Lecture 13: CRISPR
Segment 1: Introduction to CRISPR
Segment 2: Review of Homologous Recombination
Segment 3: 真核生物中的同源重組Homologous Recombination in Eukaryotes
Segment 4: Cell Cycle Terms
概覽一些生物學(xué)中重要的術(shù)語(yǔ)和概念
Segment 5: Cell Cycle
Segment 6: 減數(shù)分裂重組Meiotic Recombination, Part 1
宏觀上將減數(shù)分裂導(dǎo)致的DNA交換
Segment 7: 減數(shù)分裂重組Meiotic Recombination, Part 2
從微觀上講
Segment 8: Genome Editing and CRISPR, Part 1
CRISPR：Clustered Regularly Interspaced Short Palindromic Repeats
class I 需要多個(gè)蛋白握侧；而class II只需要1個(gè)蛋白蚯瞧，比如下圖中的cas9 蛋白，一個(gè)就能定位靶基因品擎。而且cas gene恰好跟repeat region相鄰埋合，只要找到了repeats,就能找到設(shè)計(jì)的蛋白
Segment 9: Genome Editing and CRISPR, Part 2
為什么不會(huì)定向到細(xì)菌自己的基因呢？有兩個(gè)解答萄传，現(xiàn)僅以一個(gè)為例：
如下圖所示盲再，細(xì)菌轉(zhuǎn)錄本包含一段spacer（黃色）和一段repeat（灰色）西设，左邊有tracrRNA以反式激活CRISPR RNA，若tracrRNA不表達(dá)答朋，則系統(tǒng)不工作贷揽。Cas9 蛋白實(shí)現(xiàn)剪切，Cas1和Cas2蛋白從噬菌體中獲得新的序列梦碗，PAM是2-3個(gè)核苷酸長(zhǎng)度的motif禽绪。因?yàn)榧?xì)菌中沒有PAM，所以不會(huì)被CRISPR/Cas9系統(tǒng)所剪切
Segment 10: Genome Editing and CRISPR, Part 3
下圖定位于target genome中洪规，PAM是NGG印屁，此圖中的N為C，黃色的序列是上圖中的protospacer斩例，又稱作guide RNA雄人，而灰色的是repeat序列，又稱作linker念赶。藍(lán)色的是tracrRNA础钠。所以可以通過改變guide RNA 來定向識(shí)別想要編輯的基因。箭頭所指向的是Nuclease Sites核酸酶位點(diǎn)叉谜。
You wish to use Cas9 to activate expression of a gene of interest. How should you modify your CRISPR gene?
- mutate the gene encoding Cas9 to eliminate the nuclease activity核酸酶活性很钓，這樣就不會(huì)切割目的基因了香府。
- Express Cas9 as a fusion protein融合蛋白 with a transcriptional activator轉(zhuǎn)錄激活因子。這樣可以將轉(zhuǎn)錄激活因子帶到想要表達(dá)的基因處码倦，以激活該基因企孩。
Additional CRISPR Video Resources from Our Colleagues
What is CRISPR? (2014)
Feng Zhang Explains CRISPR
Feng Zhang Lecture
CRISPR Applications to Human Health: SHERLOCK
一些文章
Dynamics and Impact of Homologous Recombination on the Evolution of Legionella pneumophila
CRISPR/Cas9 Targeting Events Cause Complex Deletions and Insertions at 17 sites in the Mouse Genome
CRISPR/Cas9-mediated Mutation of Tyrosinase (Tyr) 3′ UTR Induce Graying in Rabbit
CRISPR/Cas9-APEX-Mediated Proximity Labeling Enables Discovery of Proteins Associated with a Predefined Genomic Locus in Living Cells
