dTAG蛋白降解系统
非常火的一个由DFCI开发的药物系统，原理就是蛋白降解，在实验室里就在目标蛋白后面加一个尾巴，它会连接到泛素化降解系统，从而降解目标蛋白。
dragging a protein of interest (POI) to a E3 ligase for degradation like dTAGv1，fusing it to the FKBP domain
优点：
- 几乎可以作用于任何蛋白，很多蛋白没有小分子抑制剂从而无法研究；
- 非常灵敏，两小时即可发挥作用；
- 剂量效应，可以通过剂量来控制蛋白被降解的程度；
- 短期内可恢复，去掉药物即可恢复；
dTAG系统构建步骤：
- CRISPR敲除stop codon；
- 直接订购gRNA，注意strand，一定是直接带NGG的那条链，注意RNA的顺序，是5‘到3’；
可能会用到的小工具：
- https://www.genscript.com/sms2/rev_comp.html
- https://chopchop.cbu.uib.no/
- https://genome.ucsc.edu/index.html
- https://portals.broadinstitute.org/gppx/crispick/public
参考文献：
- Generation of locus-specific degradable tag knock-ins in mouse and human cell lines - PROTOCOL | JUNE 3, 2021
- A protocol for rapid degradation of endogenous transcription factors in mammalian cells and identification of direct regulatory targets - Volume 2, Issue 2, 18 June 2021, 100530
待续~
gRNA设计工具：
- https://portals.broadinstitute.org/gppx/crispick/public
- https://genome.ucsc.edu/index.html
- https://chopchop.cbu.uib.no/
Where are double strand breaks (DSBs) induced, compared to where the target site sequence (protospacer+PAM) is located? If there are other PAMs in the region, will they also be targeted?
The Cas9 cuts 3-4bp upstream of the PAM sequence. There can be some off-target DSBs using wildtype Cas9. The degree of off-target effects depends on a number of factors, including: how closely homologous the off-target sites are compared to the on-target site, the specific site sequence, and the concentration of Cas9 and guide RNA (gRNA). These considerations only matter if the PAM sequence is immediately adjacent to the nearly-homologous target sites. The mere presence of additional PAM sequences should not be sufficient to generate off-target DSBs; there needs to be extensive homology of the protospacer followed by PAM.
NON-HOMOLOGOUS END JOINING (NHEJ)
Non-homologous end-joining (NHEJ) is the best known and probably the most common repair mechanism that can make changes (mutations) in the DNA [3]. The NHEJ pathway joins DSB ends without the need for a homologous template. During the processes of ligating the cut ends of the DNA, NHEJ can often introduce small insertions or deletions (indels) at the break point. Frequently, an indel causes a frameshift mutation leading to a stop codon, which disrupts a gene’s expression and function—very useful to knock out a target gene.
