CRISPR ribonucleoprotein-mediated genetic engineering in plants

Plant Communications, Journal Year: 2021, Volume and Issue: 2(2), P. 100168 - 100168

Published: Feb. 10, 2021

CRISPR-derived biotechnologies have revolutionized the genetic engineering field and been widely applied in basic plant research crop improvement. Commonly used

Language: Английский

---

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

---

Next-Generation Breeding Strategies for Climate-Ready Crops

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

Published: July 21, 2021

Climate change is a threat to global food security due the reduction of crop productivity around globe. Food matter concern for stakeholders and policymakers as population predicted bypass 10 billion in coming years. Crop improvement via modern breeding techniques along with efficient agronomic practices innovations microbiome applications, exploiting natural variations underutilized crops an excellent way forward fulfill future requirements. In this review, we describe next-generation tools that can be used increase production by developing climate-resilient superior genotypes cope challenges security. Recent genomic-assisted (GAB) strategies allow construction highly annotated pan-genomes give snapshot full landscape genetic diversity (GD) recapture lost gene repertoire species. Pan-genomes provide new platforms exploit these unique genes or variation optimizing programs. The advent clustered regularly interspaced short palindromic repeat/CRISPR-associated (CRISPR/Cas) systems, such prime editing, base de nova domestication, has institutionalized idea genome editing revamped improvement. Also, availability versatile Cas orthologs, including Cas9, Cas12, Cas13, Cas14, improved efficiency. Now, CRISPR/Cas systems have numerous applications research successfully edit major develop resistance against abiotic biotic stress. By adopting high-throughput phenotyping approaches big data analytics like artificial intelligence (AI) machine learning (ML), agriculture heading toward automation digitalization. integration speed genomic phenomic rapid identifications ultimately accelerate addition, multidisciplinary open exciting avenues climate-ready

Language: Английский

---

Targeted genome-modification tools and their advanced applications in crop breeding

Nature Reviews Genetics, Journal Year: 2024, Volume and Issue: 25(9), P. 603 - 622

Published: April 24, 2024

Language: Английский

---

CRISPR-Cas9 Gene Therapy: Non-Viral Delivery and Stimuli-Responsive Nanoformulations

Molecules, Journal Year: 2025, Volume and Issue: 30(3), P. 542 - 542

Published: Jan. 24, 2025

The CRISPR-Cas9 technology, one of the groundbreaking genome editing methods for addressing genetic disorders, has emerged as a powerful, precise, and efficient tool. However, its clinical translation remains hindered by challenges in delivery efficiency targeting specificity. This review provides comprehensive analysis structural features, advantages, potential applications various non-viral stimuli-responsive systems, examining recent progress to emphasize address these limitations advance therapeutics. We describe how reports that nonviral vectors, including lipid-based nanoparticles, extracellular vesicles, polymeric gold mesoporous silica can offer diverse advantages enhance stability, cellular uptake, biocompatibility, based on their structures physio-chemical stability. also summarize nanoformulations, type vector, introduce precision control delivery. Stimuli-responsive nanoformulations are designed respond pH, redox states, external triggers, facilitate controlled targeted delivery, minimize off-target effects. insights our suggest future gene therapy technologies highlight systems CRISPR-Cas9’s efficacy, positioning them pivotal tools gene-editing therapies.

Language: Английский

---

CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

Frontiers in Plant Science, Journal Year: 2020, Volume and Issue: 11

Published: Oct. 22, 2020

Global population is predicted to approach 10 billion by 2050, an increase of over 2 from today. To meet the demands growing, geographically and socio-economically diversified nations, we need diversity expand agricultural production. This expansion productivity will occur under increasing biotic, environmental constraints driven climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) similar genome editing technologies likely be key enablers future needs. While application CRISPR-Cas9 mediated has led way, use CRISPR-Cas12a also significantly for engineering plants. The popularity CRISPR-Cas12a, type V (class-II) system, gaining momentum because its versatility simplified features. These include a small guide RNA devoid trans-activating crispr RNA, targeting T-rich regions where Cas9 not suitable use, processing capability facilitating simpler multiplexing, ability generate double strand breaks (DSB) with staggered ends. Many monocot dicot species have been successfully edited using this Cas12a system further research ongoing improve efficiency in plants, including improving temperature stability enzyme, identifying new variants or synthetically producing flexible PAM sequences. In review provide comparative survey Cas9, perspective on applications CRISPR-Cas12 agriculture.

Language: Английский

---

SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition

Genome biology, Journal Year: 2021, Volume and Issue: 22(1)

Published: Jan. 4, 2021

Abstract Background Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts editable range of plant genome. Results In this study, we thoroughly investigate nuclease activity PAM preference two structurally engineered SpCas9 variants, SpG SpRY, in transgenic rice. Our study shows that favors NGD PAMs, albeit less efficiently than previously described SpCas9-NG, SpRY achieves efficient editing across a wide genomic loci, exhibiting well NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ adenosine TadA8e are generated, respectively. These constructs induce C-to-T A-to-G conversions genes toward non-canonical including non-G Remarkably, high-frequency self-editing events (indels DNA fragments deletion) integrated T-DNA result observed, whereas nickase-mediated base editor is quite low rice lines. Conclusions The broad compatibility greatly expands targeting scope engineering.

Language: Английский

---

Increasing the efficiency and precision of prime editing with guide RNA pairs

Nature Chemical Biology, Journal Year: 2021, Volume and Issue: 18(1), P. 29 - 37

Published: Oct. 28, 2021

Language: Английский

---

Targeted DNA insertion in plants

Proceedings of the National Academy of Sciences, Journal Year: 2021, Volume and Issue: 118(22)

Published: April 30, 2021

Conventional methods of DNA sequence insertion into plants, using Agrobacterium -mediated transformation or microprojectile bombardment, result in the integration at random sites genome. These plants may exhibit altered agronomic traits as a consequence disruption silencing genes that serve critical function. Also, interest inserted are often not expressed desired level. For these reasons, targeted suitable genomic is desirable alternative. In this paper we review approaches plant genomes, discuss current technical challenges, and describe promising applications for crop genetic improvement.

Language: Английский

---

Advances in Genome Editing With CRISPR Systems and Transformation Technologies for Plant DNA Manipulation

Frontiers in Plant Science, Journal Year: 2021, Volume and Issue: 11

Published: Jan. 14, 2021

The year 2020 marks a decade since the first gene-edited plants were generated using homing endonucleases and zinc finger nucleases. advent of CRISPR/Cas9 for gene-editing in 2012 was major science breakthrough that revolutionized both basic applied research various organisms including consequently honored with “The Nobel Prize Chemistry, 2020.” CRISPR technology is rapidly evolving field multiple CRISPR-Cas derived reagents collectively offer wide range applications beyond. While most these technological advances are successfully adopted to advance functional genomics development innovative crops, others await optimization. One biggest bottlenecks plant has been delivery reagents, genetic transformation methods only established limited number species. Recently, alternative delivering being explored. This review mainly focuses on recent (1) current Cas effectors variants target range, reduced size increased specificity along tissue specific genome editing tool kit (2) cytosine, adenine, glycosylase base editors can precisely install all possible transition transversion mutations sites (3) prime directly copy desired edit into DNA by search replace method (4) mechanisms bypass culture regeneration procedures de novo meristem induction, viral vectors prospects nanotechnology-based approaches.

Language: Английский

---