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楼主从西大MESC毕业滚回国了，一直想分享一下西大的情况
西安大略大学坐落在小伦敦，离多伦多，大瀑布都是两个小时的车程。每次别人问在哪里上学都满满的尴尬，伦敦，西安。。。伦敦不大也不小，公交算方便了，学生的话坐公交都是免费的，有三个中国超市，两个Costco，两个大shopping mall，还有好多好吃的餐厅，凉皮肉夹馍都能吃到。学校记得是最古老的学校，整体校园建筑都蛮有特色的，除了有个图书馆，不想吐槽了。学校一直号称是加拿大学生最满意的学校，至少对MENG确实很好（后面会提到），白人比例比较高，虽然中国人绝对数量哪里都多，但是感觉学校里颜值很高的白人帅哥美女还是不少的，学校最厉害的是商科，在main campus外面有独立的新大楼，逼格满满；工科就感觉呵呵了，跟加拿大别的学校差距还是很大的。
MENG： MENG号称是6个term，目的是为了让你理所应当拿三年工签，其实几乎所有人都是3term毕业，个别延期4term。你拿到的学签都是两年，最最最重要的是你一年读完能拿到3年的工签！！！这一点确实非常人性化，据我认识的，只要通过网上申请的，即使一年毕业，都拿到的是三年工签；去边境办理的，除非人品太差，拿到的也是三年。一年三学期，暑假也有一个学期，主要是上一些工程管理呀，工程交流啊非专业课。总共10门课毕业。也可以选择做一个project，可以抵两门课，project水不水就看你选的导师，一般中国学生都找的是中国老师，恩。。ECE系有个software engineering track，可以选CS的课，ECE本身开的课就呵呵了，一学期开不了几门，有用的就更少了。。
MESC：一般是4门课加毕业论文还有答辩，一般是两年6个学期毕业，具体时间就看导师了。
找工作：楼主认识的人大部分都找到工作了，机械在安省还是能找到的，有一毕业就找到的，也有毕业半年内找到的，具体就看个人能力了。好多毕业都搬去密西沙加啊，多伦多去了，也有坚守在伦敦的。当然计算机统计是最好找的，跟计算机统计的人聊天，从没说担心找不到工作，别的专业就呵呵了。虽然计算机统计一枝独秀，但是也并不是别的专业就找不到，楼主的朋友在多大读教育学，按版上的格调肯定是毕业就失业的节奏，但是她和同学全部找到工作了，毕竟多伦多市场大，教育需求也大，毕竟中国的本科高中小土豪需要培训补习啊啥的吧。。楼主能力太差了，专业又坑，就滚回国了。想起来了，一直有人问研究型硕士和授课型硕士区别在哪里，哪个更好什么的，研究型硕士就是大部分有奖学金，授课型自费，但是找工作差别真的不大，除非是你的导师的人脉很广能拿给你推荐工作，要么是你的研究经历跟你找工作是吻合的，加分的，要不然真没啥区别。。举个例子，现在很多机械系的老师搞什么纳米生物，听起来贼高大上呢，硕士毕业根本找不到相关工作，还是只能接着机械硕士头衔找一些机械画图啊相关的工作，有可能你研究型硕士毕业出来还没人家授课型硕士能力强，说的就是我自己。。。。
感想：答主当时准备去美国的，阴差阳错的来到了加拿大，感觉加拿大和美国的差距还是蛮大的，当然各有各的优点啦，加拿大黑人少啊更安全啊，医疗保险便宜啊，跟美国同学聊天，感觉各种机会都少很多，不管是学术上的还是工作上的。加拿大都是公立大学，本来学费就低，所以都很穷，除非拿到外面的奖学金，对国际学生来说机会非常少，基本就是加元一个月，基本上只够吃饭住宿了，想养车可能都要啃老。不像美国排名很差的学校的博士都至少2000美元一个月。而且加拿大博士毕业更难找工作，学术界更小，工业界也没啥去处，不像美国有各种研究所国家实验室大公司。比如美国化学博士去因特尔的很多，但是加拿大化学博士毕业去哪呢？而且加拿大取消博士移民通道了，即使读博，跟硕士毕业移民也并没有明显优势，在美国，如果牛逼，直接EB1A绿卡走起，但是加拿大你发文章再多，对绿卡也没啥帮助吧~~ 总之对一般人来说加拿大拿PR比美国容易太多了，但是别的就。。。加拿大也确实很适合过小日子的人，福利好，环境好。。。并无意抹黑加拿大吹捧美国，也不想撕逼，之前说了，加美各有各的好处，美国靠读书和工作比加拿大难了N个数量级。。。先写到这里吧，以后想到什么再来更新
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MESC是啥？
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lz 提到的教育学就业的事情 幸存者偏差太大 一般来说是只有找到工作的才会吱声 所以在lz看来才是“她和同学全部找到工作了” 但是lz也不敢说你们学校教育学就业100%吧？
另外找到工作 能不能移民 就是另外一回事了
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wrath 发表于
MESC是啥？
Master of Engineering Science 就是别的学校的MSc，研究型硕士
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wrath 发表于
lz 提到的教育学就业的事情 幸存者偏差太大 一般来说是只有找到工作的才会吱声 所以在lz看来才是“她和同学 ...
是的是的呢，我只是说并不是完全找不到，多伦多机会多，当然教育学整体就业率肯定就。。
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我觉得楼主对加拿大有偏见呢，加拿大机会是少，美国机会是多，但是美国那些高质量的机会你觉得是为你准备的吗？加拿大中等机会比美国还是容易找不少，特别是加拿大基本不怎么care 你身份问题，只要你有工签，美国你是只看到别人好的时候，不知道在美国留学拿绿卡是有多少辛酸和泪水的过程;
我读金融的，ivey的，要说差距大，加拿大金融这块跟美国比你的领域不知道大多少，哪怕多伦多，baystreet 跟 wallstreet也是几何级差距，但一样，美国那边的机会多不等于都给你的，你身份不解决那边机会不比加拿大多多少，而且你既然选择加拿大廉价和身份优势，那必然有得必有失的，所以我也没想马上去美国，至少要在加拿大好好工作一段时间再考虑美国
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本帖最后由 expectnewlife 于
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kuyigougou 发表于
我觉得楼主对加拿大有偏见呢，加拿大机会是少，美国机会是多，但是美国那些高质量的机会你觉得是为你准备的 ...
你说的跟我说的有区别么。。。没啥有没有偏见，我只是说事实是这样。。。我的意思就是想快速拿移民就去加拿大啊。。。。。美国靠读书工作拿身份比加拿大难了好几个数量级。。。。
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expectnewlife 发表于
你说的跟我说的有区别么。。。没啥有没有偏见，我只是说事实是这样。。。我的意思就是想快速拿移民就去 ...
我的point是实际上美国的对于中国留学有效的机会比加拿大可能多不出来太多，不过有点还是要承认如果你真的是大牛级的留学生，美国毫无疑问是最好的，差距主要是在每个领域的top端
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有几个问题想问下楼主~
1、机械meng的同学有工作经验的多还是应届的多？找工作的时候区别大吗？
2、3学期读完的话平时上课会不会很紧张？有时间出去打工吗？
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expectnewlife 发表于
你说的跟我说的有区别么。。。没啥有没有偏见，我只是说事实是这样。。。我的意思就是想快速拿移民就去 ...
事实是 对于大部分拿不到H1B的同学来说 米国等于没有机会
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楼主，你好想问下UWO的ECE很水么...
这边拿到了个UWO的ECE MENG的AD。
本来是比较担心工签的，但是看到楼主的帖子貌似挺不错的。
另外还是那边ECE真的很水么，想转M.Sc的项目容易么~
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楼主的帖子好positive啊，我想申西大的机械meng，想问一下楼主对那个wind engineering 了解吗？我是新能源方向的
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哎呀吓死我了 发表于
有几个问题想问下楼主~
1、机械meng的同学有工作经验的多还是应届的多？找工作的时候区别大吗？
2、3学期 ...
基本都是应届的，当然你之前有工作经验找工作会好很多；
不会，基本上学期间没打工的吧，想去打也可以去吧；
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hui_bnu 发表于
楼主，你好想问下UWO的ECE很水么...
这边拿到了个UWO的ECE MENG的AD。
本来是比较担心工签的，但是看到楼 ...
ECE转MESC好像不容易，但是只要有老师要你就可以转，课程设置感觉很一般，对于大部分人来说就是个找工作的跳板
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独孤浩 发表于
楼主的帖子好positive啊，我想申西大的机械meng，想问一下楼主对那个wind engineering 了解吗？我是新能源方 ...
听说过，西大wind 实力还是很强的
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Powered byImproved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX
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Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAXXin YuXiquan LiangHuimin XieShantanu KumarNamritha RavinderJason PotterXavier de Mollerat du JeuJonathan D. ChesnutOpen AccessOriginal Research PaperDOI:
10.-016-2064-9Cite this article as: Yu, X., Liang, X., Xie, H. et al. Biotechnol Lett (9. doi:10.-016-2064-9
To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection.Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells.Lipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.Cas9 proteinCell engineeringCRISPRCRISPRMAXGenome editingHomologous recombinationThe use of cell-penetrating peptides (CPPs) facilitates the delivery of active proteins into cells (Copolovici et al. ; Erazo-Oliveras et al. ; Jo et al. ). The delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) has gained increasing attention due to the high cleavage efficiency and potentially lower off-target effect compared with plasmid DNA transfection (Kim et al. ; Zuris et al. ; Liang et al. ). Although Cas9 RNPs can be delivered into mammalian cells via electroporation with relatively high efficiency, lipid-mediated transfection remains popular due to ease of use, low cost, and adaptation to high throughput system. Several methods for delivery of Cas9 RNPs have been reported. Through conjugation of a cell-penetrating peptide to Cas9 protein and complexation of gRNA with CPPs, the subsequent Cas9 RNPs were able to deliver into HEK293T, HeLa, NCCIT (human embryonal carcinoma cell line), HDF (human dermal fibroblasts), and H9 human embryonic stem cells with up to 36 % genome modification efficiency (Ramakrishna et al. ). A method termed iTOP (induced transduction by osmocytosis and propanebetaine) allows the highly efficient transduction of native proteins into a wide variety of primary cell types (D’Astolfo et al. ). Furthermore, by fusing to negatively supercharged GFP proteins or binding to anionic nucleic acids, functional proteins could be delivered into mammalian cells via cationic lipid-mediated transfection. As described, Cas9 RNPs delivered into U2OS cells using Lipofectamine 2000 resulted in up to 80 % modification efficiency at an integrated GFP reporter and Cas9 RNPs delivered into the mouse inner ear in vivo using Lipofectamine RNAiMAX resulted in 20 % loss of GFP expression in auditory sensory cells (Zuris et al. ). However, significant toxicity from Lipofectamine 2000 was also reported in U2OS cells. In the present study, we screened a large set of transfection reagents using relatively easy and hard-to-transfect cell lines and identified a new transfection reagent, Lipofectamine CRISPRMAX, which worked significantly better than Lipofectamine 3000 or Lipofectamine RNAiMAX with very low cell toxicity.The screening of transfection reagents was conducted in a 96-well format. 1 day prior to transfection, six commonly used cell lines, A549, HEK293, HeLa, HepG2, MCF-7, and U2OS, were seeded in 96-well plates at 10,000–20,000 cells per well. On the day of transfection, a master mix of Cas9 protein and HPRT1 gRNA was prepared in Opti-MEM medium and incubated for 5 min at 25 °C to form the Cas9 RNPs. The amount of Cas9 RNPs was held constant at 40 ng GeneArt Platinum Cas9 nuclease and 8.5 ng gRNA per well. On the other hand, the amount of each transfection reagent, which was also prepared in Opti-MEM medium, varied from 0.1, 0.2, 0.4 and 0.6 ul per well. Lipofectamine 3000 and Lipofectamine RNAiMAX served as controls. The Cas9 RNPs in Opti-MEM medium were added to the transfection reagents diluted in Opti-MEM medium. The mixture was incubated at 25 °C for 10–15 min to form the Cas9 RNPs and transfection reagent complexes, followed by addition to the cells. After incubation for 48 h, the cells were lysed and percentage of Indel (insertion and deletion) was measured by GeneArt Genomic Cleavage Detection Kit. The experimental data was then analyzed using JMP, Version 11. SAS Institute Inc. (Cary, NC, USA).One day prior to transfection, adherent cells were plated onto 24-well plates at 0.4 to 1.5 × 105 cells per well in 500 ul of growth medium so that the cells reached 30–70 % confluence at the time of transfection. On the day of transfection, 25 ul of Opti-MEM medium was added to a 1.5 ml sterile Eppendorf tube, followed by the addition of 500 ng GeneArt Platinum Cas9 nuclease and 125 ng gRNA. Upon mixing by vortexing briefly, 1 ul Cas9 Plus reagent was added to the solution containing Cas9 protein and gRNA. After briefly vortexing, the mixture was incubated at 25 °C for 5 min to allow the formation of Cas9 RNPs. The Cas9 RNPs remained active at 25 °C for up to 2 h. For co-delivery of donor DNA, 500 ng single strand DNA oligonucleotide or 300 ng linear PCR fragment was added to the Cas9 RNPs at this point. Meanwhile, 25 ul Opti-MEM medium was added to a separate sterile Eppendorf tube, followed by addition of 1.5 ul of Lipofectamine CRISPRMAX. After briefly vortexing, the Lipofectamine CRISPRMAX solution was incubated at 25 °C for approx. 5 min. After incubation, the Cas9 RNPs were then added to the Lipofectamine CRISPRMAX solution. The reverse addition of Lipofectamine CRISPRMAX solution to the Cas9 RNPs was found to decrease the editing efficiency in certain cell lines. Upon mixing, the sample was incubated at 25 °C for 10–15 min to form Cas9 RNPs and Lipofectamine CRISPRMAX complexes and then added to the cells. At 48–72 h post-transfection, the cells were harvested for analysis of genome modification efficiency using GeneArt Genomic Cleavage Detection kit. Alternatively, cells were analyzed by flow cytometry to determine the percentage of EmGFP positive cells.For transfection of human iPSC, the cells were treated with TrypLE and plated onto Geltrex-coated 24-well plates at 40,000 cells per well, leading to approx. 30–40 % confluence at the time of transfection. One ug GeneArt Platinum Cas9 nuclease, 250 ng gRNA and 6 ul Cas9 Plus reagent were used to prepare the Cas9 RNPs instead, while the amount of Lipofectamine CRISPRMAX reagent remained constant at 1.5 ul. At around 6 h post-transfection, the media containing the transfection reagent was removed and replaced with fresh Essential 8 Medium. The cells were analyzed at 48 h post-transfection.A ‘reverse’ transfection protocol was used to transfect MCF-7 and HepG2 cells. In this case, the Cas9 RNPs and Lipofectamine CRISPRMAX reagent were prepared in two separate tubes as described above with 500 ng GeneArt Platinum Cas9 nuclease, 125 ng gRNA, and 1 ul Cas9 Plus reagent in Tube-1, and 1.5 ul Lipofectamine CRISPRMAX reagent in Tube-2, respectively. The Cas9 RNPs solution was then transferred to the Lipofectamine CRISPRMAX solution. Upon vortexing, the mixture was incubated at 25 °C for 10–15 min to form the Cas9 RNPs and Lipofectamine CRISPRMAX complexes. Meanwhile, MCF-7 and HepG2 cells were detached with TrypLE and counted, followed by seeding at 2 × 105 and 1.0 × 105 cells per well, respectively. The Cas9 RNP/Lipofectamine CRISPRMAX solution was then added directly to the cell suspension and incubated for 48–72 h prior to analysis.For Neon electroporation, adherent cells were detached from culture dishes and counted. In general, 105 adherent cells or 2 × 105 suspension cells were used per 10 ul reaction. For the Neon 24-well optimization protocol, 24 ug Cas9 protein and 6 ug gRNA were added to 120 ul Resuspension Buffer R, followed by mixing and incubation at 25 °C for 5 min to form the Cas9 RNPs. 2.4 × 106 adherent cells or 4.8 × 106 suspension cells were harvested and washed with DPBS. After aspiration, the cell pellets were re-suspended in 120 ul Resuspension Buffer R and then mixed with Cas9 RNPs. A 10 ul sample was taken for each electroporation using one of the Neon 24-well optimization conditions. The electroporated cells were transferred immediately to a 24 well containing 500 ul corresponding growth medium and incubated for 48 h prior to analysis. Upon optimization, the use of a higher dose of Cas9 RNPs (for example, 2 ug Cas9 protein and 500 ng gRNA per reaction) could further increase the cleavage efficiency (Liang et al. ).The genomic modification efficiency was determined by GeneArt Genomic Cleavage Detection kit as described in the manual. The targeting gRNA sequence: 5′-gcatttctcagtcctaaacaggg-3′ was used to edit HPRT1 locus. At 48 h post-transfection, suspension cells were harvested by centrifugation and then washed with DPBS, whereas adherent cells were washed directly with DPBS. Cells were lysed with 20 ul cell lysis buffer per 96-well or 50 ul cell lysis buffer per 24-well. Upon treatment with Proteinase K at 68 °C for 15 min, the mixture was held at 95 °C for 10 min. One to 3 ul of cell lysate was then used for PCR amplification with AmpliTaq Gold 360 Master Mix in the presence of the corresponding forward and reverse primers. For the HPRT1 target, a forward primer: 5′-acatcagcagctgttctg-3′ and a reverse primer: 5′- ggctgaaaggagagaact-3′ were used. The PCR program was set at 95 °C for 3 min for one cycle, then at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s for a total of 40 cycles. Final extension was set at 72 °C for 5 min. The resulting PCR product (3 ul) was mixed with 1 ul of 10x Detection Reaction Buffer and 5 ul water, and then subjected to denaturation and re-annealing at 95 °C for 5 min, 4 °C for 5 min, 37 °C for 5 min, and then 4 °C for 5 min. Finally, 1 μl 10× detection enzyme was added to the sample and then incubated at 37 °C for 1 h. The digested product was analyzed with a 2 % E-gel EX agarose. The percentage of cleavage was quantified using an AlphaImager gel documentation system running AlphaView, Version 3.4.0.0. ProteinSimple (San Jose, CA, USA).GripTite HEK293 stable cells expressing EmGFP were prepared via the Jump-In system as described in the manual (Thermo Fisher Scientific). To generate a disrupted EmGFP mutant stable cell line, 1.5 ug of GeneArt Platinum Cas9 nuclease was associated with 300 ng gRNA targeting the 5′-ctcgtgaccaccttcacctacgg-3′ sequence in the EmGFP reporter gene (T1) and were then transfected into wild type EmGFP cells via electroporation, followed by limiting dilution to isolate clonal cell lines. Upon DNA sequencing, a disrupted EmGFP stable cell line with the 5′-CACCTT-3′ deletion was selected for a homologous recombination assay (Supplementary Table 1).To create homologous recombination assays, a gRNA targeting the 5′-gaagcactgcacgccgtaggtgg-3′sequence within the disrupted EmGFP reporter gene (T2) was designed and synthesized. This gRNA only recognized the disrupted EmGFP gene but not the wild type EmGFP gene. One day prior to transfection, the cells were seeded on a 24 well plate at 105 per well. 500 ng of GeneArt Platinum Cas9 nuclease and 125 ng gRNA were transfected into the disrupted EmGFP stable cell line using 1.5 ul Lipofectamine CRISPRMAX in the presence or absence of 500 ng of a 97 bp single-stranded DNA oligonucleotide (5′-catgtggtcggggtagcgggcgaagcactgcacgccgtaggtgaaggtggtcacgagggtgggccagggcacgggcagcttgccggtggtgcagatg-3′) or 300 ng of a 400 bp linear wild type PCR fragment amplified using a forward primer 5′-atggtgagcaagggcgaggagctg-3′ and a reverse primer 5′-gtcctccttgaagtcgatgccc-3′ (Supplementary Table 2). At 48 h post-transfection, the restoration of EmGFP function was determined by flow cytometric analysis with an Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific).To identify the best transfection reagents for delivery of Cas9 RNPs, initially we compared several commercially available protein transfection reagents, including Lipofectamine 2000, Lipofectamine 3000, Lipofectamine RNAiMAX, Lipofectamine MeassengerMax, TurboFect, and Xfect Protein transfection reagent. Cas9 protein and HPRT1 gRNA were transfected into HEK293 and HCT116 cell lines according to manufacturer’s protocol. Upon 48 h post-transfection, the cells were harvested to analyze the genome modification efficiencies. As depicted in Fig. a, the Indel (insertions and deletions) production efficiencies were highest when using Lipofectamine 3000 or Lipofectamine RNAiMAX transfection reagents. Next, we used Lipofectamine 3000 and Lipofectamine RNAiMAX as controls and screened more than 60 transfection reagents. Screening was conducted in a 96-well format using three easy-to-transfect cell lines (HEK293, HeLa, and U2OS) and three hard-to-transfect cell lines (HepG2, A549, and MCF-7). Cells were seeded on 96-well plates 1 day prior to transfection. On the day of transfection, the Cas9 protein was incubated with HPRT1 gRNA at 25 °C for 5 min in Opti-MEM medium to form the Cas9 RNPs, followed by mixing with various amount of transfection reagents in Opti-MEM medium. The percentage of Indels was determined using the GeneArt genomic cleavage detection assay at 48 h post-transfection. Among all the formulations, only a few worked equally well or better than Lipofectamine RNAiMAX with one formulation (Lipofectamine CRISPRMAX) standing out.Fig. 1Identification of Lipofectamine CRISPRMAX. a One day prior to transfection, HEK293 and HCT116 were seeded on a 24 well plate at 105 cells per well. On the day of transfection, 500 ng Cas9 protein and 120 ng HPRT1 gRNA were transfected with either Lipofectamine 2000 (LF2K), Lipofectamine 3000 (LF3K), Lipofectamine MessengerMAX, Lipofectamine RNAiMAX, TurboFect or Xfect transfection reagent according to manufacturer’s protocol. At 48 h post-transfection, cells were harvested to perform genomic cleavage assays. The % of Indel was quantified using AlphaView software. The data was analyzed using JMP statistical software. The bar graph represented the mean and standard deviation of three independent experiments of two cell lines. b, c A549, HEK293, HepG2, HeLa, MCF-7 and U2OS were seeded on 96-well plates at approx. 20,000 cells per well and then transfected with 40 ng Cas9 protein and 8.5 ng HPRT gRNA using either Lipofectamine CRISPRMAX, Lipofectamine RNAiMAX or Lipofectamine 3000. After 48 h post-transfection, the cells were lysed and subjected to a genomic cleavage assay. The results were processed using JMP11 software. b was the Box Plot of Indel frequencies comparing Lipofectamine CRISPRMAX to Lipofectamine RNAiMAX in six different cell lines. c was one-way ANOVA analysis of % Indel by type of lipid with blocking of cell type. Each dot represented one experimental data point. The diamond represented the 95 % confidence interval for the mean of each group. The circles were visual representations of group mean comparisons using Student’s t-tests. The p value was less than 0.05As shown in Fig. b, the percentage of Indels in cells transfected with Lipofectamine CRISPRMAX was higher than those with Lipofectamine RNAiMAX, especially in A549, HeLa and HepG2 cell lines. Based on statistical analysis, the genome cleavage efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were significantly higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, with an average increase of 40 or 15 %, respectively, across six different cell lines (Fig. c). The visual comparison of group means using Student’s t-tests, represented by the circles, showed that Lipofectamine CRISPRMAX, Lipofectamine 3000 and RNAiMAX were significantly different from each other as the circles did not intersect.Upon identification of Lipofectamine CRISPRMAX as the best transfection reagent, we determined the functional activity of Cas9 RNPs by examining the times required for complexation of Cas9 protein with gRNA (Cas9 RNPs) and Cas9 RNPs with Lipofectamine CRISPRMAX in A549, HEK293, and HeLa cells. As shown in Fig. a, the Cas9 RNPs remained active at 25 °C for up to 3 h based on the genome cleavage assay. On the other hand, the diluted Lipofectamine CRISPRMAX were only active at 25 °C for 5–15 min as a longer incubation time had a decreased cleavage efficiency (Fig. b). When the Cas9 RNPs were mixed with Lipofectamine CRISPRMAX the editing efficiency dropped after 10–15 min indicating that the addition of Lipofectamine CRISPRMAX was the limiting step (Fig. c).Fig. 2Activities of Cas9 RNP complexes and Cas9 RNP/Lipofectamine CRISPRMAX complexes. One day prior to transfection, cells were seeded on a 96 well plates. A master mixes of Cas9 RNP complexes and Lipofectamine CRISPRMAX were prepared in Opti-MEM media based upon 40 ng Cas9 protein, 8.5 ng gRNA, and 0.3 ul Lipofectamine CRISPRMAX per well. The incubation times of Cas9 RNP (a), Lipofectamine CRISPRMAX (b), and Cas9 RNP/Lipofectamine CRISPRMAX complexes (c) served as dependent variables. At the indicated time point, aliquots of Cas9 RNP complexes in Opti-MEM were added to aliquots of Lipofectamine CRISPRMAX solution and incubated for indicated time prior to addition to A549, HEK293, and HeLa cells, respectively. Upon 48 h post-transfection, the genome cleavage efficiencies were determinedNext, we examined the key factors that governed the transfection efficiency by varying the dose of transfection reagent, the amount of Cas9 RNPs, and cell density. Six commonly used cell lines were transfected with increasing amount of Cas9 RNPs, followed by genome cleavage assay. As shown in Fig. a, the efficiency of Indel production in transfected cells increased with an increase of Cas9 RNPs. The differences between the doses were statistically significant because the group mean comparisons represented by the circles did not intersect and the p value was &0.05. To transfect cells in a 96-well plate, the optimal amounts of Cas9 protein and gRNA were approx. 120 and 25.5 ng respectively. Cell seeding density plays an important role in regulating the transfection efficiency. As depicted in Fig. b, the average genome modification efficiency across six different cell lines was significantly higher at 60 % cell confluence than at 80 % cell confluence at the time of transfection with a p value less than 0.05. However, no significant difference in editing efficiency was observed between low and high lipid doses (Fig. c). Other factors, such as cell passage and dissociation, also contributed to daily variation in cell transfection and Indel efficiency.Fig. 3Factors regulating transfection efficiencies. a A549, HEK293, HepG2, HeLa, MCF-7 and U2OS were seeded on 96-well plates at two cell densities and then transfected with either 40 ng Cas9 protein and 8.5 ng gRNA (1×), 80 ng Cas9 protein and 17 ng gRNA (2×) or 120 ng Cas9 protein and 25.5 ng gRNA (3×) using either 0.2 or 0.4 ul Lipofectamine CRISPRMAX. The editing efficiency was determined at 48 h post-transfection. The Indel percentage was determined using the AlphaView software and the resulting data was processed using JMP11 software. Analysis of variance (ANOVA) analysis of % Indel by dose of Cas9 RNP (a), cell density (b), and amount of transfection reagent (c) were carried out across six different cell lines. Each dot represented one data point, whereas the diamond represented the 95 % confidence interval. The circles were visual views of group mean comparisons using Student’s t-tests. The p value was less than 0.05We then scaled up to 24 wells to test a set of 23 cell lines, including a variety of adherent and suspension cells from different species. The morphologies of more than a dozen adherent cell lines were recorded prior to transfection and at 48 h post-transfection (Supplementary Fig. 1). Most of the cells looked healthy under the microscope with examples shown in Fig. a, very little floating dead cells were observed upon 48 h post-transfection for A549, HeLa, HEK293, and human epidermal keratinocytes (HEKa). Cell viability assays with Trypan Blue indicated that the viable cells only decreased moderately after transfection compared to control cells, suggesting that the cell toxicity induced by Lipofectamine CRISPRMAX was relatively low (Fig. b). We observed around 68, 71, 80, and 35 % genome cleavage efficiencies in A549, HeLa, HEK293, and HEKa primary cell lines, respectively (Fig. c). The low cell toxicity of Lipofectamine CRISPRMAX prompted us to transfect cells at much lower cell density so as to increase the transfection efficiency (Table
and Supplementary Table 3). For example, N2A, mouse ESC and iPSC were grown to 35, 25 and 30 % confluence at the time of transfection (Supplementary Table 3) and achieved 70, 75 and 55 % genome editing efficiencies at mouse Rosa26 and human HPRT1 loci, respectively (Table ). The improved efficiencies were probably due to the higher accessibility of transfection reagents at low cell density. However, the optimal cell density was highly dependent on cell type and needed to be determined experimentally.Fig. 4Cell toxicity using Lipofectamine CRISPRMAX. a Prior to transfection and at 48 h post-transfection, the morphologies of A549, HeLa, HEK293, and human epidermal keratinocytes (HEKa) were examined by an IncuCyte instrument, Essen BioScience Inc. (Ann Arbor, MI, USA). b Cell viabilities were measured by Trypan Blue staining before 0 and after 48 h post-transfection. c The genome modification efficiencies were determined at 48 h post-transfectionTable 1Genome editing efficiency in a variety of cell lines Cell lineSourceLipofectamine CRISPRMAX (% Indel)Neon electroporation (% Indel)1mESCMouse embryonic stem cell75 ± 374 ± 42N2AMouse liver carcinoma70 ± 581 ± 233T3Mouse embryonic fibroblast57 ± 450 ± 24CHOHamster ovary57 ± 1–5COS-7Monkey kidney44 ± 3–6A549Human lung carcinoma48 ± 366 ± 37293FTHuman kidney85 ± 588 ± 38HEK293Human kidney75 ± 5–9HCT116Human colon carcinoma85 ± 5–10HEKaHuman primary epidermal keratinocytes14 ± 232 ± 211HeLaHuman cervical cancer50 ± 7–12HepG2Human liver cancer30 ± 352 ± 313HUVECHuman umbilical vein endothelium9 ± 326 ± 214iPSCHuman induced pluripotent stem cell55 ± 385 ± 215MCF-7Human mammary gland8 ± 422 ± 516MDA-MB-231Human breast cancer39 ± 5–17U2OSHuman osteosarcoma55 ± 470 ± 318JurkatHuman T cell leukemia19 ± 394 ± 219K562Human lymphoblastoid20 ± 291 ± 120THP-1Human monocytes12 ± 331 ± 321SC-1Human B lymphoblasts044 ± 222RajiHuman B lymphocyte050 ± 523NK-92Human peripheral blood031 ± 5Suspension cells, especially hematopoietic cells, are difficult to transfect by conventional lipid reagents (Papapetrou et al. ). We also found that hematopoietic cells were hard to transfect using Lipofectamine 3000, Lipofectamine RNAiMAX, and Lipofectamine CRISPRMAX. For each hard-to-transfect cell line, we tested the delivery of Cas9 RNPs using the Neon 24-well optimization protocol (Supplementary Table 4). For example, using electroporation we achieved 94, 91 and 44 % Indel production efficiencies in Jurkat T cells, K562 and SC-1 cells respectively at the HPRT1 locus, whereas relatively low genome modification efficiencies were observed using Lipofectamine CRISPRMAX in these suspension cell lines (Table ).Precise gene modification, such as single-nucleotide polymorphism (SNP) correction in cancer cells, is an important aspect in biomedical and clinical applications (Lee ). For a proof of concept, we tested the co-delivery of various amount of donor DNA with Cas9 RNPs into a stable GripTite HEK293 cell line harboring a disrupted EmGFP gene using Lipofectamine CRISPRMAX. Delivery of Cas9 RNPs alone or Cas9 protein plus donor DNA was used as controls. After 48 h post-transfection, the cells were examined by a fluorescence microscope and images were recorded (Fig. a). The transfected cells were subjected to flow cytometric analysis to determine the percentage of EmGFP positive cells. As shown in Fig. b, approx. 17 % of the cells restored the function of EmGFP when 500 ng 97 bp ssDNA oligonucleotide (ssODN) was used, whereas approx. 6.5 % of EmGFP positive cells were observed when 300 ng of a 400 bp dsDNA fragment was used. The homologous recombination efficiency we obtained is comparable to those observed by other research groups. For example, the delivery of Cas9 RNPs and ssODN into unsynchronized HEK293T cells via nucleofection yielded around 10 % HDR frequencies, although up to 38 % HDR efficiency was observed in synchronized cells (Lin et al. ). The delivery of Cas9 RNPs and ssODN into an EGFP-repair reporter cell line using Lipofectamine 2000 resulted in 8–11 % of HDR frequencies (Zuris et al. ). Although the HDR efficiency is getting better, there is undoubtedly room for further improvement.Fig. 5Co-delivery of Cas9 RNP and donor DNA. Various amount of a 97 bp single-stranded DNA oligonucleotide (ssDNA) or a 400 bp double-stranded DNA fragment (dsDNA) was co-delivered with Cas9 RNPs into a disrupted EmGFP stable cell line in 24-well culture plates. After 48 h post-transfection, the percentages of GFP positive cells were quantified using flow cytometric analysis. Delivery of Cas9 RNP or Cas9 plus donor DNA (Cas9/D) served as controls. The experiments were performed in triplicateLipofectamine CRISPRMAX is more robust than Lipofectamine RNAiMAX and several other transfection reagents in delivery of Cas9 protein/gRNA complexes into a variety of cell lines. However, delivery of the Cas9 RNPs by Neon electroporation was effective across all different cell lines tested and often was the only method that could produce Indels in difficult to transfect suspension cell lines. Because of the ease of use and low toxicity, Lipofectamine CRISPRMAX will further facilitate high throughput drug screening and genome editing where electroporation is less applicable.
We acknowledged Natasha Roark, Jason Carte, Mahalakshmi Sridharan, Yanfei Zou, and Wen Chen for their technical supports and Jarrod Clark for his critical review of the manuscript.Supplementary Table 1—EmGFP sequence.Supplementary Table 2—CRISPR target sequences and PCR primers.Supplementary Table 3—Genome editing efficiency in a variety of cell lines (24-well Format).Supplementary Table 4—Neon optimization protocols.Supplementary Fig. 1—Cell morphology. (191 kb)Supplementary material 1 (PDF 191 kb)Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kühn R (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33:543–548Copolovici DM, Langel K, Eriste E, Langel ? (2014) Cell-penetrating peptides: design, synthesis, and applications. ACS Nano 8:D’Astolfo DS, Pagliero RJ, Pras A, Karthaus WR, Clevers H, Prasad V, Lebbink RJ, Rehmann H, Geijsen N (2015) Efficient intracellular delivery of native proteins. Cell 161:674–690Erazo-Oliveras A, Najjar K, Dayani L, Wang TY, Johnson GA, Pellois JP (2014) Protein delivery into live cells by incubation with an endosomolytic agent. Nat Methods 11:861–867Jo J, Hong S, Choi WY, Lee DR (2014) Cell-penetrating peptide (CPP)-conjugated proteins is an efficient tool for manipulation of human mesenchymal stromal cells. Sci Rep 4:4378Kim S, Kim D, Cho SW, Kim J, Kim JS (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:Lee SJ (2012) Clinical application of CYP2C19 pharmacogenetics toward more personalized medicine. Front Genet 3:318Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD (2015) Rapid and highly efficient cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53Lin S, Staahl BT, Alla RK, Doudna JA (2014) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3:e04766Papapetrou EP, Zoumbos NC, Athanassiadou A (2005) Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects. Gene Ther 12:S118–S130Ramakrishna S, Dad AK, Beloor J, Gopalappa R, Lee SK, Kim H (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24:Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33:73–80Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Xin Yu1Xiquan Liang1Huimin Xie1Shantanu Kumar1Namritha Ravinder1Jason Potter1Xavier de Mollerat du Jeu1Jonathan D. Chesnut11.Synthetic Biology DepartmentThermo Fisher ScientificCarlsbadUSA
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