Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature-tolerant CRISPR/LbCas12

Huang, T.-K.

Merker, L.

Puchta, H.

Schindele, P.

Wolter, F.

English

Merker, Laura

Schindele, Patrick

Huang, Teng‐Kuei

Wolter, Felix

Puchta, Holger

NEUROSURGERY ENTHUSIASTIC WOMEN SOCIETY

Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature‐tolerant CRISPR/LbCas12a

KITopen

Brief CommunicationEnhancing in planta gene targeting efficiencies inArabidopsis using temperature-tolerant CRISPR/LbCas12aLaura Merker , Patrick Schindele, Teng-Kuei Huang, Felix Wolter and Holger Puchta*Botanical Institute, Karlsruhe Institute of Technology, Karlsruhe, GermanyReceived 15 April 2020;revised 20 May 2020;accepted 22 May 2020.*Correspondence (Tel +49721 608-48894; fax +49 721 608-44874; emailholger.puchta@kit.edu)Keywords: CRISPR, Cas, Cas12, Cpf1, temperature-tolerant,homologous recombination, double-strand break repair.The application of the CRISPR/Cas-system paved the way for theera of genome engineering and quickly became the standardlyapplied tool for targeted non-homologous end joining (NHEJ)based mutagenesis. Despite various efforts, homologous recom-bination (HR) based gene targeting (GT) still requires furtherimprovement regarding efficiency for general applicability inplants (Huang and Puchta, 2019). We developed the in planta GT(ipGT) method aimed to establish a technology for crops withmeagre transformation efficiencies (Fauser et al., 2012). Here, thetargeting vector including the nuclease is integrated into thegenome and excised at the same time as the target site isactivated by double-strand break (DSB) induction (Figure 1a). Wesuccessfully adopted Cas9 for this application (Schiml et al., 2014)and improved GT efficiencies by replacing Cas9 from Strepto-coccus pyogenes (SpCas9) with Cas9 from Staphylococcus aureus(SaCas9), which is more efficient in DSB induction in Arabidopsis(Steinert et al., 2015). Egg-cell specific expression and screeningfor the most efficient transgenic lines were further key points forGT improvement (Miki et al., 2018; Wolter et al., 2018). Mostrecently, we tested the CRISPR/LbCas12a-system for ipGT andcould demonstrate a further increase in GT efficiencies despitelower InDel rates induced by LbCas12a compared with SaCas9 atthe ALS target locus (Wolter and Puchta, 2019). We speculatedthat the higher GT efficiency is caused by Cas12a-mediatedcleavage on the PAM distal site, leaving the seed sequenceunaffected by mutagenesis. Thus, further cleavage of NHEJrepaired junctions might be much more frequent with Cas12athan with Cas9, enhancing the probability for HR to take overDSB repair. This is also in line with very recently published resultson enhancing GT efficiency using LbCas12a in rice and tomato (Liet al., 2019; van Vu et al., 2020). Recently, by introducing thesingle amino acid substitution D156R, we were able to obtain aLbCas12a nuclease variant with improved, temperature-tolerantcutting efficiency (ttLbCas12a) for plant gene editing, outper-forming LbCas12a at 22°C and 28°C in mutation induction(Schindele and Puchta, 2019). Based on those findings, we wereinterested to test whether replacing LbCas12a by ttLbCas12acould further improve GT.Therefore, we performed ipGT experiments in Arabidopsistesting LbCas12a and ttLbCas12a at 22°C as well as at 28°C inparallel for a direct comparison. As described before, theacetolactate synthase gene (ALS) was selected as target andboth nucleases were expressed by an egg-cell specific promoterto assure efficient germline transmission (Wolter et al., 2018). ForGT induction, a ribozyme-flanked crRNA cassette was used with aspacer targeting ALS, a respective GT donor molecule and aKanamycin expression cassette for selection. The donor moleculecontains the modification (S653N) conferring Imazapyr resistanceand one silent point mutation to avoid DSB induction within thedonor. For donor excision, the donor molecule is flanked by therespective spacer sequence (Figure 1a). The GT constructs, eithercontaining LbCas12a or ttLbCas12a, were transformed as T-DNAsinto Arabidopsis thaliana Col-0 plants via the floral dip method.After transformation, the T0 plants were grown either at 22°C orat 28°C until maturity and the harvested seeds subsequentlysown out on Kanamycin selection medium. The resistant T1primary transformants were grown until maturity at either 22°Cor 28°C, respectively. The harvested T2 seeds were sown out onImazapyr medium to verify ipGT events. At least 90 lines weretested per approach (Figure 1b). As expected, it turned out thatraising the temperature from 22°C to 28°C is indeed enhancingGT efficiency. If we simply take the number of lines that producedresistant seedlings into account, ttLbCas12a outperforms thestandard enzyme at 22°C by almost twofold. At 28°C, still onethird more lines with GT events were detected for ttLbCas12a.The effect is even more pronounced if we take the number of GTevents per line into account. For all lines, the individual GTfrequencies were calculated by setting the total number of seedsinto relation with the number of Imazapyr resistant seedlings.Mean GT frequencies were determined over all lines perapproach. At 22°C, ttLbCas12a outperforms the native enzymeby 2.4-fold and at 28°C still by 1.7-fold. This is also obvious whenwe compare the targeting frequencies for the single linesindividually for 28°C and for 22°C (Figure 1c and d). Thedifference in numbers and frequencies are especially impressivefor the lower temperature but still clearly visible for the higher.Additionally, molecular analysis was performed to check thenature of GT events. DSB induced GT depends on the synthesis-dependent strand annealing (SDSA) mechanism of HR (Huangand Puchta, 2019). In principle, besides perfect GT events, usingHR at both junctions at the target locus also with a combinationof HR and NHEJ, can lead to the restoration of the marker gene.Previously, we were able to demonstrate that in a large numberPlease cite this article as: Merker, L., Schindele, P., Huang, T.-K., Wolter, F. and Puchta, H. (2020) Enhancing in planta gene targeting efficiencies in Arabidopsisusing temperature-tolerant CRISPR/LbCas12a. Plant Biotechnol. J., https://doi.org/10.1111/pbi.13426ª 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons LtdThis is an open access article under the terms of the Creative Commons Attribution License, which permits use,distribution and reproduction in any medium, provided the original work is properly cited.1Plant Biotechnology Journal (2020), pp. 1–3 doi: 10.1111/pbi.13426ª 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd, 1–3Laura Merker et al.2of cases the marker gene is first restored by a homologousinteraction with the target locus but then the vector integrateselsewhere in the genome by NHEJ. These ectopic targeting eventscan be discriminated from perfect GT events by PCR analysis ofboth donor-genome junctions. We applied the same kind ofanalysis that we established in previous studies using the ALSgene as target (Wolter et al., 2018; Wolter and Puchta, 2019). Allin all, over one hundred Imazapyr-resistant plants, all representingindependent GT events, were analysed. The respective result isshown in Figure 1e: the GT events induced by ttLbCas12a are inabout half of the cases true GT events indicating that themodified enzyme, ttLbCas12a, is at least as efficient as the nativeone, which showed perfect GT events in about one third of thecases. Furthermore, for ttLbCas12a at 28°C molecular analysisindicated that 3% of the analysed T2 lines already comprised ahomozygous GT event, which did not occur in any otherapproach in this study.Recent results already demonstrated the beneficial effect ofincreased temperature on LbCas12a-mediated GT in plants (vanVu et al., 2020). Here, we showed that the application of therecently developed ttLbCas12a (Schindele and Puchta, 2019) isnot only an alternative way to boost GT for plants that cannotcope with high temperatures. GT can be elevated even further byits use in combination with a high-temperature treatment. Yet,the potential of ttLbCas12a for GT might be even morepromising as our data indicate. We were recently able to showthat ttLbCas12a provides access to target sites that could hardlybe edited at all before by LbCas12a even with a high-temper-ature treatment (Schindele and Puchta, 2019). As we used atarget locus that was accessible for native LbCas12a (Wolter andPuchta, 2019), the use of ttLbCas12a might increase GTefficiencies at those loci that are more difficult to access evenmuch stronger.AcknowledgementsWe thank Anna Imhof for excellent technical assistance. Thiswork was funded by the EU Horizon 2020 Project 760331-2‘NEWCOTIANA’. The ttLbCas12a construct is freely available onrequest.Conflict of InterestThe authors declare no conflict of interest.Author contributionsH.P. conceived the research. P.S., F.W., T-K.H. and L.M. designedand L.M. executed the experiments. H.P., P.S. and L.M. wrote thearticle.ReferencesFauser, F., Roth, N., Pacher, M., Ilg, G., Sanchez-Fernandez, R., Biesgen, C. andPuchta, H. (2012) In planta gene targeting. Proc. Natl. Acad. Sci. USA, 109,7535–7540.Huang, T.-K. and Puchta, H. (2019) CRISPR/Cas-mediated gene targeting inplants: finally a turn for the better for homologous recombination. Plant CellRep., 38, 443–453.Li, S., Zhang, Y., Xia, L. and Qi, Y. (2019) CRISPR-Cas12a enables efficientbiallelic gene targeting in rice. Plant Biotechnol. J. 18, 1351–1353.Miki, D., Zhang, W., Zeng, W., Feng, Z. and Zhu, J.-K. (2018) CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation. Nat.Commun. 9, 1967.Schiml, S., Fauser, F. and Puchta, H. (2014) The CRISPR/Cas system can be usedas nuclease for in planta gene targeting and as paired nickases for directedmutagenesis in Arabidopsis resulting in heritable progeny. Plant J. 80, 1139–1150.Schindele, P. and Puchta, H. (2019) Engineering CRISPR/LbCas12a for highlyefficient, temperature-tolerant plant gene editing. Plant Biotechnol. J. 18,1118–1120.Steinert, J., Schiml, S., Fauser, F. and Puchta, H. (2015) Highly efficient heritableplant genome engineering using Cas9 orthologues from Streptococcusthermophilus and Staphylococcus aureus. Plant J. 84, 1295–1305.van Vu, T., Sivankalyani, V., Kim, E.-J., Doan, D.T.H., Tran, M.T., Kim, J., Sung,Y.W. et al. (2020) Highly efficient homology-directed repair using CRISPR/Cpf1-geminiviral replicon in tomato. Plant Biotechnol. J. https://doi.org/10.1111/pbi.13373 [Epub ahead of print].Wolter, F. and Puchta, H. (2019) In planta gene targeting can be enhanced bythe use of CRISPR/Cas12a. Plant J. 100, 1083–1094.Wolter, F., Klemm, J. and Puchta, H. (2018) Efficient in planta gene targeting inArabidopsis using egg cell-specific expression of the Cas9 nuclease ofStaphylococcus aureus. Plant J. 94, 735–746.Figure 1 GT efficiencies and molecular analysis. (a) Schematic representation of the ipGT assay. The T-DNA construct consists of an egg cell-specificCas12a expression cassette, followed by a guide RNA expression cassette, the HR donor sequence and a Kanamycin resistance. The HR donor harbours theS653N mutation conferring Imazapyr resistance and a silent mutation preventing cleavage. Homologous regions of 637 bp and 905 bp, both including onemismatch each, are present upstream and downstream of the target DSB on the HR donor. After inducing three DSB, the released HR donor can be used asa template for HR-mediated DSB repair. (b) Quantitative outcome of the four GT approaches. The amount of analysed independent T1 lines are shown inthe first column and the number and percentage of positive lines generating heritable GT events as well as the total GT efficiency in the following. The totalGT efficiency describes the average of the GT rates of each single line, including lines without resistant plants. Each GT efficiency was calculated by dividingthe number of resistant plants by the total number of T2 seeds. (c) Distributions of GT efficiencies of single lines for ttLbCas12a and LbCas12a at 28°C. Thepositive single lines of the two approaches are arranged from highest to lowest efficiencies, whereby each bar demonstrates the GT efficiency of a singleline. GT events were detected in 68 out of 97 lines for ttLbCas12a and for LbCas12a in 52 out of 90 analysed lines. (d) Distributions of GT efficiencies ofsingle lines for ttLbCas12a and LbCas12a at 22°C. The representation is equivalent to d. GT events were detected in 40 out of 90 analysed lines forttLbCas12a and for LbCas12a in 26 out of 99 analysed lines. (e) Molecular analysis of GT events. The numbers of analysed Imazapyr resistant plants areshown in the first column, followed by the number and percentage of plants, in which a perfect GT event was detected.ª 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd, 1–3Enhancing ipGT using temperature-tolerant LbCas12a 3

Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature-tolerant CRISPR/LbCas12a

https://publikationen.bibliothek.kit.edu/1000123519/88290979

Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature-tolerant CRISPR/LbCas12a

Abstract

Similar works

Full text

Available Versions

NEUROSURGERY ENTHUSIASTIC WOMEN SOCIETY

KITopen