite production and expanding genetic diversity remains underexplored. Therefore, this review aims to provide a comprehensive analysis of TALEN-mediated genome editing in plants, focusing on their role in enhancing secondary metabolite biosynthetic pathways and improving genetic diversity. The mechanisms underlying TALENs are examined, including their ability to target specific genes involved in the synthesis of bioactive compounds, highlighting comparisons with other genome editing tools such as CRISPR/Cas9. This review further highlights key applications in medicinal plants, particularly the modification of pathways responsible for alkaloids, flavonoids, terpenoids, and phenolic compounds. Furthermore, the role of TALENs in inducing genetic variation, improving stress tolerance, and facilitating hybridization in plant breeding programs is highlighted. Recent advances, challenges, and limitations associated with using TALENs for enhancing secondary metabolite production are critically evaluated. In this review, gaps in current research are identified, particularly regarding the integration of TALENs with multi-omics technologies and synthetic biology approaches. The findings suggest that while underutilized, TALENs offer sustainable strategies for producing high-value secondary metabolites in medicinal plants. Future research should focus on optimizing TALEN systems for commercial applications and integrating them with advanced biotechnological platforms to enhance the yield and resilience of medicinal plants.

Targeted base editing in rice with CRISPR/ScCas9 system

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

The clustered regularly interspaced short palindromic repeats (CRISPR-Cas9), a genome editing tool, has gained a tremendous position due to its therapeutic efficacy, ability to counteract abiotic/biotic stresses in plants, environmental remediation and sustainable agriculture with the aim of food security. This is mainly due to their potential of precised genome modification and numerous genetic engineering protocols with versatility as well as simplicity. This technique is quite useful for crop refinement and overcoming the agricultural losses and regaining the soil fertility hampered by hazardous chemicals. Since CRISPR/Cas9 has been widely accepted in genome editing in plants, however, their revolutionised nature and progress enable genetic engineers to face numerous challenges in plant biotechnology. Therefore, nanoparticles have addressed these challenges and improved cargo delivery and genomic editing processes. Henceforth, this barrier prevents CRISPR-based genetic engineering in plants in order to show efficacy in full potential and eliminate all the barriers. This advancement accelerates the genome editing process and its applications in plant biotechnology enable us to sustain and feed the massive population under varying environments. Genome editing tools using CRISPR/Cas9 and nanotechnology are advantageous that produce transgenic-free plants that overcome global food demands. Here, in this review, we have aimed towards the mechanisms/delivery systems linked with CRISPR/Cas9 system. We have elaborated on the applications of CRISPR/Cas9 and nanotechnology-based systems for sustainable agriculture. Moreover, the challenges and limitations associated with genome editing and delivery systems have also been discussed with a special emphasis on crop improvement.

The tissue culture process is usually involved in gene transfer and genome editing in plants. Like other species, there is enormous variation among wheat genotypes in tissue culture response. In the rapidly advancing system of CRISPR/Cas9 for genome editing, particle bombardment has received increasing attention as a delivery method for a large amount of nucleic acids and RNA-protein complexes. However, the efficiency of transformation by particle bombardment has been low in wheat, and only a limited number of varieties have been transformed. In this study, replacement of maltose with trehalose as an osmolyte for high osmotic treatment for the protection of tissues from physical impacts improved callus formation in immature wheat embryos and efficiency of transformation and genome editing in varieties that are relatively poor in tissue culture response. The range of varieties amenable to biolistic transformation and genome editing may be expanded by this modification.

Prime editing is a novel and universal CRISPR-Cas-derived precise genome-editing technology and has great potentials for applications in basic plant research and crop molecular breeding (Anzalone et al., 2019Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (1346) Google Scholar). Although low efficiency has restrained the original prime editors (PEs) from being used as a routine tool for precise genome editing in plants, an iterative update of the PEs is removing this obstacle (Lin et al., 2021Lin Q. Jin S. Zong Y. Yu H. Zhu Z. Liu G. Kou L. Wang Y. Qiu J.L. Li J. et al.High-efficiency prime editing with optimized, paired pegRNAs in plants.Nat. Biotechnol. 2021; 39: 923-927Crossref PubMed Scopus (73) Google Scholar; Xu et al., 2022Xu W. Yang Y. Yang B. Krueger C.J. Xiao Q. Zhao S. Zhang L. Kang G. Wang F. Yi H. et al.A design optimized prime editor with expanded scope and capability in plants.Nat. Plants. 2022; 8: 45-52Crossref PubMed Scopus (16) Google Scholar). Recently, the Liu group reported three optimization strategies for improving prime editing efficiency (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar; Nelson et al., 2022Nelson J.W. Randolph P.B. Shen S.P. Everette K.A. Chen P.J. Anzalone A.V. An M. Newby G.A. Chen J.C. Hsu A. et al.Engineered pegRNAs improve prime editing efficiency.Nat. Biotechnol. 2022; 40: 402-410Crossref PubMed Scopus (61) Google Scholar). The first strategy is based on engineered prime editing guide RNAs (epegRNAs), which were generated by incorporating structured RNA motifs to the 3′ terminus of pegRNAs. This strategy enhances pegRNA stability and prevents degradation of the 3′ extension (Nelson et al., 2022Nelson J.W. Randolph P.B. Shen S.P. Everette K.A. Chen P.J. Anzalone A.V. An M. Newby G.A. Chen J.C. Hsu A. et al.Engineered pegRNAs improve prime editing efficiency.Nat. Biotechnol. 2022; 40: 402-410Crossref PubMed Scopus (61) Google Scholar). The second strategy is based on the optimized PE2 protein (PEmax), which harbors a SpCas9 variant with increased nuclease activity, an additional nuclear localization signal (NLS) sequences, and a new linker between nCas9 and reverse transcriptase (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The third strategy is based on inhibition of DNA mismatch repair (MMR) in cells (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In this work, we tested the optimized PEs generated with these three strategies in rice, demonstrating that the optimized PEs greatly improved prime editing efficiency in rice. We named the two optimized PEs ePE3max and ePE5max: the former is comprised of the PEmax protein, an epegRNA with evopreQ1 appended to its 3′ end, and a nicking sgRNA; the latter is comprised of the ePE3max system and a dominant negative OsMLH1 variant for inhibiting MMR. Using the two optimized PEs, we efficiently generated homozygous and heterozygous T173I, A174V, and P177S (TAP-IVS) mutation in EPSPS in rice, which lays a solid foundation for rice non-transgenic glyphosate-resistance breeding.TAP-IVS mutation (T102I, A103V, and P106S) is a naturally occurring EPSPS triple substitution found in the highly glyphosate resistant Amaranthus hybridus population from Argentina (Perotti et al., 2019Perotti V.E. Larran A.S. Palmieri V.E. Martinatto A.K. Alvarez C.E. Tuesca D. Permingeat H.R. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate-resistant Amaranthus hybridus population from Argentina.Pest Manag. Sci. 2019; 75: 1242-1251Crossref PubMed Scopus (44) Google Scholar). Less or little fitness cost associated with the homozygous TAP-IVS mutation accounted for the proliferation of the mutants within the population, even in absence of glyphosate (Perotti et al., 2019Perotti V.E. Larran A.S. Palmieri V.E. Martinatto A.K. Alvarez C.E. Tuesca D. Permingeat H.R. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate-resistant Amaranthus hybridus population from Argentina.Pest Manag. Sci. 2019; 75: 1242-1251Crossref PubMed Scopus (44) Google Scholar). Since non-transgenic glyphosate-resistance rice varieties have great values in agriculture, we wondered whether we were able to use the optimized PEs to efficiently generate the TAP-IVS mutation in OsEPSPS for rice non-transgenic glyphosate-resistance breeding (Perotti et al., 2019Perotti V.E. Larran A.S. Palmieri V.E. Martinatto A.K. Alvarez C.E. Tuesca D. Permingeat H.R. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate-resistant Amaranthus hybridus population from Argentina.Pest Manag. Sci. 2019; 75: 1242-1251Crossref PubMed Scopus (44) Google Scholar; Li et al., 2020Li H. Li J. Chen J. Yan L. Xia L. Precise modifications of both exogenous and endogenous genes in rice by prime editing.Mol. Plant. 2020; 13: 671-674Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Thus, we tested the optimized PEs for installation of the TAP-IVS edits in EPSPS in rice protoplasts. Our results indicate that the PEmax architecture improved prime editing efficiency and could synergize with the epegRNA (pegRNA-evopreQ1) in rice protoplasts (Figure 1A ).Since the ePE3max, which is comprised of the PEmax protein, an epegRNA, and a nicking sgRNA, achieved the highest editing efficiency (Figure 1A), we then asked whether we were able to further improve editing efficiency of the ePE3max by co-expressing dominant negative OsMLH1 variants. We generated codon-optimized OsMLH1dn, OsMLH1 E44A, OsMLH1dn E44A, and OsMLH1 1-393-2xNLS, corresponding to human MLH1dn, MLH1 E34A, MLH1dn E34A, and MLH1 1-335-NLS, respectively (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). These variants could integrate into the MMR complex in replacement of OsMLH1 with normal function and interfere the function of MMR, leading to inhibition of MMR. These OsMLH1 variants and the ePE3max constitute the ePE5max system. The ePE5max system did not further improve editing efficiency of the ePE3max in rice protoplasts (Figure 1A).To generate rice lines harboring the TAP-IVS mutation, we generated two optimized PEs, ePE3max-TAP and ePE5max-TAP, for rice transformation (Figure 1B). We also generated two control PEs, PE3-TAP and PE5-TAP, which harbor the original PE protein and pegRNA, for comparison with the two optimized PEs in rice transgenic lines. We transformed rice with these four PEs. We analyzed the edits by the Sanger sequencing of PCR fragments amplified from the transgenic lines (Figure 1C) and next-generation sequencing of PCR amplicons. Next-generation sequencing results indicate that the ePE3max and ePE5max significantly improved editing efficiency in rice transgenic lines compared with PE3 and PE5, respectively (Figure 1D). The ePE5max had similar efficiency of heritable mutations (homozygous and heterozygous) to the ePE3max but had higher efficiency of homozygous mutations than the ePE3max. Using the ePE5max, we achieved 6.1% (10/163) homozygous TAP-IVS mutant lines (Figure 1D; Supplemental Table 1).We transplanted the homozygous TAP-IVS mutant lines and transgenic lines without edits (control) to soil and sprayed 10 mM glyphosate on the seedlings. As expected, the mutant plants showed reliable tolerance compared with the control (Figure 1E). As we previously reported (Jiang et al., 2020Jiang Y.Y. Chai Y.P. Lu M.H. Han X.L. Lin Q. Zhang Y. Zhang Q. Zhou Y. Wang X.C. Gao C. et al.Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.Genome Biol. 2020; 21: 257Crossref PubMed Scopus (71) Google Scholar), in this study, once again we observed the new type of byproducts in rice protoplasts and transgenic lines (Figure 1A; Supplemental Figure 1). This type of undesired edits, which we previously described as unbiased heteroduplex DNA repair-derived byproducts (Re-byproducts), was produced upon installation of multiple-base substitution edits (Jiang et al., 2020Jiang Y.Y. Chai Y.P. Lu M.H. Han X.L. Lin Q. Zhang Y. Zhang Q. Zhou Y. Wang X.C. Gao C. et al.Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.Genome Biol. 2020; 21: 257Crossref PubMed Scopus (71) Google Scholar). Consistent with the role of nicking sgRNAs in PE3 (Anzalone et al., 2019Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (1346) Google Scholar), the Re-byproducts relative to the desired edits induced by the ePE3max were much less than those by the ePE2max (Figure 1A). Since prime editing efficiency in the generation of homozygous TAP-IVS mutants has been sufficiently high, the optimized PEs enable researchers to perform in planta-directed evolution of the TAP site of EPSPS, which facilitates overcoming the potential fitness cost, if present, associated with the TAP-IVS mutation (Xu et al., 2021Xu R. Liu X. Li J. Qin R. Wei P. Identification of herbicide resistance OsACC1 mutations via in planta prime-editing-library screening in rice.Nat. Plants. 2021; 7: 888-892Crossref PubMed Scopus (16) Google Scholar).To further evaluate effects of dominant negative OsMLH1 variants on prime editing efficiency, we tested them at more target sites in rice protoplasts. On the whole, we still did not observe significant improvement of prime editing efficiency in rice protoplasts when PEs were co-expressed with OsMLH1 variants (Figures 1F and 1G; Supplemental Figures 2 and 3). Nevertheless, installation of the desired edits at additional 20 target sites demonstrated that both the PEmax architecture and epegRNAs greatly improved prime editing efficiency and that these two strategies could synergize with each other (Figures 1H and 1I; Supplemental Figures 4, 5, and 6). Not surprisingly, we observed that improved prime editing efficiency also led to high-frequency byproducts derived from the pegRNA scaffold (Figure 1H; Supplemental Figures 4, 5, and 6). To overcome this problem, in our previous report, we put forward the rule of termination for design of the reverse transcriptase template (rtT): 5′ terminus of genomic DNA sequences corresponding to the rtT should be terminated behind one to three specific bases in genomic DNA, and the bases should be C, GC, or TGC, which correspond to the 3′ terminus of the sgRNA scaffold (Jiang et al., 2020Jiang Y.Y. Chai Y.P. Lu M.H. Han X.L. Lin Q. Zhang Y. Zhang Q. Zhou Y. Wang X.C. Gao C. et al.Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.Genome Biol. 2020; 21: 257Crossref PubMed Scopus (71) Google Scholar). We demonstrate that by using the rule of termination to re-design pegRNAs, we were able to eliminate the byproducts (Supplemental Figures 7 and 8). Since most of the pegRNA-derived byproducts resulted from mistaken reverse transcription of the only one base (C) of the sgRNA scaffold adjoining the rtT, the rule for design of the rtT was able to change most pegRNA-derived byproducts, if present, into desired sequences matching genomic DNA completely.Interestingly, we found that PE3 outperformed PE2 at most sites (Figures 1H and 1I; Supplemental Figures 4, 5, and 6), which seems inconsistent with previous reports (Lin et al., 2020Lin Q. Zong Y. Xue C. Wang S. Jin S. Zhu Z. Wang Y. Anzalone A.V. Raguram A. Doman J.L. et al.Prime genome editing in rice and wheat.Nat. Biotechnol. 2020; 38: 582-585Crossref PubMed Scopus (287) Google Scholar). We reasoned that the 35S-CmYLCV-U6 (35C) composite promoter (Jiang et al., 2020Jiang Y.Y. Chai Y.P. Lu M.H. Han X.L. Lin Q. Zhang Y. Zhang Q. Zhou Y. Wang X.C. Gao C. et al.Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.Genome Biol. 2020; 21: 257Crossref PubMed Scopus (71) Google Scholar) we used in this study may account for the inconsistency. To provide evidence, we compared editing efficiency of PE2s and PE3s with pegRNAs driven by the U3 or 35C promoter at four target sites in rice protoplasts (Supplemental Figure 9A and 9B). As expected, the results indicated that the 35C promoter exhibited higher editing efficiency in the two types of PEs and a larger ratio of PE3 to PE2 in efficiency than the U3 promoter. We further reasoned that it was because we selected appropriate nicking sgRNAs that we achieved higher editing efficiency of PE3s than that of PE2s. To provide evidence, we tested the same PE2 in combination with different nicking sgRNAs to constitute PE3 in rice protoplasts. As expected, editing efficiency of PE3 depended on appropriate nicking sgRNAs, which should have the high nicking activity and appropriate positions (Supplemental Figure 9C).To provide evidence that PE3 outperforms PE2 in transgenic lines and that OsMLH1dn is functional at some in transgenic we generated four types of PEs at four target sites for rice to the results in PE3 and outperformed PE2 and in transgenic lines (Supplemental Figure Supplemental Table Since outperformed PE2 at the four (Supplemental Figure Supplemental Table the results that as in OsMLH1dn is more or less at at some in transgenic lines. Consistent with the previous (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), of MLH1dn the of or byproducts of PE3 (Figure 1D; Supplemental Figure Supplemental Table further evidence that OsMLH1dn is more or less at at some Since the MMR system is for genome stability (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), the MMR activity from the of MLH1dn may and substitution of bases at the This potential to be the ePE5max system in we demonstrate that the PEmax architecture and the epegRNA greatly improved prime editing efficiency in rice, and inhibition of cellular activity of MMR by co-expressing dominant negative OsMLH1 could synergize with the PEmax architecture and the epegRNA at some We also demonstrate that the pegRNA byproducts, which increased with prime editing efficiency, could be by using the termination rule for design of pegRNAs. Our results that the optimized PEs able to the obstacle to applications of prime editing in rice. In by using the optimized PE we efficiently generated heritable TAP-IVS mutation in EPSPS in rice, which lays a solid foundation for rice non-transgenic glyphosate-resistance The optimized PE also facilitates overcoming the potential fitness cost associated with the TAP-IVS mutation by evolution of the TAP site of Y. and the and Y. Zhang the Y. and the the was by from the of and and the Prime editing is a novel and universal CRISPR-Cas-derived precise genome-editing technology and has great potentials for applications in basic plant research and crop molecular breeding (Anzalone et al., 2019Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (1346) Google Scholar). Although low efficiency has restrained the original prime editors (PEs) from being used as a routine tool for precise genome editing in plants, an iterative update of the PEs is removing this obstacle (Lin et al., 2021Lin Q. Jin S. Zong Y. Yu H. Zhu Z. Liu G. Kou L. Wang Y. Qiu J.L. Li J. et al.High-efficiency prime editing with optimized, paired pegRNAs in plants.Nat. Biotechnol. 2021; 39: 923-927Crossref PubMed Scopus (73) Google Scholar; Xu et al., 2022Xu W. Yang Y. Yang B. Krueger C.J. Xiao Q. Zhao S. Zhang L. Kang G. Wang F. Yi H. et al.A design optimized prime editor with expanded scope and capability in plants.Nat. Plants. 2022; 8: 45-52Crossref PubMed Scopus (16) Google Scholar). Recently, the Liu group reported three optimization strategies for improving prime editing efficiency (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar; Nelson et al., 2022Nelson J.W. Randolph P.B. Shen S.P. Everette K.A. Chen P.J. Anzalone A.V. An M. Newby G.A. Chen J.C. Hsu A. et al.Engineered pegRNAs improve prime editing efficiency.Nat. Biotechnol. 2022; 40: 402-410Crossref PubMed Scopus (61) Google Scholar). The first strategy is based on engineered prime editing guide RNAs (epegRNAs), which were generated by incorporating structured RNA motifs to the 3′ terminus of pegRNAs. This strategy enhances pegRNA stability and prevents degradation of the 3′ extension (Nelson et al., 2022Nelson J.W. Randolph P.B. Shen S.P. Everette K.A. Chen P.J. Anzalone A.V. An M. Newby G.A. Chen J.C. Hsu A. et al.Engineered pegRNAs improve prime editing efficiency.Nat. Biotechnol. 2022; 40: 402-410Crossref PubMed Scopus (61) Google Scholar). The second strategy is based on the optimized PE2 protein (PEmax), which harbors a SpCas9 variant with increased nuclease activity, an additional nuclear localization signal (NLS) sequences, and a new linker between nCas9 and reverse transcriptase (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The third strategy is based on inhibition of DNA mismatch repair (MMR) in cells (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In this work, we tested the optimized PEs generated with these three strategies in rice, demonstrating that the optimized PEs greatly improved prime editing efficiency in rice. We named the two optimized PEs ePE3max and ePE5max: the former is comprised of the PEmax protein, an epegRNA with evopreQ1 appended to its 3′ end, and a nicking sgRNA; the latter is comprised of the ePE3max system and a dominant negative OsMLH1 variant for inhibiting MMR. Using the two optimized PEs, we efficiently generated homozygous and heterozygous T173I, A174V, and P177S (TAP-IVS) mutation in EPSPS in rice, which lays a solid foundation for rice non-transgenic glyphosate-resistance TAP-IVS mutation (T102I, A103V, and P106S) is a naturally occurring EPSPS triple substitution found in the highly glyphosate resistant Amaranthus hybridus population from Argentina (Perotti et al., 2019Perotti V.E. Larran A.S. Palmieri V.E. Martinatto A.K. Alvarez C.E. Tuesca D. Permingeat H.R. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate-resistant Amaranthus hybridus population from Argentina.Pest Manag. Sci. 2019; 75: 1242-1251Crossref PubMed Scopus (44) Google Scholar). Less or little fitness cost associated with the homozygous TAP-IVS mutation accounted for the proliferation of the mutants within the population, even in absence of glyphosate (Perotti et al., 2019Perotti V.E. Larran A.S. Palmieri V.E. Martinatto A.K. Alvarez C.E. Tuesca D. Permingeat H.R. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate-resistant Amaranthus hybridus population from Argentina.Pest Manag. Sci. 2019; 75: 1242-1251Crossref PubMed Scopus (44) Google Scholar). Since non-transgenic glyphosate-resistance rice varieties have great values in agriculture, we wondered whether we were able to use the optimized PEs to efficiently generate the TAP-IVS mutation in OsEPSPS for rice non-transgenic glyphosate-resistance breeding (Perotti et al., 2019Perotti V.E. Larran A.S. Palmieri V.E. Martinatto A.K. Alvarez C.E. Tuesca D. Permingeat H.R. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate-resistant Amaranthus hybridus population from Argentina.Pest Manag. Sci. 2019; 75: 1242-1251Crossref PubMed Scopus (44) Google Scholar; Li et al., 2020Li H. Li J. Chen J. Yan L. Xia L. Precise modifications of both exogenous and endogenous genes in rice by prime editing.Mol. Plant. 2020; 13: 671-674Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Thus, we tested the optimized PEs for installation of the TAP-IVS edits in EPSPS in rice protoplasts. Our results indicate that the PEmax architecture improved prime editing efficiency and could synergize with the epegRNA (pegRNA-evopreQ1) in rice protoplasts (Figure 1A Since the ePE3max, which is comprised of the PEmax protein, an epegRNA, and a nicking sgRNA, achieved the highest editing efficiency (Figure 1A), we then asked whether we were able to further improve editing efficiency of the ePE3max by co-expressing dominant negative OsMLH1 variants. We generated codon-optimized OsMLH1dn, OsMLH1 E44A, OsMLH1dn E44A, and OsMLH1 1-393-2xNLS, corresponding to human MLH1dn, MLH1 E34A, MLH1dn E34A, and MLH1 1-335-NLS, respectively (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). These variants could integrate into the MMR complex in replacement of OsMLH1 with normal function and interfere the function of MMR, leading to inhibition of MMR. These OsMLH1 variants and the ePE3max constitute the ePE5max system. The ePE5max system did not further improve editing efficiency of the ePE3max in rice protoplasts (Figure 1A). To generate rice lines harboring the TAP-IVS mutation, we generated two optimized PEs, ePE3max-TAP and ePE5max-TAP, for rice transformation (Figure 1B). We also generated two control PEs, PE3-TAP and PE5-TAP, which harbor the original PE protein and pegRNA, for comparison with the two optimized PEs in rice transgenic lines. We transformed rice with these four PEs. We analyzed the edits by the Sanger sequencing of PCR fragments amplified from the transgenic lines (Figure 1C) and next-generation sequencing of PCR amplicons. Next-generation sequencing results indicate that the ePE3max and ePE5max significantly improved editing efficiency in rice transgenic lines compared with PE3 and PE5, respectively (Figure 1D). The ePE5max had similar efficiency of heritable mutations (homozygous and heterozygous) to the ePE3max but had higher efficiency of homozygous mutations than the ePE3max. Using the ePE5max, we achieved 6.1% (10/163) homozygous TAP-IVS mutant lines (Figure 1D; Supplemental Table 1). We transplanted the homozygous TAP-IVS mutant lines and transgenic lines without edits (control) to soil and sprayed 10 mM glyphosate on the seedlings. As expected, the mutant plants showed reliable tolerance compared with the control (Figure 1E). As we previously reported (Jiang et al., 2020Jiang Y.Y. Chai Y.P. Lu M.H. Han X.L. Lin Q. Zhang Y. Zhang Q. Zhou Y. Wang X.C. Gao C. et al.Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.Genome Biol. 2020; 21: 257Crossref PubMed Scopus (71) Google Scholar), in this study, once again we observed the new type of byproducts in rice protoplasts and transgenic lines (Figure 1A; Supplemental Figure 1). This type of undesired edits, which we previously described as unbiased heteroduplex DNA repair-derived byproducts (Re-byproducts), was produced upon installation of multiple-base substitution edits (Jiang et al., 2020Jiang Y.Y. Chai Y.P. Lu M.H. Han X.L. Lin Q. Zhang Y. Zhang Q. Zhou Y. Wang X.C. Gao C. et al.Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.Genome Biol. 2020; 21: 257Crossref PubMed Scopus (71) Google Scholar). Consistent with the role of nicking sgRNAs in PE3 (Anzalone et al., 2019Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (1346) Google Scholar), the Re-byproducts relative to the desired edits induced by the ePE3max were much less than those by the ePE2max (Figure 1A). Since prime editing efficiency in the generation of homozygous TAP-IVS mutants has been sufficiently high, the optimized PEs enable researchers to perform in planta-directed evolution of the TAP site of EPSPS, which facilitates overcoming the potential fitness cost, if present, associated with the TAP-IVS mutation (Xu et al., 2021Xu R. Liu X. Li J. Qin R. Wei P. Identification of herbicide resistance OsACC1 mutations via in planta prime-editing-library screening in rice.Nat. Plants. 2021; 7: 888-892Crossref PubMed Scopus (16) Google Scholar). To further evaluate effects of dominant negative OsMLH1 variants on prime editing efficiency, we tested them at more target sites in rice protoplasts. On the whole, we still did not observe significant improvement of prime editing efficiency in rice protoplasts when PEs were co-expressed with OsMLH1 variants (Figures 1F and 1G; Supplemental Figures 2 and 3). Nevertheless, installation of the desired edits at additional 20 target sites demonstrated that both the PEmax architecture and epegRNAs greatly improved prime editing efficiency and that these two strategies could synergize with each other (Figures 1H and 1I; Supplemental Figures 4, 5, and 6). Not surprisingly, we observed that improved prime editing efficiency also led to high-frequency byproducts derived from the pegRNA scaffold (Figure 1H; Supplemental Figures 4, 5, and 6). To overcome this problem, in our previous report, we put forward the rule of termination for design of the reverse transcriptase template (rtT): 5′ terminus of genomic DNA sequences corresponding to the rtT should be terminated behind one to three specific bases in genomic DNA, and the bases should be C, GC, or TGC, which correspond to the 3′ terminus of the sgRNA scaffold (Jiang et al., 2020Jiang Y.Y. Chai Y.P. Lu M.H. Han X.L. Lin Q. Zhang Y. Zhang Q. Zhou Y. Wang X.C. Gao C. et al.Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.Genome Biol. 2020; 21: 257Crossref PubMed Scopus (71) Google Scholar). We demonstrate that by using the rule of termination to re-design pegRNAs, we were able to eliminate the byproducts (Supplemental Figures 7 and 8). Since most of the pegRNA-derived byproducts resulted from mistaken reverse transcription of the only one base (C) of the sgRNA scaffold adjoining the rtT, the rule for design of the rtT was able to change most pegRNA-derived byproducts, if present, into desired sequences matching genomic DNA we found that PE3 outperformed PE2 at most sites (Figures 1H and 1I; Supplemental Figures 4, 5, and 6), which seems inconsistent with previous reports (Lin et al., 2020Lin Q. Zong Y. Xue C. Wang S. Jin S. Zhu Z. Wang Y. Anzalone A.V. Raguram A. Doman J.L. et al.Prime genome editing in rice and wheat.Nat. Biotechnol. 2020; 38: 582-585Crossref PubMed Scopus (287) Google Scholar). We reasoned that the 35S-CmYLCV-U6 (35C) composite promoter (Jiang et al., 2020Jiang Y.Y. Chai Y.P. Lu M.H. Han X.L. Lin Q. Zhang Y. Zhang Q. Zhou Y. Wang X.C. Gao C. et al.Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.Genome Biol. 2020; 21: 257Crossref PubMed Scopus (71) Google Scholar) we used in this study may account for the inconsistency. To provide evidence, we compared editing efficiency of PE2s and PE3s with pegRNAs driven by the U3 or 35C promoter at four target sites in rice protoplasts (Supplemental Figure 9A and 9B). As expected, the results indicated that the 35C promoter exhibited higher editing efficiency in the two types of PEs and a larger ratio of PE3 to PE2 in efficiency than the U3 promoter. We further reasoned that it was because we selected appropriate nicking sgRNAs that we achieved higher editing efficiency of PE3s than that of PE2s. To provide evidence, we tested the same PE2 in combination with different nicking sgRNAs to constitute PE3 in rice protoplasts. As expected, editing efficiency of PE3 depended on appropriate nicking sgRNAs, which should have the high nicking activity and appropriate positions (Supplemental Figure To provide evidence that PE3 outperforms PE2 in transgenic lines and that OsMLH1dn is functional at some in transgenic we generated four types of PEs at four target sites for rice to the results in PE3 and outperformed PE2 and in transgenic lines (Supplemental Figure Supplemental Table Since outperformed PE2 at the four (Supplemental Figure Supplemental Table the results that as in OsMLH1dn is more or less at at some in transgenic lines. Consistent with the previous (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), of MLH1dn the of or byproducts of PE3 (Figure 1D; Supplemental Figure Supplemental Table further evidence that OsMLH1dn is more or less at at some Since the MMR system is for genome stability (Chen et al., 2021Chen P.J. Hussmann J.A. Yan J. Knipping F. Ravisankar P. Chen P.F. Chen C. Nelson J.W. Newby G.A. Sahin M. et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.Cell. 2021; 184: 5635-5652.e29Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), the MMR activity from the of MLH1dn may and substitution of bases at the This potential to be the ePE5max system in In we demonstrate that the PEmax architecture and the epegRNA greatly improved prime editing efficiency in rice, and inhibition of cellular activity of MMR by co-expressing dominant negative OsMLH1 could synergize with the PEmax architecture and the epegRNA at some We also demonstrate that the pegRNA byproducts, which increased with prime editing efficiency, could be by using the termination rule for design of pegRNAs. Our results that the optimized PEs able to the obstacle to applications of prime editing in rice. In by using the optimized PE we efficiently generated heritable TAP-IVS mutation in EPSPS in rice, which lays a solid foundation for rice non-transgenic glyphosate-resistance The optimized PE also facilitates overcoming the potential fitness cost associated with the TAP-IVS mutation by evolution of the TAP site of Y. and the and Y. Zhang the Y. and the the Y. and the and Y. Zhang the Y. and the the was by from the of and and the

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genetic engineering that uses cas9 to target the sequence-specific region and introduce a double-stranded break in the target area. This simple RNA-guided genome editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. This technique helps to make precise genome modification in many different tissues and organisms. Development of genetically edited crops will assist sustainable productive agriculture for better feeding of the rapidly growing population in a changing climate. The emerging areas of research for the genome editing in plants include rewiring the regulatory signaling networks, and interrogating gene functions and sgRNA library for high-throughput loss-of-function screening. This chapter deals with the strengths and weaknesses of Cas9 nuclease-mediated genome editing in plants for development of designer crops like sugarcane. With this powerful and innovative technique, the genetically engineered non-GM plants will support the sustainable agriculture and maximize yield by combating abiotic and biotic stresses.

Recently the engineered nucleases have revolutionized genome editing to perturb gene expression at specific sites in complex eukaryotic genomes. Three important classes of these genome editing tools are Moreover, the more recent type II Clustered Regularly Inter-spaced Short Palindromic Repeats/Crispr associated protein (CRISPR/Cas9) system has become the most favorite plant genome editing tool for its precision and RNA based specificity unlike its counterparts which rely on protein based specificity. Plasmid-mediated co-delivery of multiple sgRNAs and Cas9 to the Plant cell can simultaneously alter more than one target loci which enable multiplex genome editing. In this review, we discuss recent advancements in the CRISPR/ Cas9 technology mechanism, theory and its applications in plants and agriculture. We also suggest that the CRISPR/ Cas9 as an effective genome editing tool, has vast potential for crop improvement and studying gene regulation mechanism and chromatin remodeling.

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Genome editing and plant transformation are crucial techniques in plant biotechnology, allowing for the precise modification of plant genomes to enhance agronomically essential traits. The advancement of CRISPR-based genome editing tools in plants is limited, among others, by developing novel in vitro tissue culture methodologies for efficient plant genetic transformation. In-planta methodologies offer a promising alternative to overcome tissue culture limitations and facilitate crops’ genetic improvement. The in-planta transformation methods can be categorized under the definition of means of plant genetic transformation with no or minimal tissue culture steps meeting the conditions for minimal steps: short duration with a limited number of transfers, high technical simplicity, limited list of hormones, and that the regeneration does not undergo callus development. In this review, we analyzed over 250 articles. We identified studies that follow an in-planta transformation methodology for delivering CRISPR/Cas9 components focusing on crop plants, as model species have been previously reviewed in detail. This approach has been successfully applied for genome editing in crop plants: camelina, cotton, lemon, melon, orange, peanut, rice, soybean, and wheat. Overall, this study underscores the importance of in-planta methodologies in overcoming the limitations of tissue culture and advancing the field of plant genome editing.

Genome editing has revolutionized plant research and plant breeding by enabling precise genome manipulation. In particular, the application of type II CRISPR-Cas9 systems to genome editing has proved an important milestone, accelerating genetic engineering and the analysis of gene function. On the other hand, the potential of other types of CRISPR-Cas systems, especially many of the most abundant type I CRISPR-Cas systems, remains unexplored. We recently developed a novel genome editing tool, TiD, based on the type I-D CRISPR-Cas system. In this chapter, we describe a protocol for genome editing of plant cells using TiD. This protocol allows the application of TiD to induce short insertion and deletions (indels) or long-range deletions at target sites with high specificity in tomato cells.

<p indent="0mm">CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) nucleases were originally identified in bacteria and archaea as their immune protection system to destroy phage and exogenous DNA. Relatively simple and powerful genome editing technologies have been developed based on the CRISPR/Cas system. In the last decade, CRISPR/Cas has been utilized as an effective genome editing tool in microorganisms, animals and plants. In this review, we will first discuss how the CRISPR/Cas system (e.g., CRISPR/Cas9) is used to edit plant genomes through various developed tools including small and large size DNA fragment deletions, homology-directed recombination, cytosine base editor, adenine base editor, dual base editor and prime editor. Details about these CRISPR/Cas plant genome editing tools including their working mechanisms, the construction of these tools using various functional elements, and several examples of applications will be provided. Indeed, the successful development of these tools allows researchers to modify plant genomes, functionally dissect plant genes, conduct molecular-design based breeding. Although the CRISPR/Cas T-DNA can be screened out either through back-crossing to wild type or self-crossing, the DNA-free CRISPR/Cas plant genome editing technologies bring obvious advantages in crop molecular breeding, especially for vegetative breeding crops or long juvenile stage plants. Therefore, secondly, we will describe how DNA-free CRISPR/Cas plant genome editing technology works. DNA-free CRISPR/Cas plant genome editing technology, which is especially useful in crop molecular breeding, can be achieved through the following approaches: Transiently expressed CRISPR/Cas DNA can be delivered into immature embryos using particle bombardment, or into explants using <italic>Agrobacterium tumefacien</italic> mediated method, or into protoplasts through polyethylene glycol mediation; <italic>in vitro</italic> transcripts of sgRNA and Cas nuclease can be imported into immature embryos using particle bombardment. BothsgRNA and Cas nuclease can be cloned into RNA virus vector before they are used to transform explants. Ribonucleoproteins<italic> </italic>complexes formed by <italic>in vitro </italic>transcribed<italic> </italic>sgRNA and Cas nuclease can be delivered into protoplasts, immature embryos through polyethylene glycol, lipofection or nanoparticle mediated methods, respectively. DNA-free edited plants can be identified from plants regenerated on medium without selective reagent. Thirdly, we will cover the recent successful applications of CRISPR/Cas as a plant genome-editing tool in crop improvements including crop yield and quality improvement, biotic and abiotic stresses resistance, de novo domestication, and crop directed improvement. These examples demonstrate that CRISPR/Cas plant genome editing technology is effective and valuable to crop molecular breeding. Lastly, we will discuss the future development of CRISPR/Cas technology in plant genome editing, the role of appropriate national management policies and favorable social environment in promoting this field. The editing efficiency of these tools can be further improved and the off-target rate reduced. Further enhancement of the CRISPR/Cas technology will depend on improved functional elements (e.g., CasX, SuperFi-Cas9, engineered APOBEC3A and TadA, etc.) and good vector design. Successful applications of CRISPR/Cas plant genome editing technologies in crop improvement will depend on appropriate regulatory policies and public acceptance of genome-edited crops. This paper aims to provide important insights into how CRISPR/Cas as a powerful plant genome-editing technology can be used in improving crop varieties, accelerating seed industry development, and fulfilling our national strategy of food storage in technology.

BackgroundReporter methods to quantitatively measure the efficiency and specificity of genome editing tools are important for the development of novel editing techniques and successful applications of available ones. However, the existing methods have major limitations in sensitivity, accuracy, and/or readiness for in vivo applications. Here, we aim to develop a straight-forward method by using nucleotide insertion/deletion resulted from genome editing. In this system, a target sequence with frame-shifting length is inserted after the start codon of a cerulean fluorescence protein (CFP) to inactivate its fluorescence. As such, only a new insertion/deletion event in the target sequence will reactivate the fluorescence. This reporter is therefore termed as “Insertion/deletion-activated frame-shift fluorescence protein”. To increase its traceability, an internal ribosome entry site and a red fluorescence protein mCherryFP are placed downstream of the reporter. The percentage of CFP-positive cells can be quantified by fluorescence measuring devices such as flow cytometer as the readout for genome editing frequency.ResultsTo test the background noise level, sensitivity, and quantitative capacity of this new reporter, we applied this approach to examine the efficiency of genome editing of CRISPR/Cas9 on two different targeting sequences and in three different cell lines, in the presence or absence of guide-RNAs with or without efficiency-compromising mutations. We found that the insertion/deletion-activated frame-shift fluorescence protein has very low background signal, can detect low-efficiency genome editing events driven by mutated guideRNAs, and can quantitatively distinguish genome editing by normal or mutated guideRNA. To further test whether the positive editing event detected by this reporter indeed correspond to genuine insertion/deletion on the genome, we enriched the CFP-positive cells to examine their fluorescence under confocal microscope and to analyze the DNA sequence of the reporter in the genome by Sanger sequencing. We found that the positive events captured by this reporter indeed correlates with genuine DNA insertion/deletion in the expected genome location.ConclusionThe insertion/deletion-activated frame-shift fluorescence protein reporter has very low background, high sensitivity, and is quantitative in nature. It will be able to facilitate the development of new genome editing tools as well as the application of existing tools.