此前，网传杨辉曾对质疑作出正面回应，以下为网传杨辉之前的回应 （尚未经自己证实，仅供参考 对比）：这是之前几天回复质疑的信，抱歉，附件和原邮件不能给你，response 还在审稿中。对我每篇学术文章的质疑，欢迎发信给杂志社或许我来评论，我只会回复学术的部分，谢谢！关于我在 MIT 的文章，Genome Biology 文章 (即质疑文章) 刚在线的时分，我现已和 editor 取得联系，她欢迎咱们写 response 回复，由于涉及到一些试验，最近刚刚和文章一起榜首作者 Haoyi Wang 与通讯作者 Rudolf Jaenisch 准备好，文章正在投稿中。以下是回复，详见附件：Gurumurthyet al. [1]recently reported that a method developed by Yang et al. togenerate floxed allele (designated as“two donor method”by Gurumurthyet al.) [2] had poor reproducibility. They claimed that three centerscould not reproduce our results on generating conditional alleles of the Mecp2 locus and that the“two-donor method”had very low successful rate onother loci.Here, weprovide our responses to these claims:1.Our results on Mecp2 locus published byYang et al have beenreproducedby independent experiments in the Jaenisch (8-10% correct alleles), Yang(8% correct alleles) and Hatada’s groups (2-6% correct alleles) [3] ,respectively.2.The conditions used by Gurumurthy et al.[1] do not correspond to the conditionsused in our paper. The concentrations of CRISPR reagents used in the Gurumurthy et al.’s study [1]on the Mecp2 locus (10 ng/μl for Cas9 mRNA, 10ng/μl for sgRNA and 10 ng/μl for oligos) were much lower (10 fold lowerRNA and 20 fold lower oligo donor concentration) than those used in the Yang et al.’s experiments (Cas9 100 ng/μl, sgRNA 50 ng/μl and 100 ng/ μl foreach oligo) [2] and Yang et al.’s previous [4] and following publications[5-8]. It is well known that the concentrations of CRISPR reagentsare well correlated with the genome editing efficiency.3.We utilized piezo-driven zygote injectionmethod in our original paper , whichallows for injecting CRISPR components at much higher concentration. The differencebetween this method and pronuclear injection method used by Gurumurthyet al. might also contribute to the difference of successful rates.4.Multiple peer-reviewed publications [3,9-12] have successfully used ourmethod to create conditional knockout (CKO) mice (9 out of 11 loci succeeded,2.5% to 18% efficiency). We note that the efficiency of generating CKO mice by CRISPR/Cas9 is highly dependent on the professional skillsand well-built platforms. Thus, it is inappropriate to calculate the editingefficiency based on data from different labs and“core facilities”with varying capability and using different methodologies.5.With any genome editing method or strategy being used, theefficienciesat different genomic loci are often highly variable. In the2013proof of concept paper, we showed the feasibility of generating floxed alleleat Mecp2 locus using CRISPR. As a X-linked gene, Mecp2 has a higher chance of having two independent loxP insertion events residing on the same Xchromosome, since half of the embryos are males. To assume the efficiency we demonstrated at Mecp2 locus will be directly translated to the successful rate atother genomic loci seems premature.We agree with the Gurumurthy et al’s comment that the“one-donor method”offershigher success rate for generating floxed alleles in general, while the efficiency of“one-donor method”is also variable depending on the genomic loci and donor plasmid design. Before the publication of Gurumurthy et al., we also noted this, and developed a“one-donor method”, termed“Tild-CRISPR”method [8], and demonstrated the feasibility and high efficiencyin generating CKO mice.With thefast improvement of genome editing technologies, we and many others constantly optimize our protocols. We welcome all discussions about the choiceof optimal strategy for particular applications, however, we think thereproducibility of any published work can only be validated by using the exactsame experimental methods and technical parameters.PS: 和 Genome Biology 的 editor 暗里沟通，这篇文章原本在 Nature Methods 审稿，但有一个 reviewer 便是用咱们这种办法日常做 CKO 小鼠，所以文章拒了。转投 GenomeBiology 的时分，也有一个 reviewer 用咱们这种办法日常做 CKO 小鼠，所以他们的文章重视点改为比较咱们的 CKO 办法和最新开展的 ”one-donor method”，才宣布出来。我一向不明白，许多人现已重复出咱们的试验成果，也有许多人用该办法做 CKO 小鼠，这篇文章也能说造假吗？数据不可信吗？当然更多人做不出来，我以为很大原因是由于自身的试验体系欠好，这篇文章的一切试验室（绝大大数是 core facility）都没有先严格重复咱们的试验成果，再在此基础上规划新的试验。我以为这不是受过好的科研练习的人应有的干事办法，我也没有收到他们的任何来信问询我该试验的试验细节和 tricks。关于我“其他文章也有许多问题”，我将我之前宣布的文章都放到附件中，欢迎质疑者发信给我和咱们，一一指出，我会做一一答复。我也将我自博士期间以来宣布的一些重要文章做一个简明阐述：博士期间：Publication list 30: 孤雄单倍体的树立，这个已有一些试验室相同树立，比方周琪教师组，一起李劲松教师组也有许多后续的作业。但我信任更多试验室树立不了这个体系，技能要求太高，所以很难像 CRISPR 那样广泛运用。不过李劲松教师树立的孤雄单倍体平台会促进该技能的进一步使用。博后期间：Publication list 27, 28: 初次报导运用 CRISPR 技能可以制作各种基因修饰小鼠，两篇文章都有数千次引用，也有无数个试验室重复和使用。当然技能还在不断优化和前进，我树立试验室后也宣布过相应的文章，见 Publication list14, 20.独立 PI 期间：Publicationlist 18: 初次报导运用 CRISPR 可以敲除全体染色体。同期其他试验室也有报导，得到验证。Publication list 7, 11：初次报导单碱基修改技能的脱靶安全性问题，这两个作业都是 back to back 宣布，相互很好的印证了互相的定论。一起咱们经过生物学改造取得高真单碱基修改东西的文章也现已在 Nature Methods 接纳，同期 Nature Biotechnology 的 David Liu 的文章的成果也跟咱们有很好的印证。值得一提的是，咱们在 Science 文章中树立的高敏感检测脱靶的 GOTI 技能，技能要求很高，只要少量试验室具备这个试验条件，但这些并不影响咱们试验数据和定论的可靠性。Publication list 1, 15：运用各种基因修改技能做胶质细胞向神经元转分化研究，并且在最近的 Cell 文章中运用该办法医治帕金森及 RGC loss 的小鼠模型。这篇文章两种疾病的医治作用皆是该范畴最好的，必然引起很大重视和争议，但咱们信任在不久之后，许多试验室都能在自己各自的体系中得到验证。最终说说我对最近一些作业的全体观点：我个人关于外界的评论和质疑其实并不重视或看重，只要不是杂志修改部或许同行邮件的问询质疑，都尽量置之不理。我十分爱惜现在中国杰出的科研环境，所以分外爱惜每一点时刻。只需一心在科研上面，不断有好的作业展现给同行，就足够了。也很敬佩像蒲教师、王晓东教师、施一公教师那样，呵护着咱们这群青年 PI，不用花费过多时刻申请经费，有很好的 core facility，在文章思路或写作上给予咱们一定的指导。一切这些让咱们有更多时刻聚集于科学自身。此外，“最近一堆年轻人以为杨辉在 MIT 的文章涉嫌造假”。我信任绝大多数不是这个范畴的专家，我以为正确的办法是写信给 Cell 的 editor，质疑这篇文章的真实性、可靠性，而不是靠公众的言论来挑取自己想要接纳的信息。他们好像不在乎质疑咱们作业的文章的数据可靠性，也更承受 10 个月（其实不到半年时刻）发两篇 Cell 只要造假才有的可能。然后近一年又把 CNS 都发了一遍，造假就实锤了。我觉得这不是一个好的风气，好像许多人承受不了同代人比自己出色太多。张锋一向是我追敢的方针，从 PhD 开始就一向很出色，很快的 fellow 经历，刚独立就宣布了最重要的作业，后续更是一系列的重要文章，也树立了自己的公司，将 CRISPR 技能榜初次使用于人医治失明。而我，PhD 和 Postdoc 相对都很顺，但是回国独立头三年没有宣布一篇文章，直到第 5 年才连续有重要作业呈现。也开始向临床跨进，期望两年内可以国内上临床，终究谋福国内的患者。尽管时刻有点落后张锋了，但我仍然深信有赶上和超过他的一天。所以期望““最近一堆年轻人”能找到自己追赶的方针，再为之努力，而不是传一些不在自己学术水平范围内可以 judgement 的作业。学术圈毕竟不是娱乐圈。杨辉以下为Nature Methods 5 月 17 日对杨辉的报导英文原文：Hui YangA better base editor with fewer off-target changes, from a die-hard Manchester United fan.When he was in high school in southern China, a teacher told him,“The 21st century is the century of life sciences,”says Hui Yang, a researcher at the Institute of Neuroscience at Shanghai Institutes for Biological Sciences, which is part of the Chinese Academy of Sciences. It’s why he chose to do his PhD research in biology. Yang studied at Shanghai Jiao Tong University and then completed his PhD research at the Shanghai Institute of Biochemistry and Cell Biology, focusing on developmental biology. He learned about gene editing while working on a project to generate androgenetic haploid stem cells, but he found the traditional strategies they used to be inefficient.During a postdoctoral fellowship with Rudolf Jaenisch at the Whitehead Institute, Yang learned about CRISPR-based editing, and his first research project involved stem cells and reprogramming. It was nothing he had previously encountered in his textbooks, he says.“But I quickly fell in love with this.”With another postdoc, Haoyi Wang, he worked on generating gene-modified mice and found CRISPR powerful and easier to use than other approaches. In 2014 Yang was recruited back to China as part of the Youth Thousand Talents Program and started a lab. He shifted from creating genetically modified animal models to somatic editing focused on gene editing to one day treat human diseases.Yang“is a talented young scientist fascinated by new technology,”says Mu-ming Poo, who directs the Institute of Neuroscience.“He has the audacity to pursue wild ideas, many of which began with a few sparks in his mind.”Yang fearlessly dives into the highly competitive field of gene editing, says Poo, in line with a Chinese saying:“a newborn calf is not afraid of the tiger.”Poo met Yang when the latter was a PhD student and they worked on a project involving gene manipulation in monkeys. After Yang returned from his postdoctoral fellowship, he joined the institute, where he enjoys Poo’s mentorship.“My hobby is just learning new things and seeking new challenges,”says Yang.In his latest work, Yang and his team present base editor variants with improved fidelity and efficiency. He believes they have potential for clinical use, such as in human stem cells and ex vivo applications. But, for these and other clinical applications,“we need more accuracy,”he says. CRISPR–Cas-based editing usually involves double-stranded breaks. In base editing, which generally uses the rat cytidine deaminase APOBEC1 fused to the Cas9 nickase, only single stranded breaks are made. The result at the targeted site is conversion of cytosine to thymine. Yang and his colleagues have tuned base editors: they engineered the deaminase to achieve better expression and nuclear localization, says Yang. Their three base-editing variants generate fewer off-target changes and unwanted insertions and deletions than other base editors.And they have a narrower base-editing window.“We do not want to correct one mutation and induce another mutation,”he says. But when cytosines are near the targeted base, bystander mutations can lead to unwanted on-target effects.Using structure as a guide, the team focused on engineering the DNA-binding domain of the deaminase. The deaminase has its own DNA-binding domain, but it’s not needed; the Cas9 DNA-binding domain can be used instead, says Yang. They looked into which amino acids matter most, chose those, and screened for variants that affect DNA binding but that do not negatively affect the other desired traits: on-target efficiency and low off-target rates. Because the DNA-binding domain clusters with the RNA-binding domain, he believes the variants can be used for DNA and RNA editing.“Some amino acids are key for both,”he says. Some variants had higher off-target rates for RNA editing and others for DNA editing, but the researchers managed to find some with good efficiency and low off-target levels for both RNA and DNA editing.For in vivo editing, he prefers RNA editing. The appeal is the smaller size of these editors and their higher efficiency. To him, RNA editing appears safer than DNA editing, although some concerns remain about non-specific cutting.“But I believe this could also be resolved,”he says.For drug screening in which a broad editing window is acceptable, CRISPR is likely the right choice, says Yang. Base editing’s clinical promise is connected to the fact that many diseases are caused by point mutations, he says. He and a former student have founded a company called Hui-Gene Therapeutics to explore gene editing and human disease. Much work remains to be done, especially related to safety, he says.“My hobby is just learning new things and seeking new challenges.”Yang often gets ideas for the lab through interaction on social media, usually WeChat, but he prefers face-to-face discussion, he says. In the Jaenisch lab, he liked how valued independent thinking and interaction were and has styled his lab in that vein. Lab members can knock on his door anytime, he says. He helps students and postdocs and encourages more experienced lab members to guide others in designing and doing experiments. In lab meetings,“we just share ideas.”One of his students is in a joint program with a Danish university, and he wants to attract others from outside China to the lab. Once a week Yang plays basketball with friends, but he adores soccer. Since high school he’s been a die-hard Manchester United fan.
