Construct design for CRISPR/Cas-based genome editing in plants

Trends in Plant Science, Journal Year: 2021, Volume and Issue: 26(11), P. 1133 - 1152

Published: July 31, 2021

Many Cas nucleases (e.g., SpCas9-NRRH, SpG, SpCas9-NG) that can target non-canonical protospacer adjacent motifs (PAMs) have been developed for plant genome editing.Near-PAMless nuclease SpRY has optimized editing to increase the flexibility of gRNA design.A next-generation technology, prime editing, tested in many plants, including Arabidopsis, rice, maize, potato, and tomato.Multiplex clustered regularly interspaced short palindromic repeat (CRISPR) systems based on tRNA/gRNA or Csy4 work better Cas9 a hammerhead hepatitis delta virus (HH-HDV)-based system works Cas12a.A multiplex CRISPR expressing up 24 gRNAs plants.Use multiple introns gene dramatically improves efficacy.Improved pegRNA design significantly efficiency editor. construct is key step practice which includes identification appropriate proteins, selection guide RNAs (gRNAs), regulatory elements express proteins. Here, we review choices CRISPR-based editors suited different needs applications. We consider technical aspects associated computational tools. also discuss strategies constructs high-throughput manipulation complex biological processes polygenic traits. provide recommendations remaining challenges optimization editing. Genome be defined as targeted intervention genetic materials (i.e., DNA RNA) living organisms deliberately alter their sequences. Although both RNA, here only mainly relies introduction vivo double-stranded breaks (DSBs) induced by engineered sequence-specific (SSNs) programmed recognize predefined sites genome. The DSBs are then repaired cellular repair mechanisms, namely non-homologous end-joining (NHEJ) homology-directed (HDR) (Figure 1). NHEJ results mutation at break site, largely via imprecise sequence insertions deletions (indels), disrupting native structure function sequences genes, promoters). In addition, mediate insertion replacement when suitable fragment provided [1.Lu Y. et al.Targeted, efficient rice.Nat. Biotechnol. 2020; 38: 1402-1407Crossref PubMed Scopus (28) Google Scholar]. By contrast, HDR precisely introduce carried donor template SSNs, with capacity DSB DNA, referred technologies include meganucleases [2.Bogdanove A.J. al.Engineering altered protein-DNA recognition specificity.Nucleic Acids Res. 2018; 46: 4845-4871Crossref (14) Scholar], zinc finger (ZFNs) [3.Bibikova M. al.Targeted chromosomal cleavage mutagenesis Drosophila using zinc-finger nucleases.Genetics. 2002; 161: 1169-1175Crossref transcription activator-like effector (TALENs) [4.Christian al.Targeting double-strand TAL 2010; 186: 757-761Crossref (1163) [5.Zetsche B. al.Cpf1 single RNA-guided endonuclease class 2 CRISPR-Cas system.Cell. 2015; 163: 759-771Abstract Full Text PDF Scholar, 6.Mali P. al.RNA-guided human engineering Cas9.Science. 2013; 339: 823-826Crossref (5536) 7.Cong L. al.Multiplex CRISPR/Cas systems.Science. 819-823Crossref (8358) 8.Jinek al.A programmable dual-RNA-guided adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref (7291) Unlike ZFNs TALENs, rely protein–DNA interaction define specificity, use RNA–DNA targeting cleavage, making it simple, efficient, inexpensive technology manipulation. now become leading applied wide variety species. Efficient achieved dicot monocot species diverse fundamental research crop improvement application plants increased over past few years [9.Huang T.-K. Puchta H. Novel applications plants: from chromosome engineering.Transgenic 2021; (Published online March 1, 2021. https://doi.org/10.1007/s11248-021-00238-x)Crossref (2) 10.Zhu al.Applications agriculture biotechnology.Nat. Rev. Mol. Cell Biol. 21: 661-677Crossref (80) 11.Haque E. al.Application CRISPR/Cas9 crops cultivated tropical climates: recent progress, prospects, challenges.Front. Plant Sci. 9: 617Crossref (44) 12.Jaganathan D. al.CRISPR improvement: an update review.Front. 985Crossref (147) Three classes currently available genomes [10.Zhu Scholar,13.Gao C. future agriculture.Cell. 184: 1621-1635Abstract (29) These nucleases, base editors, editors. require inducing DSB, whereas primer do not edit genomes. Over years, there tremendous progress development technologies. rapid discovery toolboxes thus make prospect selecting tool desired daunting, particularly researchers new technology. Besides right tools, delivery reagents cells challenging. some such mammalian cells, purified protein mRNA protein, well (see Glossary), simultaneously delivered zygotic cell. this way, possibility improved controlling dosage proteins gRNAs. This approach shown but still significant overcome. Thus, most frequently, into harboring least one along components required expression promoter, terminator) through Agrobacterium-mediated transformation particle bombardment. Hence, critical conduct experiment. Different influence outcome often achieve [14.Johnson R.A. al.Comparative assessments nucleases' planta.Plant 87: 143-156Crossref (55) 15.Mikami al.Parameters affecting frequency mediated rice.Plant Rep. 34: 1807-1815Crossref (62) 16.Mikami al.Comparison 88: 561-572Crossref (141) 17.Ng Dean N. Dramatic Candida albicans RNA expression.mSphere. 2017; 2e00385-16Crossref (37) 18.Long al.Optimization cotton sgRNA expression.Plant Methods. 14: 85Crossref (24) 19.Yamamoto A. al.Developing heritable mutations Arabidopsis thaliana modified toolkit comprising PAM-altered variants gRNAs.Plant Physiol. 2019; 60: 2255-2262Crossref (10) Specifically, following three factors need considered constructs: (i) (ii) gRNAs, (iii) (GREs) used aim users optimizing various restrict our discussion refer readers excellent reviews other transcriptional regulation [20.Pan al.CRISPR/dCas-mediated epigenetic plants.Curr. Opin. 101980Crossref Scholar,21.Moradpour Abdulah S.N.A. CRISPR/dCas9 platforms beyond editing.Plant J. 18: 32-44Crossref Scholar] [22.Miglani G.S. al.Plant control genome- epigenome-editing technologies.J. Crop Improv. 1-63Crossref (1) section, developments progressively applicability effectiveness plants. will help identify select widely studies, isolated Streptococcus pyogenes (SpCas9). It complexes (sgRNA) requires stretch nucleotides known motif (PAM) downstream its 1A). PAM SpCas9 5′-NGG-3′ (N = A, T, C, G). Once recognizes sequence, Cas9-sgRNA binds generates site 1D). activity combined effort two parts called domain domains (RuvC HNH). senses complementary cleave [23.Jiang F. Doudna J.A. CRISPR-Cas9 structures mechanisms.Annu. Biophys. 505-529Crossref (503) Despite widespread proven efficacy purpose across range organisms, does certain limitations. Firstly, share high identity resulting off-target Secondly, stringent NGG requirement limits manipulated SpCas9. Thirdly, cell viral-based vector difficult due relatively large size exceeds cargo virus-based vector. To overcome these limitations, several natural alternative PAMs (Table Among them, Staphylococcus aureus (SaCa9) variant notable [24.Ran F.A. al.In Cas9.Nature. 520: 186-191Crossref (1431) 5′-NNGRRT coding ~1.0 kb shorter than SpCas9, being vectors [25.Kaya al.Highly specific Cas9.Sci. 2016; 6: 26871Crossref Scholar,26.Steinert orthologues thermophilus aureus.Plant 84: 1295-1305Crossref Cas9-NG xCas9 [27.Ge Z. al.Engineered SpCas9-NG broaden generate plants.Plant 17: 1865-1867Crossref (27) 28.Hua K. al.Genome rice NG sequences.Mol. Plant. 12: 1003-1014Abstract (50) 29.Li expanded compatibility.J. Genet. Genomics. 277-280Crossref (12) 30.Negishi al.An adenine editor scope SpCas9-NGv1 1476-1478Crossref (26) 31.Ren al.Cas9-NG greatly expands genome-editing recognizing atypical rice.Mol. 1015-1026Abstract 32.Wang al.Optimizing 1697-1699Crossref (21) 33.Wang al.xCas9 reduced 709-711Crossref 34.Zhong al.Improving high-fidelity PAM-targeting Cas9-NG.Mol. 1027-1036Abstract (67) 35.Endo PAM.Nat. Plants. 5: 14-17Crossref (72) iSpyMacCas9 [36.Sretenovic S. al.Expanding A-rich sequences.Plant Commun. 2: 100101Abstract (6) A remarkable SpRY, capable almost all (NRN>NYN) [37.Walton R.T. al.Unconstrained near-PAMless variants.Science. 368: 290-296Crossref (189) [38.Ren Q. al.PAM-less CRISPR-SpRY toolbox.Nat. 7: 25-33Crossref (0) Scholar,39.Xu al.SpRY highly flexible recognition.Genome 22: 6Crossref (19) low Off-target issues paired nickase [40.Schiml al.The planta nickases directed progeny.Plant 2014; 80: 1139-1150Crossref (211) Recently, number SpCas9-NRRH) [41.Li broad compatibility plants.Mol. 352-360Abstract Scholar].Table 1CRISPR-Cas editingCas nucleasePAMMutationKey featuresRefsSpCas9NGGWTHighly efficient[10.Zhu Scholar,143.Zhang emerging uncultivated potential science.Nat. 778-794Crossref (113) Scholar]SpCas9-VQRNGAD1135V/R1335Q/T1337RAlternate PAM[19.Yamamoto Scholar,108.Hu X. al.Increasing CRISPR-Cas9-VQR precise 16: 292-297Crossref (42) Scholar,144.Hu 943-945Abstract (64) Scholar]SpCas9-EQRNGAGD1135E/R1335Q/T1337RAlternate Scholar]SpCas9-VRERNGCGD1135V/G1218R/R1335E/T1337RAlternate PAM[144.Hu Scholar]SpCas9-NGNGR1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337RHighly relaxed PAM[28.Hua Scholar,34.Zhong Scholar,35.Endo Scholar,43.Qin R. al.SpCas9-NG self-targets editing.Nat. 197-201Crossref (15) Scholar]iSpymacCas9NAAR221K/N394KGood site[36.Sretenovic Scholar]SpCas9-HF1NGGN497A/R661A/Q695A/Q926ALow off-target[145.Zhang al.Potential high-frequency prevention.Plant 96: 445-456Crossref (76) 146.Zhang al.Perfectly matched 20-nucleotide enable robust nucleases.Genome 191Crossref (68) 147.Xu W. nucleotide rice.BMC 19: 511Crossref (8) Scholar]eSpCas9NGGK810A/K1003A/R1060ALow Scholar]HypaCas9NGGN692A/M694A/Q695A/H698ALow off-target[147.Xu Scholar,148.Liang al.Genotyping genome-edited ribonucleoprotein complexes.Plant 2053-2062Crossref (31) Scholar]eHF1-Cas9NGGN497A/R661A/Q695A/K848A/Q926A/K1003A/R1060ALow off-target[148.Liang Scholar]eHypa-Cas9NGGN692A/M694A/Q695A/H698A/K848A/K1003A/R1060ALow Scholar]HiFi Cas9NGGR691ALow off-target[149.Banakar CRISPR-Cas9/Cas12a phytoene desaturase (OsPDS) gene.Rice Y). 13: 4Crossref (11) Scholar]xCas9NG, GAA GATA262T/R324L/S409I/E480K/E543D/M694I/E1219VLow Flexible PAM[27.Ge Scholar,29.Li Scholar,33.Wang Scholar,150.Zeng tools expand 1348-1350Crossref (17) Scholar]SaCas9NNGRRTNatural variantLow High efficiency[26.Steinert Scholar,110.Wolter al.Efficient egg cell-specific 94: 735-746Crossref (56) Scholar]SaCas9-KKHNNNRRTE782K/N968K/R1015HFlexible PAM[151.Qin wildly CRISPR-SaCas9 toolset 706-708Crossref Scholar]St1Cas9NNAGAAWNatural variantAlternate PAM[26.Steinert Scholar]ScCas9NNGNatural variantFlexible PAM[152.Wang CRISPR/ScCas9 system.Plant 1645-1647Crossref (18) Scholar]XNG-Cas9R1335V/A262T/R324L/S409I/E480K/E543D/M694I/L1111R/D1135V/G1218R/E1219V/E1219F/A1322R/T1337RHighly PAM[153.Niu CRISPR/Cas9-mediated hybrid.J. Integr. 62: 398-402Crossref Scholar]SpRYNGD, NAND1135L/S1136W/G1218K/E1219Q/R1335Q/T1337RHighly PAM[38.Ren Scholar,41.Li Scholar]SpGNGD1135L/S1136W/G1218K/E1219Q/R1335Q/T1337RHighly PAMSpCas9-NRRHNRRHI322V/S409I/E427G/R654L/R753G/R1114G/D1135N/V1139A/D1180G/E1219V/Q1221H/A1320V/R1333KFlexible PAM[41.Li Scholar]SpCas9-NRCHNRCHI322V/S409I/E427G/R654L/R753G/R1114G/D1135N/E1219V/D1332N/R1335Q/T1337N/S1338T/H1349RFlexible Scholar]SpCas9-NRTHNRTHI322V/S409I/E427G/R654L/R753G/R1114G/D1135N/D1180G/G1218S/E1219V/Q1221H/P1249S/E1253K/P1321S/D1322G/R1335LFlexible Scholar]AsCas12aTTTVNatural variantT-rich PAM[154.Malzahn A.A. CRISPR-Cas12a temperature sensitivity Arabidopsis.BMC 9Crossref (53) Scholar,155.Bernabé-Orts J.M. al.Assessment Cas12a-mediated 1971-1984Crossref Scholar]LbCas12aTTTVNatural Scholar,156.Schindele Engineering CRISPR/LbCas12a temperature-tolerant 1118-1120Crossref Scholar]LbCas12a-RRTYCV, CCCCG532R/K595RAlternate PAM[157.Li CRISPR/Cpf1-mediated 11: 995-998Abstract Scholar,158.Zhong fncpf1 lbcpf1 redefined sites.Mol. 999-1002Abstract Scholar]LbCas12a-RVRTATVG532R/K538V/Y542RAlternate Scholar]FnCas12a-RVRTATGN607R/K613V/N617RAlternate PAM[158.Zhong Scholar]enLbCas12aTTTVD156R/G532R/K538RTemperature tolerant[156.Schindele Scholar]ttLbCas12aTTTVD156RTemperature Scholar,159.Huang Nicotiana tabacum CRISPR/SaCas9 tolerant LbCas12a.Plant January 28, 899 https://doi.org/10.1111/pbi.13546)Crossref Scholar]AacCas12bVTTVNatural variantTemperature tolerant[160.Ming al.CRISPR-Cas12b enables engineering.Nat. 202-208Crossref (30) Scholar,161.Wang heat-inducible CRISPR/Cas12b (C2c1) tetraploid (G. hirsutum) 2436-2443Crossref Scholar]AaCas12bVTTVNatural variantHigh efficiency[160.Ming Scholar]BthCas12bATTNNatural PAM[160.Ming Scholar]BhCas12b v4ATTNNatural PAM[162.Wu CRISPR-Cas12b/C2c1.J. 1653-1658Crossref (4) Scholar]BvCas12bATTNNatural thal

Language: Английский

---

CRISPR/Cas9-Mediated Generation of Pathogen-Resistant Tomato against Tomato Yellow Leaf Curl Virus and Powdery Mildew

International Journal of Molecular Sciences, Journal Year: 2021, Volume and Issue: 22(4), P. 1878 - 1878

Published: Feb. 13, 2021

Tomato is one of the major vegetable crops consumed worldwide. yellow leaf curl virus (TYLCV) and fungal Oidium sp. are devastating pathogens causing disease powdery mildew. Such viral reduce tomato crop yields cause substantial economic losses every year. Several commercial varieties include Ty-5 (SlPelo) Mildew resistance locus o 1 (SlMlo1) that carries susceptibility (S-gene) factors for TYLCV mildew, respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) a valuable genome editing tool to develop disease-resistant varieties. In this regard, targeting encoded by host plant instead promising approach achieve pathogen without need stable inheritance CRISPR components. study, CRISPR/Cas9 system was employed target SlPelo SlMlo1 trait introgression in elite cultivar BN-86 confer host-mediated immunity against pathogens. SlPelo-knockout lines were successfully generated, carrying biallelic indel mutations. assays mutant confirmed suppressed accumulation restricted spread non-inoculated parts. Generated knockout showed complete mildew fungus. Overall, our results demonstrate efficiency introduce targeted mutagenesis rapid development pathogen-resistant tomato.

Language: Английский

---

Brassinosteroid gene regulatory networks at cellular resolution in the Arabidopsis root

Science, Journal Year: 2023, Volume and Issue: 379(6639)

Published: March 31, 2023

Brassinosteroids are plant steroid hormones that regulate diverse processes, such as cell division and elongation, through gene regulatory networks vary in space time. By using time series single-cell RNA sequencing to profile brassinosteroid-responsive expression specific different types developmental stages of the Arabidopsis root, we identified elongating cortex a site where brassinosteroids trigger shift from proliferation elongation associated with increased wall–related genes. Our analysis revealed HOMEOBOX FROM ARABIDOPSIS THALIANA 7 ( HAT7 ) GT-2-LIKE 1 GTL1 transcription factors elongation. These results establish brassinosteroid-mediated growth unveil brassinosteroid signaling network regulating transition which illuminates aspects spatiotemporal hormone responses.

Language: Английский

---

Oligomerization-mediated autoinhibition and cofactor binding of a plant NLR

Nature, Journal Year: 2024, Volume and Issue: 632(8026), P. 869 - 876

Published: June 12, 2024

Abstract Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors 1,2 . Maintaining balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity 3,4 Unlike most NLRs, the required for cell death 2 (NRC2) belongs small group characterized constitutively high without self-activation 5 The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) ( Sl NRC2) forms dimers tetramers higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of these oligomers. Dimerization oligomerization only stabilize state but also sequester from assembling into active form. Mutations dimeric or interdimeric interfaces enhance pathogen-induced Nicotiana benthamiana cryo-electron structures unexpectedly inositol hexakisphosphate (IP 6 ) pentakisphosphate bound inner surface C-terminal domain NRC2, confirmed mass spectrometry. phosphate-binding site impair phosphate binding NRC2-mediated N. Our study indicates negative regulatory mechanism suggests phosphates cofactors NRCs.

Language: Английский

---

Jasmonates and Plant Salt Stress: Molecular Players, Physiological Effects, and Improving Tolerance by Using Genome-Associated Tools

International Journal of Molecular Sciences, Journal Year: 2021, Volume and Issue: 22(6), P. 3082 - 3082

Published: March 17, 2021

Soil salinity is one of the most limiting stresses for crop productivity and quality worldwide. In this sense, jasmonates (JAs) have emerged as phytohormones that play essential roles in mediating plant response to abiotic stresses, including salt stress. Here, we reviewed mechanisms underlying activation JA-biosynthesis JA-signaling pathways under saline conditions Arabidopsis several crops. molecular components such MYC2 transcription factor JASMONATE ZIM-DOMAIN (JAZ) repressors are key players JA-associated response. Moreover, review antagonist synergistic effects between JA other hormones abscisic acid (ABA). From an applied point view, reports shown exogenous applications increase antioxidant plants alleviate Finally, discuss latest advances genomic techniques improvement tolerance stress with a focus on jasmonates.

Language: Английский

---

Challenges Facing CRISPR/Cas9-Based Genome Editing in Plants

Frontiers in Plant Science, Journal Year: 2022, Volume and Issue: 13

Published: May 18, 2022

The development of plant varieties with desired traits is imperative to ensure future food security. revolution genome editing technologies based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has ushered in a new era breeding. Cas9 and single-guide RNA (sgRNA) form an effective targeting complex locus or loci interest, enabling all plants high accuracy efficiency. Therefore, CRISPR/Cas9 can save both time labor relative what typically associated traditional breeding methods. However, despite improvements gene editing, several challenges remain that limit application CRISPR/Cas9-based plants. Here, we focus four issues relevant editing: (1) organelle editing; (2) transgene-free (3) virus-induced (4) recalcitrant elite crop inbred lines. This review provides up-to-date summary state CRISPR/Cas9-mediated will push this technique forward.

Language: Английский

---

Multiplex Genome-Editing Technologies for Revolutionizing Plant Biology and Crop Improvement

Frontiers in Plant Science, Journal Year: 2021, Volume and Issue: 12

Published: Oct. 6, 2021

Multiplex genome-editing (MGE) technologies are recently developed versatile bioengineering tools for modifying two or more specific DNA loci in a genome with high precision. These have greatly increased the feasibility of introducing desired changes at multiple nucleotide levels into target genome. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) [CRISPR/Cas] system-based MGE allow simultaneous generation direct mutations precisely gene genes. is enhancing field plant molecular biology and providing capabilities revolutionizing modern crop-breeding methods as it was virtually impossible to edit genomes so single base-pair level prior tools, such zinc-finger nucleases (ZFNs) transcription activator-like effector (TALENs). Recently, researchers not only started using advance applications certain science fields but also attempted decipher answer basic questions related biology. this review, we discuss current progress that has been made toward development utilization an emphasis on improvements after discovery CRISPR/Cas9. Furthermore, most recent advancements involving CRISPR/Cas editing genes described. Finally, insights strengths importance technology advancing crop-improvement programs presented.

Language: Английский

---

Dynamic evolution of small signalling peptide compensation in plant stem cell control

Nature Plants, Journal Year: 2022, Volume and Issue: 8(4), P. 346 - 355

Published: March 28, 2022

Language: Английский

---

Pervasive downstream RNA hairpins dynamically dictate start-codon selection

Nature, Journal Year: 2023, Volume and Issue: 621(7978), P. 423 - 430

Published: Sept. 6, 2023

Translational reprogramming allows organisms to adapt changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles regulating translation by providing alternative sites1-4. However, what determines this selective initiation of between conditions remains unclear. Here, integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity Arabidopsis, we found that transcripts with immune-induced enriched upstream open reading frames (uORFs). Without infection, these uORFs selectively translated owing hairpins immediately downstream uAUGs, presumably slowing engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for recognizable double-stranded RNA structures uAUGs (which term uAUG-ds) being responsible prediction rational design translating uAUG-ds. We uAUG-ds-mediated regulation can be generalized human cells. Moreover, start-codon selection is dynamically regulated. After immune challenge plants, induced helicases homologous Ded1p yeast DDX3X humans resolve structures, allowing ribosomes bypass translate defence proteins. This study shows mRNA regulate selection. The prevalence feature conservation across kingdoms suggest remodelling a general reprogramming.

Language: Английский

---

Diversification of JAZ‐MYC signaling function in immune metabolism

New Phytologist, Journal Year: 2023, Volume and Issue: 239(6), P. 2277 - 2291

Published: July 4, 2023

Jasmonate (JA) re-programs metabolism to confer resistance diverse environmental threats. stimulates the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins that repress activity MYC transcription factors. In Arabidopsis thaliana, and JAZ are encoded by 4 13 genes, respectively. The extent which expansion families has contributed functional diversification JA responses is not well understood. Here, we investigated role paralogs in controlling production defense compounds derived from aromatic amino acids (AAAs). Analysis loss-of-function dominant myc mutations identified MYC3 MYC4 as major regulators JA-induced tryptophan metabolism. We developed a family-based, forward genetics approach screen randomized jaz polymutants for allelic combinations enhance biosynthetic capacity. found mutants defective all members (JAZ1/2/5/6) group I over-accumulate AAA-derived compounds, constitutively express marker genes JA-ethylene branch immunity more resistant necrotrophic pathogens but insect herbivores. defining regulate amino-acid-derived our results provide insight into specificity signaling immunity.

Language: Английский

---