PrimeDesign software for rapid and simplified design of prime editing guide RNAs

Nature Communications, Journal Year: 2021, Volume and Issue: 12(1)

Published: Feb. 15, 2021

Prime editing (PE) is a versatile genome technology, but design of the required guide RNAs more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, user-friendly, end-to-end web application and command-line tool PE experiments. PrimeDesign can be used single combination applications, as well genome-wide saturation mutagenesis screens. Using construct PrimeVar, comprehensive searchable database that includes candidate prime RNA (pegRNA) nicking sgRNA (ngRNA) combinations installing correcting >68,500 pathogenic human genetic variants from ClinVar database. Finally, use to pegRNAs/ngRNAs install variety in cells.

Language: Английский

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CRISPR-based genome editing through the lens of DNA repair

Molecular Cell, Journal Year: 2022, Volume and Issue: 82(2), P. 348 - 388

Published: Jan. 1, 2022

Language: Английский

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Deletion and replacement of long genomic sequences using prime editing

Nature Biotechnology, Journal Year: 2021, Volume and Issue: 40(2), P. 227 - 234

Published: Oct. 14, 2021

Language: Английский

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Saturation variant interpretation using CRISPR prime editing

Nature Biotechnology, Journal Year: 2022, Volume and Issue: 40(6), P. 885 - 895

Published: Feb. 21, 2022

Language: Английский

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Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases

Nature Biomedical Engineering, Journal Year: 2021, Volume and Issue: 6(2), P. 181 - 194

Published: Aug. 26, 2021

Language: Английский

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Phage-assisted evolution and protein engineering yield compact, efficient prime editors

Cell, Journal Year: 2023, Volume and Issue: 186(18), P. 3983 - 4002.e26

Published: Aug. 1, 2023

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size improved efficiency. Using phage-assisted evolution, efficiencies compact reverse transcriptases by up 22-fold generated that are 516–810 base pairs smaller than the current-generation editor PEmax. We discovered different specialize types used this insight outperform PEmax PEmaxΔRNaseH, truncated dual-AAV delivery systems. Finally, Cas9 domains improve editing. These resulting (PE6a-g) enhance therapeutically relevant patient-derived fibroblasts primary human T-cells. PE6 variants also enable longer insertions be installed vivo following delivery, achieving 40% loxP insertion cortex murine brain, 24-fold improvement compared previous state-of-the-art editors.

Language: Английский

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Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair

Nature Communications, Journal Year: 2022, Volume and Issue: 13(1)

Published: Feb. 9, 2022

Abstract Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. However, efficiency PE varies widely depends not only on region targeted, but also genetic background edited cell. Here, to determine which cellular factors affect efficiency, we carry out focused screen targeting 32 DNA repair factors, spanning all reported pathways. We show that, depending cell line type edit, ablation mismatch (MMR) affords 2–17 fold increase in across several human lines, types edits loci. The accumulation key MMR MLH1 MSH2 at sites argues for direct involvement control. Our results shed new light mechanism suggest how its might be optimised.

Language: Английский

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Dual-AAV delivering split prime editor system for in vivo genome editing

Molecular Therapy, Journal Year: 2021, Volume and Issue: 30(1), P. 283 - 294

Published: July 21, 2021

Prime editor (PE), a new genome editing tool, can generate all 12 possible base-to-base conversions, insertion, and deletion of short fragment DNA. PE has the potential to correct majority known human genetic disease-related mutations. Adeno-associated viruses (AAVs), safe vector widely used in clinics, are not capable delivering (∼6.3 kb) single because limited loading capacity (∼4.8 kb). To accommodate AAVs, we constructed four split-PE (split-PE994, split-PE1005, split-PE1024, split-PE1032) using Rma intein (Rhodothermus marinus). With use GFP-mutated reporter system, reconstituting activities were screened, two efficient split-PEs (split-PE1005 split-PE1024) identified. We then demonstrated that delivered by dual-AAV1, especially could mediate base transversion insertion at endogenous sites cells. test performance vivo, split-PE1024 was into adult mouse retina dual-AAV8. successful Dnmt1 retina. Our study provides method deliver tissue, paving way for vivo gene-editing therapy PE.

Language: Английский

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Prime editing – an update on the field

Gene Therapy, Journal Year: 2021, Volume and Issue: 28(7-8), P. 396 - 401

Published: May 24, 2021

Language: Английский

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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

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