Zhilong Jia@plaghHospital 09:37 PM
ABEmax是base editing的？怎么筛选靶向hbf的区域用于ABEmax实验？成人hbf高有其他副作用吗？谢谢。
ycheng 10:08 PM
1）是的。2） 通常要基于基因方面的知识。3）在红细胞中应该没有。在一些血癌中， 高hbf 是一个高风险指标
Great talk, I am wondering why CBE have higher efficiency compared with ABE? Thanks.
ycheng 10:09 PM
First, in our cell system, ABE activities are higher. However, I am not sure about the exact mechanism. But we know that CBE has higher off-target RNA editing activites.
Anonymous Attendee 09:57 PM
老师您好，二级结构这么重要的话，guide是不是就不是越长越好？因为越长越容易产生二级结构。是不是尽量用23个nt就好呢。
This question has been answered live
Anonymous Attendee 09:58 PM
这个活性不是只有Cas13a才有吗 Cas13b和d好像是没有这样的活性。
Wei Li 10:12 PM
我们在Cas13a,b,d 观测到，但是13a的intrinsic target effect是最强的. Cas13d最弱，但是也有。
Dongdong Wang 10:00 PM
Hi Wei, Just a small question. according to your survival probability, it seems the target is not so promising, which is probably because of low number of patients. Just wondering which database did you use? probably you could use TCGA database.
Wei Li 10:13 PM
我们用了TANRIC数据库已经处理过的TCGA data.
Houjian Cai 10:04 PM
It has been reported that RNA has 3D structure. Could that influence your prediction.?
Wei Li 10:16 PM
We only look at gRNA structure which is short and easy to predict 2nd structure (<40bp). But the 3D structure of target RNA may affect the results, which we haven’t looked into (but that’s a great suggestion)!
Suxuan Wang 10:06 PM
想请教一下李老师关于发现Cas13的limitation中，有没有哪些改进方法（e.g. directed evolution) 来减少这些limitation呢？对这方面的改进请问有什么见解么？
Wei Li 10:17 PM
有的。我们bioRxiv ms有做domain mutation. 其他的优化也可以做(应该其他组已经有做过类似的工作).
Wenhui Hu 10:06 PM
Dr. Wei LI, Great talk. For your lentivirus disruption, does it apply to HIV native virus?
Wei Li 10:17 PM
I guess so. There are works to use Cas13 to detect HIV virus
Xiaojun Lance Lian 10:27 PM
Hi Wei Li, in your DeepCas13 website, does gRNA with a high Deep score have better knockdown efficiency?
Wei Li 10:28 PM
Yes, that’s right
Houjian Cai 10:32 PM
Han, interesting talk. The editing specificty is correlated with the concentration of CRISPR machinery and time staying intracellularly. Am I right?
Han Xu 10:54 PM
Thank you. Yes, you are right. At the same time, there is a balance between efficiency and specificity. The important thing is that whether or not we can find a window in which both efficiency and specificity are satisfied.
From Kin Fai Au to Everyone 08:57 PM
这个报告已经是第三个liwei了 哈哈哈
From Me to Everyone 09:13 PM
To date, almost 2600 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide.
From Wei Li to Everyone 09:19 PM
大家如果有问题的话可以通过Q&A提出来。
Kaifu Chen to Me, All Panelists 09:29 PM
👍👍👍
From Xiaojun Lance Lian to All Panelists 10:03 PM
Are you saying that Cas13d, even without crRNA, can degrade some RNAs in cells ?
From Kaifu Chen to Everyone 10:04 PM
有没有一些RNA被editing后变得更加稳定的？ 另外一个问题是，细胞里面不同RNA原本的stability也不一样，这回不回导致原本不stable的RNA更容易看到效果？
From Xiaoyu Yang to All Panelists 10:05 PM
请问老师如何看这篇文章：https://www.biorxiv.org/content/10.1101/2021.09.14.460134v1
From Me to All Panelists 10:12 PM
Genomics England recently released thousands of germline mutation causing rare diseases. I am wondering is there any systemic way to validate functions for these mutation in a large scale with CRISPR?
From Kaifu Chen to Everyone 10:14 PM
欢迎大家raise hand进入panelist，方便直接语音或视频提问、讨论
Wei Li to Xiaoyu Yang, All Panelists 10:24 PM
这个是我们的competitor. biorxiv文章我们两组差不多同时放出来🙂
From sunx to Everyone 10:33 PM
老师，您讲的荧光报告系统，WT reporter， V600E reporter具体原理是什么？
From Wenhui Hu to Everyone 11:27 PM
Wonderful talk. Do you have best ways for in vivo delivery?
From Lu to Everyone 11:27 PM
请问环形不影响线性的RNA，是已经发文章了吗，请问是哪一篇？我想去学习一下。
那么lncRNA呢？snoRNA也是吗？snoRNA的载体 滞留在核仁内，是否可能核移位到核质？
snoRNA载体设计的时候，启动子是否考虑基因序列中连着4个T用哪种类型的启动子，我想请问一下，因为我们找公司设计的回复是：
咱们组之前做过表达snorna用过gv235和gv342这两个载体，唯一区别是启动子不同，前者是hU6-MCS-CMV-Puromycin ，后者是Ubi-MCS-SV40-puromycin。
在核内他充当了管家基因的角色？
这是之前的普遍认知，认为它稳定存在，功能固定。
From Suxuan Wang to All Panelists 11:27 PM
刚刚的群二维码人数超过200了，现在没有里面的人邀请好像加入不进去，请问有群小助手可以加来入群么？
From shunzhang to Everyone 11:28 PM
杨力老师您好 想请问在pegRNA上增加同义突变的数量为什么会增加效率 是降低gRNA在靶位点的结合难度吗 那这样的同义突变引入的数量和效率的提高上怎么样达到一个很好的平衡？
Computational Biology in Gene Editing, 06/25/2022
Zhilong Jia@plaghHospital 09:37 PM
ABEmax是base editing的？怎么筛选靶向hbf的区域用于ABEmax实验？成人hbf高有其他副作用吗？谢谢。 ycheng 10:08 PM 1）是的。2） 通常要基于基因方面的知识。3）在红细胞中应该没有。在一些血癌中， 高hbf 是一个高风险指标 Great talk, I am wondering why CBE have higher efficiency compared with ABE? Thanks. ycheng 10:09 PM First, in our cell system, ABE activities are higher. However, I am not sure about the exact mechanism. But we know that CBE has higher off-target RNA editing activites. Anonymous Attendee 09:57 PM
老师您好，二级结构这么重要的话，guide是不是就不是越长越好？因为越长越容易产生二级结构。是不是尽量用23个nt就好呢。 This question has been answered live Anonymous Attendee 09:58 PM
这个活性不是只有Cas13a才有吗 Cas13b和d好像是没有这样的活性。 Wei Li 10:12 PM 我们在Cas13a,b,d 观测到，但是13a的intrinsic target effect是最强的. Cas13d最弱，但是也有。 Dongdong Wang 10:00 PM
Hi Wei, Just a small question. according to your survival probability, it seems the target is not so promising, which is probably because of low number of patients. Just wondering which database did you use? probably you could use TCGA database. Wei Li 10:13 PM 我们用了TANRIC数据库已经处理过的TCGA data. Houjian Cai 10:04 PM
It has been reported that RNA has 3D structure. Could that influence your prediction.? Wei Li 10:16 PM We only look at gRNA structure which is short and easy to predict 2nd structure (<40bp). But the 3D structure of target RNA may affect the results, which we haven’t looked into (but that’s a great suggestion)! Suxuan Wang 10:06 PM
想请教一下李老师关于发现Cas13的limitation中，有没有哪些改进方法（e.g. directed evolution) 来减少这些limitation呢？对这方面的改进请问有什么见解么？ Wei Li 10:17 PM 有的。我们bioRxiv ms有做domain mutation. 其他的优化也可以做(应该其他组已经有做过类似的工作). Wenhui Hu 10:06 PM
Dr. Wei LI, Great talk. For your lentivirus disruption, does it apply to HIV native virus? Wei Li 10:17 PM I guess so. There are works to use Cas13 to detect HIV virus Xiaojun Lance Lian 10:27 PM
Hi Wei Li, in your DeepCas13 website, does gRNA with a high Deep score have better knockdown efficiency? Wei Li 10:28 PM Yes, that’s right Houjian Cai 10:32 PM
Han, interesting talk. The editing specificty is correlated with the concentration of CRISPR machinery and time staying intracellularly. Am I right? Han Xu 10:54 PM Thank you. Yes, you are right. At the same time, there is a balance between efficiency and specificity. The important thing is that whether or not we can find a window in which both efficiency and specificity are satisfied.
From Kin Fai Au to Everyone 08:57 PM 这个报告已经是第三个liwei了 哈哈哈 From Me to Everyone 09:13 PM To date, almost 2600 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. From Wei Li to Everyone 09:19 PM 大家如果有问题的话可以通过Q&A提出来。 Kaifu Chen to Me, All Panelists 09:29 PM 👍👍👍 From Xiaojun Lance Lian to All Panelists 10:03 PM Are you saying that Cas13d, even without crRNA, can degrade some RNAs in cells ? From Kaifu Chen to Everyone 10:04 PM 有没有一些RNA被editing后变得更加稳定的？ 另外一个问题是，细胞里面不同RNA原本的stability也不一样，这回不回导致原本不stable的RNA更容易看到效果？ From Xiaoyu Yang to All Panelists 10:05 PM 请问老师如何看这篇文章：https://www.biorxiv.org/content/10.1101/2021.09.14.460134v1 From Me to All Panelists 10:12 PM Genomics England recently released thousands of germline mutation causing rare diseases. I am wondering is there any systemic way to validate functions for these mutation in a large scale with CRISPR? From Kaifu Chen to Everyone 10:14 PM 欢迎大家raise hand进入panelist，方便直接语音或视频提问、讨论 Wei Li to Xiaoyu Yang, All Panelists 10:24 PM 这个是我们的competitor. biorxiv文章我们两组差不多同时放出来🙂 From sunx to Everyone 10:33 PM 老师，您讲的荧光报告系统，WT reporter， V600E reporter具体原理是什么？ From Wenhui Hu to Everyone 11:27 PM Wonderful talk. Do you have best ways for in vivo delivery? From Lu to Everyone 11:27 PM 请问环形不影响线性的RNA，是已经发文章了吗，请问是哪一篇？我想去学习一下。 那么lncRNA呢？snoRNA也是吗？snoRNA的载体 滞留在核仁内，是否可能核移位到核质？ snoRNA载体设计的时候，启动子是否考虑基因序列中连着4个T用哪种类型的启动子，我想请问一下，因为我们找公司设计的回复是： 咱们组之前做过表达snorna用过gv235和gv342这两个载体，唯一区别是启动子不同，前者是hU6-MCS-CMV-Puromycin ，后者是Ubi-MCS-SV40-puromycin。
因为序列中有连续4个t时，无法使用3型启动子即u6，需要更换为2型启动子ubi，故为保持一致，本方案中的6个序列技术都推荐的是gv342载体。
引物设计方面：比如我看陈玲玲老师发的一篇文章中SNORD116-14序列探针，上游引物有四十多个，是不是试了很多次之后发现这么加才能扩增出来，二十几个也扩不出来？ 如果用snovector载体(2014),确实是把sno定位在核内了， 这里你表达的不准确，不是说定在这了，是指为了避免外源载体表达后运送到胞浆了（改变内源性表达原本就在核内，而非在胞浆的原初定位。）所以这么设计的 这一点是为了保障至少在构建体系的时候，与真实生物发生过程是一致的。
但是未知的是：这是否影响snoRNA成功在细胞系中表达后的长期定位？ 这个问题可以拆解为： 1.这个载体到底如何定位到和内源性表达一样的核内的？ 2.怎么维持这种效果？ 3.真实生命中的snoRNA如果出核，是怎么出核的?依靠什么出核？ 4.本身的结构与核内是否有，有什么区别？
但是做不出功能， （首先我们并不知道它的功能是什么，推测与ATM相关，因此至少会对DNA损伤有所响应吧）
是不是可以说明这个sno发挥功能是通过出盒发挥功能？
（不能通过在核内"做不出功能"就说通过出核发挥功能，这需要看到它有功能了才去分析它是在哪里发挥的功能。）
在核内他充当了管家基因的角色？ 这是之前的普遍认知，认为它稳定存在，功能固定。 From Suxuan Wang to All Panelists 11:27 PM 刚刚的群二维码人数超过200了，现在没有里面的人邀请好像加入不进去，请问有群小助手可以加来入群么？ From shunzhang to Everyone 11:28 PM 杨力老师您好 想请问在pegRNA上增加同义突变的数量为什么会增加效率 是降低gRNA在靶位点的结合难度吗 那这样的同义突变引入的数量和效率的提高上怎么样达到一个很好的平衡？