0000005661 00000 n RNP complexes were assembled by combining the CRISPRCas nuclease (Alt-R S.p. Renaud, J. This kit has been determined to be optimal for transfecting following cell types: 32D 3T3-L1 pre-ad A20 A375 A7r5 AGS ARPE-19 B16-F10 BA/F3 (DSMZ) C2C12 C6 Capan-1 CHO [suspension] CHO-S [suspension] (Life Technologies) COS-1 HaCaT HCT116 HEK293 HepG2 HL-60 HUV-EC-C Jurkat K-562 (DSMZ) MCF7 MDA-MB-231 MDA-MB-468 NB-4 (DSMZ) NCTC clone 929 Neuro-2a [N2a] NG108-15 P19 PC-12 PC-3 Raji Ramos RAW 264.7 RBL-1 S49 Saos-2 Schneiders Drosophila Line 2 SH-SY5Y SK-N-SH SK-OV-3 SW480 T/C-28 a2 T/G HA-VSMC T-47D THP-1 U-2 OS Vero. 83, 409439. In our study, we identified no bias favoring which mismatched base was used as the blocking mutation to prevent Cas9 re-cleavage, which imparts flexibility in designing appropriate silent blocking mutations so as to preserve the coding sequence. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Bothmer, A. et al. 0000083586 00000 n Google Scholar. Genomic DNA was extracted after 4872h incubation using 50 L Quick Extract DNA Extraction Solution (Lucigen, Middleton, WI, USA) following the manufacturers protocol. 0000009329 00000 n The authors thank Ashley Jacobi and Kim Lennox for editing the manuscript. Carroll, D. Genome engineering with targetable nucleases. When the EcoRI insertion was within the guide seed region or PAM, incorporating additional PAM mutations negatively impacted the HDR efficiency. Donor ssODNs containing 40-nt homology arms were designed to insert a six base EcoRI restriction digest recognition site (GAATTC) at the Cas9 cleavage site which canonically lies three bases in the 5 direction of the PAM, as illustrated in Fig. Rep. 9, 4811. https://doi.org/10.1038/s41598-019-41121-4 (2019). 24, 132141. Zetsche, B. et al. In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting. PCR primers are listed in Supplementary Table 1. In addition, at these sites WT Cas9 demonstrated a strong preference for the NT strand donor template (bottom strand for WT with left gRNA, and top strand for WT with right gRNA), especially at positions distant from the cut site, while Cas9 D10A did not show a strand preference. Data are represented as meanSEM of three biological replicates for D10A and two biological replicates for WT. The cleavage sites and associated distance to the desired insertion location (green) for each gRNA are indicated above the sequence shown. 0000002114 00000 n PubMed https://doi.org/10.1093/bioinformatics/btr507 (2011). Chemically modified guide RNAs enhance CRISPRCas genome editing in human primary cells. 4D. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Kim, D. et al. For HDR analysis using restriction enzyme digestion, 10 L of the PCR product was incubated with 2 U of EcoRI-HF in 1 CutSmart Buffer (New England BioLabs, Ipswich, MA, USA) at 37C for 60min. Using Cas12a, we observed a distinct reduction in total editing rates when the T strand was used universally. 5B). Bioinformatics 27, 29572963. These ssODN donor templates were delivered to Jurkat and HeLa cells along with their respective Cas9 RNP complexes by nucleofection, and the frequency of perfect HDR was determined by NGS with the mean HDR rate for each ssODN across the 12 genomic loci shown in Fig. Mol. Cas12a generates a DSB with 5 overhangs, requires a TTTV PAM, and enables editing in AT-rich genomes4. Nat. https://doi.org/10.1038/nmeth.2857 (2014). Ma, X. et al. We performed the same experiment in K562 cells, which demonstrated robust HDR overall with WT Cas9. Both the T and NT strand ssODN donor templates were delivered with their respective RNP complexes to Jurkat and HAP1 cells by nucleofection, and NGS was used to measure the frequency of total editing and perfect HDR. The addition of blocking mutations has demonstrated improvement in HDR depending on the selected guide RNA and its relative positioning to the desired HDR mutation. https://doi.org/10.1016/j.jbiotec.2015.04.024 (2015). Jurkat cells were transfected with eGFP-mRNA using a 4D-Nucleofector according to the manufacturer's recommendations with the SE Cell line 4D-Nucleofector kit (V4XC-1032) (Lonza, Breda, the Netherlands). 1B show that although efficient total editing is required for HDR to occur, high editing does not always lead to high HDR-mediated insertion at each site tested. Jurkat cells (ATCC TIB-152) were transfected with program 96-CL-120 and 1 g of pmaxGFP Vector. If the available NGG PAM sites are greater than 15 bases from the desired mutation, then the data presented in Fig. To investigate the balance between guide cleavage efficiency and distance of the desired HDR mutation to the cut site, we selected 13 guides flanking the stop codon of GAPDH to determine which guide led to the highest HDR insertion frequency of an EcoRI site just upstream of the TAA stop codon. RNP complexes (Alt-R A.s. Cas12a nuclease complexed with Alt-R CRISPRCas12a crRNA) were delivered at 5M along with 3M Alt-R Cpf1 Electroporation Enhancer and 3M donor template by nucleofection. In addition to the desired HDR mutation, a single blocking mutation in the seed region of the guide or PAM to prevent Cas9 re-cleavage was included in donor templates. CD95 Signaling Inhibits B Cell Receptor-Mediated Gammaherpesvirus Data are represented as meanSEM. Cas9 nuclease with similar editing outcomes. Thus, we confirmed by NGS that the optimal position for Cas12a-mediated perfect HDR is between positions 1216 of the guide, and moving an insertion outside of the protospacer can give the desired insertion, but is complicated by undesired editing. 2A). 0000003500 00000 n The set of 2432 ssODNs for the four targets were delivered along with their respective Cas9 RNP complex to Jurkat cells by nucleofection (N=120 ssODNs tested across 4 sites). 4B). Rep. 7, 2095. https://doi.org/10.1038/s41598-017-02013-7 (2017). Gene correction of HBB mutations in CD34(+) hematopoietic stem cells using Cas9 mRNA and ssODN donors. https://doi.org/10.1126/science.1231143 (2013). 4B (for each ssODN design n=9). Deltcheva, E. et al. Cong, L. et al. When the EcoRI cleavage site was inserted within the guide sequence, there was no benefit to including blocking mutations to prevent further re-cleavage, likely because the EcoRI site disrupts subsequent cleavage events (Fig. Blocking mutations improve HDR rates. 14) in HEK293 cells, which may be due to differences in the available repair machinery and capacity for HDR in each cell type (Supplemental Fig. (C) Schematic representation of donor templates used to generate PAM-proximal and PAM-distal insertions 20 bases from a Cas9 cut site with no further mutations (None), PAM mutations (PAM), or mutations in the repair track with or without additional PAM mutation. Blocking mutations placed around the Cas9 cleavage site and near the 3 end of the guide were also highly effective, with the impact reduced as the position of the blocking mutation moved PAM-distal. 24 hours post Nucleofection cells were analyzed on a FACSCalibur [Becton Dickinson]. However, the design of these donor templates remains challenging for researchers due to uncertainty about which CRISPRCas system should be applied, selection of gRNA(s) for each new HDR mutation location, and the most beneficial template strand to be used to achieve the highest frequency of HDR. Electroporation was performed using the Lonza Nucleofector 96-well Shuttle System (Lonza, Basel, Switzerland). OBrien, A. R., Wilson, L. O. W., Burgio, G. & Bauer, D. C. Unlocking HDR-mediated nucleotide editing by identifying high-efficiency target sites using machine learning. However, for PAM-distal insertions, the repair track mutations significantly improved the frequency of HDR 3.4-fold over having only a PAM mutation (p<0.01, paired t-test). Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein (RNP) complexes. The table below shows data for the cell type and Nucleofector Platform selected. Aims We herein report a convenient and affordable method based on in . Mol. Although we observed no universal donor strand preference in the experiment outlined in Fig. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. 4. When Cas9 D10A nickase RNP complexes targeting both strands in the PAM-out orientation nick the genomic DNA, a DSB with 3 overhangs is generated. Nucleofection Buffer | Lonza | Bioz Article After transfection, cells were grown for 4872h in total, after which genomic DNA was isolated using QuickExtract DNA Extraction Solution (Epicentre, Madison, WI, USA). Cas9 Nuclease V3, Alt-R S.p. However, for insertions further from the Cas9 cleavage site there is a preference for the donor strand that contains 3 sequence complementary to the overhangs generated during DSB repair44. Type in Product name, Keyword or Catalog number to see suggestions. Nat. Further studies to investigate the optimal selection of gRNAs which have the highest potential HDR frequency, including related studies to understand cell type-specific differences, are underway. Editing and HDR frequencies were calculated using the following formula: average molar concentration of the cut products/(average molar concentration of the cut products+molar concentration of the uncut product)100. 2B, top middle and right panels), consistent with our earlier findings. Human T Cell Nucleofector Kit | Lonza 14, 80968106 (1994). Cas9 Nuclease, Alt-R CRISPRCas9 crRNA and tracrRNA) for the 13 guides targeting GAPDH were delivered at 2M along with 2M Alt-R Cas9 Electroporation Enhancer and 2M donor template designed to insert an EcoRI site before the stop codon by nucleofection to K562 cells. 5C, top panel by the data points generally clustering below the line through the origin because of the increased total editing when delivered with the NT strand. Within this website, you will now have access 24 hours a day, 7 days a week to commonly requested forms, useful highlighted links, and frequently asked questions regarding your benefit information. Lipofection was performed in 96-well plates. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. In Fig. Kim, S., Kim, D., Cho, S. W., Kim, J. Commun. https://doi.org/10.1016/j.dnarep.2008.06.018 (2008). Schubert, M.S., Thommandru, B., Woodley, J. et al. Cell 163, 759771. 25, 25852598. Nat. As a comparison, neither WT Cas9 nor Cas9 H840A with the same gRNA pair demonstrated HDR insertion frequency as high as Cas9 D10A at all positions tested (Supplementary Figure S2C). I need a successful delivery of plasmids to suspension. By submitting a comment you agree to abide by our Terms and Community Guidelines. Cells were analyzed 24 hours post Nucleofection by fl ow cytometry. 24, 10121019. Paquet, D. et al. https://doi.org/10.1016/j.cell.2015.09.038 (2015). 0000022306 00000 n Systematic evaluation of CRISPRCas systems reveals design principles for genome editing in human cells. The nucleofection mixtures were then transferred into 16-well strip for nucleofection in Lonza 4D Nucleofector using the buffer and pulse code specified. RNP complexes (Alt-R S.p. Grab a coffee and check out our free webinars. Scientific Reports (Sci Rep) HAP1 cells were used for transfection at 5070% confluency. Donor oligos were hydrated using IDTE pH 7.5 (Integrated DNA Technologies). Multiplex genome engineering using CRISPR/Cas systems. https://doi.org/10.1038/nbt.3190 (2015). (D) Schematic representation of four unique HDR mutations that were designed using the Alt-R HDR Design tool either with or without the addition of silent mutations. However, when the EcoRI insertion was outside of the PAM/protospacer region, blocking mutations increased the rate of HDR from 0.7%, to 13.3% with a mutation in the PAM and up to 13.0% HDR with a mutation at position 14 of the guide (Fig. After a CRISPRCas system and gRNA(s) have been selected and the donor template has been designed, homology arm lengths remains a variable for consideration. After a DSB is generated, the ends are resected, generating 3 overhangs which are then available for base pairing with the donor DNA. However, for WT Cas9 we have shown that the preferred strand is strongly dependent upon where the desired HDR mutation is, relative to the Cas9 gRNA. Article Each position in the region indicated was changed to every possible alternate base in a unique donor template that also contained the desired HDR mutation. Bioz Stars score: 86/100, based on 1 PubMed citations. However, a significant difference in editing efficiency (p<0.0001, paired t-test) was observed in HAP1 cells where the mean editing was 80.2% when the NT strand was used and 67.8% when the T strand was used (Supplemental Fig. Reads were aligned to the target, favoring alignment choices with indels near the predicted cut site(s). CAS (A) Schematic representation of donor templates used to generate PAM-proximal and PAM-distal insertions 25 bases from a Cas9 cut site with no further mutations (None), PAM mutations (PAM), or mutations in the repair track with or without an additional PAM mutation. A final donor template design consideration that we investigated was strand preference for the donor template. Sci. For efficient transfection of cell lines, e.g. First, 25 L of Opti-MEM (Thermo Fisher Scientific) containing 1.2 L (RNP delivery) or 0.75 L (gRNA delivery) of Lipofectamine RNAiMAX (Thermo Fisher Scientific) was combined with equal volume of Opti-MEM containing RNP or gRNA and HDR donor template (when present), and incubated at room temperature for 20min. Association of Cas9 protein with a gRNA forms a ribonucleoprotein (RNP) complex, which surveys a dsDNA substrate and generates a DSB when its complementary target sequence with a PAM sequence 3 of that target is recognized by an active Cas9 RNP complex8,9,10. Cas9 D10A nickase and A.s. Cas12a nuclease and present optimized design considerations for each enzyme, including positioning of the gRNA(s) relative to the desired mutation, donor strand preference, and the incorporation of blocking mutations to improve desired HDR. To investigate if Cas12a demonstrates a universal strand preference when an EcoRI insertion was optimally placed, a set of 15 Cas12a guide RNAs was selected, and donor templates were designed to insert an EcoRI restriction digest recognition site 16 bases 3 of the PAM. The combined results from the fifteen sites comparing T and NT strand donors in two cell lines is shown in Fig. Lonza), Jurkat cells SE buffer (SE Cell Line 4D-Nucleofector X Kit, Lonza), PHH P3 buffer (P3 Primary Cell 4D-Nucleofector X Kit, Lonza . (B) Cas9 D10A with gRNA pairs (left panel), or Cas9 WT with each of the individual gRNAs (middle and right panel) RNP complexes (Alt-R S.p. (B) An EcoRI recognition site was inserted at a Cas9 cleavage site at 254 genomic loci in Jurkat and 239 genomic loci in HAP1 cells using either the T or NT strand as the donor template. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Science 337, 816821. https://doi.org/10.1126/science.aar6245 (2018). Biophys. We observed this to be beneficial for PAM-distal HDR events as shown in Fig. Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs. Cas9 Nuclease complexed with Alt-R CRISPRCas9 sgRNA) were delivered at 2M along with 2M Alt-R Cas9 Electroporation Enhancer and 0.5M donor template by nucleofection. In this work, we thoroughly investigated design features for both S.p. This case study indicates that guide efficiency is a critical factor for efficient HDR, and guide selection that is as close as possible to the desired HDR mutation is a secondary consideration. Total editing and perfect HDR was assessed via NGS. Chen, J. S. et al. Optimization of scarless human stem cell genome editing. https://doi.org/10.1038/nbt.3609 (2016). Total editing rate was measured using the Alt-R Genome Editing Detection Kit (T7EI) (Integrated DNA Technologies) following the manufacturers protocol. Mao, Z., Bozzella, M., Seluanov, A. Thank you for visiting nature.com. Slider with three articles shown per slide. 33, 985989. Does anyone know the recipe for the different Lonza nucleofection solutions and supplements? 0000002163 00000 n RNP complexes (Alt-R S.p. Cite this article, CRISPRCas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. Perfect HDR (blue), imperfect HDR (red) and total editing, which includes NHEJ events (black) are shown. 5B and follow-up in Supplemental Fig. https://doi.org/10.1126/science.1232033 (2013). Insertions outside of the nick sites were not as efficient as ones placed between the nick sites. The 21-baseregion where the gRNA targets is highlighted in green. b, Genomic coordinates of gRNAs plotted against enrichment into the 'CD69 high' sorted population. We aimed to further investigate this to define a ruleset for the placement and number of blocking mutation(s) required to maximize HDR efficiency. Paired end, 150bp reads were sequenced using V2 chemistry. Plant Cell Physiol. 33, 538542. Riesenberg, S. & Maricic, T. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells. In previous work investigating HDR improvements in the cell lines mentioned above, asymmetric homology arms did not improve HDR beyond symmetrical homology arms when arm length was30-nt from both the mutation location and the Cas9 cleavage site (data not shown). The observation that Cas9 D10A has no identifiable strand preference for the HDR donor template also held true at this site. https://doi.org/10.1016/j.molcel.2016.06.037 (2016). ISSN 2045-2322 (online). Cas9 and A.s. Cas12a nucleases relating to gRNA selection, donor strand preference, the placement and composition of blocking mutations, and the number of blocking mutations that are required for maximum HDR efficiency. Those data are either based on Lonza Optimized Protocols, Rodrigo Vazquez-Lombardi, Johanna S. Jung, Fabrice S. Schlatter, Anna Mei, Natalia Rodrigues Mantuano, Florian Bieberich, Kai-Lin Hong, Jakub Kucharczyk, Edo Kapetanovic, Erik Aznauryan, Cedric R. Weber, Alfred Zippelius, Heinz Laeubli, and Sai T. Reddy, Tacken PJ, Joosten B, Reddy A, Wu D, Eek A, Laverman P, Kretz-Rommel A, Adema GJ, Torensma R, Figdor CG, Primary Cells and Media, Classical Media and Reagents, Serum-free and Speciality Media, Murdoch AD, Grady LM, Ablett MP, Katopodi T, Meadows RS, Hardingham TE, Sekine Y, Tsuji S, Ikeda O, Sugiyma K, Oritani K, Shimoda K, Muromoto R, Ohbayashi N, Yoshimura A, Matsuda T, Mor A, Campi G, Du G, Zheng Y, Foster DA, Dustin ML, Philips MR, Ohnuma K, Uchiyama M, Yamochi T, Nishibashi K, Hosono O, Takahashi N, Kina S, Tanaka H, Lin X, Dang NH, Morimoto C, Huang Y, Yang H, Borg BB, Su X, Rhodes SL, Yang K, Tong X, Tang G, Howell CD, Rosen HR, Thio CL, Thomas DL, Alter HJ, Sapp RK, Liang TJ, Proc Natl Acad Sci USA (2007) 104(3): 985-90, Lopez-Anton N, Rudy A, Barth N, Schmitz ML, Pettit GR, Schulze-Osthoff K, Dirsch VM, Vollmar AM, Tavano R, Contento RL, Baranda SJ, Soligo M, Tuosto L, Manes S, Viola A, Vandenbussche CJ, Dakshanamurthy S, Posch PE, Hurley CK, Thurau M, Everett H, Tapernoux M, Tschopp J, Thome M, Jia W, Hegde VL, Singh NP, Sisco D, Grant S, Nagarkatti M, Nagarkatti PS, Mahmoudi T, Parra M, Vries RG, Kauder SE, Verrijzer CP, Ott M, Verdin E, von Essen M, Nielsen MW, Bonefeld CM, Boding L, Larsen JM, Leitges M, Baier G, Odum N, Geisler C, Krueger A, Fas SC, Giaisi M, Bleumink M, Merling A, Stumpf C, Baumann S, Holtkotte D, Bosch V, Krammer PH and Li-Weber M, Nguyen DH, Hurtado-Ziola N, Gagneux P, Varki A, Proc Natl Acad Sci USA (2006) 103(20): 7765-70, Zimmerman AW, Joosten B, Torensma R, Parnes JR, van Leeuwen FN and Figdor CG, Huang C, Bi E, Hu Y, Deng W, Tian Z, Dong C, Hu Y, Sun B, Lettau M, Qian J, Linkermann A, Latreille M, Larose L, Kabelitz D, Janssen O, Proc Natl Acad Sci USA (2006) 103(15): 5911-6, Booth AM, Fang Y, Fallon JK, Yang JM, Hildreth JE, Gould SJ, Weinberg MS, Villeneuve LM, Ehsani A, Amarzguioui M, Aagaard L, Chen ZX, Riggs AD, Rossi JJ and Morris KV, Lieto LD, Maasho K, West D, Borrego F and Coligan JE, Zipfel PA, Bunnell SC, Witherow DS, Gu JJ, Chislock EM, Ring C, Pendergast AM, Nejmeddine M, Barnard AL, Tanaka Y, Taylor GP and Bangham CR, Kawaida R, Yamada R, Kobayashi K, Tokuhiro S, Suzuki A, Kochi Y, Chang X, Sekine A, Tsunoda T, Sawada T, Furukawa H, Nakamura Y and Yamamoto K, Ingold K, Zumsteg A, Tardivel A, Huard B, Steiner QG, Cachero TG, Qiang F, Gorelik L, Kalled SL, Acha-Orbea H, Rennert PD, Tschopp J, Schneider P, Wirtz S, Becker C, Fantini MC, Nieuwenhuis EE, Tubbe I, Galle PR, Schild HJ, Birkenbach M, Blumberg RS and Neurath MF, Gaikwad A, Poblenz A, Haridas V, Zhang C, Duvic M and Gutterman J, Song EJ, Yim SH, Kim E, Kim NS and Lee KJ, Puissant B, Roubinet F, Dellacasagrande J, Massip P, Abbal M, Pasquier C, Izopet J, Blancher A, Zhang Y, Erdmann F, Baumgrass R, Schutkowski M and Fischer G, Dombroski D, Houghtling RA, Labno CM, Precht P, Takesono A, Caplen NJ, Billadeau DD, Wange RL, Burkhardt JK and Schwartzberg PL, Lacalle RA, Gomez-Mouton C, Barber DF, Jimenez-Baranda S, Mira E, Martinez-A C, Carrera AC, Manes S, Frazer-Abel AA, Baksh S, Fosmire SP, Willis D, Pierce AM, Meylemans H, Linthicum DS, Burakoff SJ, Coons T, Bellgrau D and Modiano JF, J Pharmacol Exp Ther (2004) 311(2): 758-769, Gerlo S, Verdood P, Gellersen B, Hooghe-Peters EL and Kooijman R, High-throughput T cell receptor engineering by functional screening identifies candidates with enhanced potency and specificity, No advantage of cell-penetrating peptides over receptor-specific antibodies in targeting antigen to human dendritic cells for cross-presentation, CEACAM1 dynamics during neisseria gonorrhoeae suppression of CD4+ T lymphocyte activation.
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