1. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair
Genome Research :142–153
Thomas O. Auer, Karine Duroure, Anne De Cian,
Jean-Paul Concordet and Filippo Del Bene
Impact factor :
這篇PAPER 主要是要利用CRISPR/Cas9 的方式 將DNA knock-in in 到zebrafish ,但這篇比較特別的是它所使用的是 homology-independent 的方式
Data:05/20/2015
Adviser: Han-Jia Lin
Speaker: Wei-Yu Yeh

2. Abstract
Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish.
Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR).
Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways.
After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA.
We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens.
In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles.
Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

3. Gene knock-in
plasmid
Involves the insertion of a protein coding DNA sequence at a particular locus in an organism's chromosome.
首先,什麼是Gene knock-in ? Gene knock-in就是一種將一段protein coding DNA sequence 嵌入生物體內染色體的特定位置
將一段外源基因嵌入生物體內基因的特定位置
那要做到knock in，必須將DNA序列斷開，造成DSB這個現象， 而能夠做到這個現象的方法有
Zinc fingers
TALEN
CRISPR/Cas9 --> 本篇使用的

4. How do knock-in in vivo ? Early stage Using1 2 3 2
maker 3
Early stage Using
HR (homologous recombination)
Transformation probability  Low
homologous
recombination 1 2
maker 3
那早期的knock-in ，也就是我們分生課本上常講到的，是用homologous recombination 來進行 ,可以透過有相似的序列來將目標基因換入
但homologous recombination 的效率很低，因為HR在生物體內的自然發生率很低
因此為了要增加效率，科學家發現了DSB 的產生有助於增加knock-in的效率 我們要用什麼方法來使DSB出現呢?
最初的KNOCK IN
轉型率低 生物體內自然發生率比較低
最近發現的方法 因為會讓他受傷 讓成功率提升
Recent studies have found DNA double strand breaks (DSB) can improve the efficiency of success

5. How to create DSB in vivo?
Zinc fingers
Zinc fingers
TALEN
CRISPR/Cas9
Guide
Artificial Protein
RNA
Effector
single strand cut
DSB
Experimential Design
Complicated
Simple
TALEN
CRISPR/Cas9
要在生物體內製造DSB,我們常用的方法有三種，分別是ZFG TALEN 和CRISPR， 首先Zinc fingers 和 TALEN 都是利用人工蛋白來設計序列，Zinc fingers 在這邊的設計上比較複雜
而TALEN 是由多達23個的胺基酸系列來設計一個DNA 序列，如果要設計很多DNA序列在應用上比較麻煩，而它們的FokI 酵素只能造成單股序列剪切，若要剪切雙股則需要在兩端都進行設計。 而
如何在生物體內造成 DSB?
ZFG TALEN 的方法
造成 DNA double strand breaks (DSB) 誘發生物體內的修復機制
Cause DNA double strand breaks (DSB) to induce Repair mechanisms in vivo

6. Genomic editing by CRISPR/Cas9 Systems
Streptococcus pyogenes
This system has two element
Cas9：Endonuclease (double strand break)
sgRNA：complementary target gene & activates Cas9 Science 337, 816 (2012)
這套系統最常被人應用的是這隻菌type ii的Cas9蛋白
那這套系統剛剛雖然講得很簡單，但其實他也有三個重要的元件。
cas9 蛋白，他是一種斷雙股的核酸內切酶
crRNA，告訴Cas9蛋白要切的位置
tracrRNA，活化cas9的活性
但是在2012年，這篇ＰＡＰＥＲ將crRNA和tracrRNA結合
形成sgRNA，並且仍保有定位以及活化cas的活性
從此這套系統只需要兩個元件又變得更好用。

7. DNA double strand breaks (DSB) repair
NHEJ (non-homologous end- joining)
Knock out
DNA cassettes DSB repair in cultured cells 10%
(Cristea et al. 2013; Maresca et al. 2013)
Highly active in early zebrafish development
(Hagmann et al. 1998; Dai et al. 2010; Liu et al. 2012)
gene knock-in 
homology-independent
HR 的修補機制在生物體內時 會利用一段同源序列來進行修補 所造成的失誤率較低
比較常用的方法
優點 :可以達到perfel rupear
缺點:
1.設計不容易 構築麻煩
2.在生物體內的自然發生率低-->機率低
有文獻(Zu et al. 2013)指出HR 在斑馬魚體內只有1.5%的成功率，代表成功率極低
那反過來想就是突變率極高的意思 高達98%
4.NHEJ
因此作者就想走另一條路 開始考慮NHEJ這種方式
NHEJ 這種修復機制並沒有另一股同源序列可以對照 因此在修復後常會產生INDEL的現象
而文獻(Cristea et al. 2013; Maresca et al. 2013)指出，有人在細胞內使用NHEJ的修補機制，但是效率?
那又有另一篇文獻指出 斑馬魚在早期發育的時候NHEJ活耀 因此會提高效率
所以本篇作者才想使用斑馬魚來嘗試進行NHEJ的knock in
Can drive targeted integration of DNA cassettes in cultured cells via homology independent DSB repair. (Cristea et al. 2013; Maresca et al. 2013)
highly active in early zebrafish development
(Hagmann et al. 1998; Dai et al. 2010; Liu et al. 2012)
HR (homologous recombination)
perfect repair
Design is not easy、Build trouble
in zebrafish 1.5%
(Zu et al. 2013)

8. NHEJ in knock-in application mode
transgenic line
plasmid
NHEJ or HR ?
Indel
Screening !
NHEJ in knock-in 的應用方式
怎麼用這個技術來達到Knock in
INDEL : Small fragments insertion or deletion of bases in the DNA of an organism
E2A  in-frame  Ribosomal skipping
KalTA4(GAL 4)  screening

9. Design plasmid: E2A linker for multicistronic expression
Equine rhinitis A virus (ERAV) 2A
genus Aphthovirus 
the N-terminal eGFP peptide is cleaved from the KalTA4 protein
linker for multicistronic expression
cotranslational ribosomal skipping.

10. Design plasmid: KalTA4 Gal4/UAS expression system
 GAL4 gene : encoding the yeast transcription activator protein GAL4
UAS (Upstream Activation Sequence) : enhancer to which GAL4 specifically binds to activate gene transcription
KalTA4
1.the result of altering the codon usage of Gal4TA4 ORF for efficient translation in zebrafish
2.adding the Vertebrate Kozak sequence GCCGCCACC at the 5' end.
the Kozak sequence :轉譯起始點
Prüßing et al. Molecular Neurodegeneration 2013 8:35   doi: /

11. GAP
1. How to use and proven    homology-independent way to achieve knock-in
2. homology-independent knock-in  Probability of success
3. homology-independent  Rate of germline transmission
如何使用和驗證 同源性無關的方式來實現敲入
成功概率
生殖率的傳輸

12. Results

13. Efficiency Test of sgRNA eGFP
sgRNA + Cas9 mRNA
Indel mutation efficiency
sgRNA eGFP1 Cas9  66.6%
sgRNA kif5aa Cas9  22.2%

14. CRISPR/Cas9-mediated knock-in of KalTA4 into the Tg (neurod:eGFP) transgenic line
Tg (neurod:eGFP) x Tg(UAS:RFP,Cry1:eGFP) transgenic line
+Cas9
Indel
+sgRNA GFP1
NHEJ or HR ?
No Indel  eGFP
Indel  No or low eGFP  RFP
Cas9 + sgRNA eGFP 1 切開 Tg(neurod:eGFP)斑馬魚體內 eGFP 和 質體上 的sgRNA eGFP 1 序列，使魚體內產生NHEJ的修復方式來將質體接進體內
Indel  no eGFP Screening
之後再對篩選到的個體進行PCR測定及定序
0.因為
1.想知道所設計的這段sgRNA eGFP 的效率以及會產生什麼樣的indel 2.
E2A peptide
linker for multicistronic expression
cotranslational ribosomal skipping
elf locus 為綠螢光表現基因
The eGFP transgene allows the direct visualization of target gene disruption and should not compromise survival upon loss of gene function.
NHEJ被證明是在早期斑馬魚發育高活性（海格曼等人1998; Dai等人，2010年; Liu等人2012）
Cas9 + sgRNA eGFP1  Cut Target sequence on Tg (neurod: eGFP) and Plasmid
NHEJ repair in Fish body  Sequence connection
Screening 
1.No or low eGFP Fluorescence (Indel )  RFP Fluorescence
2.PCR assays and sequencing

15. CRISPR/Cas9-mediated knock-in of KalTA4 into the Tg (neurod:eGFP) transgenic line
Transgenic Fish x Tool fish
Successful mating : GFP signal at brain, retinal and crystalline
Knock-in false (no sgRNA): No RFP signal
Knock-in (with sgRNA): RFP signal at brain and retinal
Knock-in strain
KalTA4 functional transcription factors  open the downstream gene UAS
Brain and Retinal
Whole tissue
Knock-in (With sgRNA)
Neurod:eGFP
UAS:RFP,Cry1:eGFP
Crystallin
1.為了瞭解knock-in 的基因有沒有接到正確位置，能夠正確的使naurod開啟表現下游的eGFP蛋白
2.利用microinject 將質體+sgRNA+cas9 送入斑馬魚胚胎裡
Tg(neurod:eGFP) :neurod promoter調控的eGFP螢光的表現，Neurod 表現在 Central Nervous System (Brain and Retinal)
Tg(UAS:RFP,cry1:eGFP):UAS promoter調控RFP螢光的表現,cry1 調控eGFP 的螢光表現。UAS存在於全身，表現在被kalTA4調控區域，Cry1 表現在 Crystallin。
圖b因為 In-frame fusion of the E2A-KalTA4-pA cassette results in a multicistronic mRNA after successful integration at the eGFP locus ，使得KalTA4 可表現產生調控因子，使UAS開啟調控下游RFP，產生紅色螢光
Brain
Retinal
Control (No sgRNA)

16. Screening and sequencing of knock-in Tg (neurod:eGFP) x Tg(UAS:RFP,Cry1:eGFP) fish
Fluorescent confirmation  no eGFP Fluorescence
PCR results 
Sequence directional
Sequencing results
Sequence inside Indelin-frame insertions?
PCR檢測
Part D is a representative gel of PCR products obtained from the founder fish shown in B, demonstrating targeted knock-in of the donor plasmid at the eGFP locus.
有加入 sgRNA eGFP1 並且成功knock in 到正確位子，經過PCR 檢測就可以看到band.
Fragment A 為控制組。
(E) Sequence analysis at the 5' and 3' junctions of five representative targeted integration events.
sgRNA Mutation or insertions. the PAM seq
RFP expression  three possible frames and two integration directions 1/3 * 1/2 = 1/6 (16.6%)

17. Comparison of circular and linearized donor plasmid injection
Retinal
Brain
linearized
(dorsal view)
circular
RFP expression
circular plasmid 76%
linearized plasmid 10%

18. Targeted knock-in of KalTA4 into the Tg(vsx2:eGFP) transgenic line
retina progenitor cells and hindbrain region
Whole tissue
Knock-in
(With sgRNA)
Crystallin
retina progenitor cells
hindbrain
region
Vsx2:eGFP
UAS:RFP,Cry1:eGFP
Sequencing results
Transgenic Fish x Tool fish
Successful mating : GFP signal at retina progenitor cells 、 hindbrain region and crystalline
Knock-in (with sgRNA): RFP signal at retina progenitor cells and hindbrain region
做一次不准 要做兩次
Vsx2
Visual system homeobox (retina progenitor cells and the hindbrain region)

19. Knock-in efficiencies at the eGFP and the kif5aa locus
DNA靶載體的簡單
提供給BAC轉基因更容易的選擇。
序列是小尺寸的，它們可以很容易地通過PCR或寡克隆產生，並且供體和目標部位之間沒有長同源拉伸是必需的。
Embryos with RFP expression : neurod:eGFP 75.6% > vsx2:eGFP 57.6%
neurod:eGFP  High efficiency
Broad RFP  pattern(>5% of retinal cells)

20. CRISPR/Cas-mediated knock-in of KalTA4 into the kif5aa locus.
Tg(UAS:RFP,Cry1:eGFP)transgenic line
+Cas9
+sgRNA kif5aa1
+sgRNA kif5aa1
NHEJ or HR ?
Indel
kif5aa-E2A-KalTA4-pA plasmid
Kif5aa (kinesin family member 5Aa)
endogenous loci
brain and spinal
This gene encodes a member of the kinesin family of proteins.
Kif5aa (kinesin family member 5Aa)  endogenous loci

21. CRISPR/Cas-mediated knock-in of KalTA4 into the kif5aa locus.
Tg(UAS:RFP,Cry1:eGFP)
In situ hybridization  confirmation the location of gene expression
Transgenic Fish x Tool fish
Successful mating : in situ hybridization signal at Spinal cord、cerebellum、 hindbrain
Knock-in (with sgRNA): RFP signal at Spinal cord、cerebellum、 hindbrain and motoneuron
(sc) Spinal cord, (cb) cerebellum, (hb) hindbrain, (mn) motoneuron
Sequencing results
Sequence inside Indelin-frame insertions?
（SC）脊髓，（CB）小腦，（HB）後腦，（MN）運動神經元

22. Knock-in efficiencies at the eGFP and the kif5aa locus
DNA靶載體的簡單
提供給BAC轉基因更容易的選擇。
序列是小尺寸的，它們可以很容易地通過PCR或寡克隆產生，並且供體和目標部位之間沒有長同源拉伸是必需的。
Embryos with RFP expression : neurod:eGFP 75.6% > vsx2:eGFP 57.6% > sgRNA kif5aa1 4%
kif5aa  Chances of success so low
Broad RFP  pattern(>5% of retinal cells)

23. Combination of multiple sgRNAs to increase knock-in efficiency
Tg(UAS:RFP,Cry1:eGFP)
transgenic line
+Cas9
+sgRNA GFP1
+sgRNA kif5aa1
sgRNA kif5aa1
NHEJ or HR ?
Indel
sgRNA eGFP 1 guides Cas9 nuclease activity to cut the donor plasmid in the eGFPbait sequence
sgRNA kif5aa 1 is used to target the endogenous target locus.
Different sgRNA bait 
Verification  Target gene freedom of choice and success rate

24. Combination of multiple sgRNAs to increase knock-in efficiency
Transgenic Fish x Tool fish
Knock-in (with sgRNA): RFP signal at Diencephalon、cerebellum、optic tectum、hindbrain、 midbrain and spinal cord.
(dc) Diencephalon, (cb) cerebellum, (ot) optic tectum, (hb) hindbrain, (mb) midbrain, (sc) spinal cord.
Note that, in this case, due to the frame difference between the kif5aa and eGFP genes, only +2 or -1 indels will produce functional fusion protein.
（DC）間腦，（CB）小腦，（OT）視頂蓋，（HB）後腦，（MB）腦，（SC）脊髓。

25. Knock-in efficiencies at the eGFP and the kif5aa locus
neurod:eGFP High(75.6%) plasmid + sgRNA kif5aa1 Low(4%) Chromosome = 9.6%
 Unexpected discovery

26. Rate of germline transmission of KalTA4 knock-in into the eGFP locus  Tg(neurod:eGFP)
Screening
NO Screening
Screening
Germline  Determination can continue

27. Germline  Determination can continue
Rate of germline transmission of KalTA4 knock-in into the eGFP locus  Tg(vsx2:eGFP)
Screening
retina progenitor cells
Kif5aa (kinesin family member 5Aa)
endogenous loci
brain and spinal
This gene encodes a member of the kinesin family of proteins
Germline  Determination can continue

28. Germline transmission of in-­frame transgene knock-­in in 5 founder fish for the neurod:Egfp locus
Tg(neurod:eGFP)
Tg(neurod:eGFP) X
(UAS:RFP , Cry1:eGFP)
Tg(neurod:eGFP) : 1.2% ~ 34.2% 、 Tg(neurod:eGFP) X (UAS:RFP , Cry1:eGFP) : 6.2%~ 16.9%
1. Variety preservation
2. Non-chimera

29. Germline transmission of knocked-in transgenes
Analysis of the stable founder C for site-specific transgene integration by Southern blot analysis.
The neurod locus-specific probe 1 detects a 2.7-kb fragment after HindIII digest in the wild-type allele.
The transgenic BAC neurod:eGFP locus is digested into a 2.6-kb fragment and, in the case of a partial digest in the BAC backbone, into a 4.4-kb fragment.
After insertion of the KalTA4 cassette, a 6.6-kb fragment is detected.
2.7K
2.6K
Southern blot analysis  locus-specific
Determining plasmid sequences inserted correctly

30. Conclusions

31. Conclusions Performing a knock-in HR  TALEN
showed germline transmission in zebrafish at rates of 1.5%, (98% indel rates). Zu et al. (2013)
NHEJ  eGFP sgRNA + Cas9
germline transmission rates for the neurod:eGFP locus up to 31%. (66% indel rates)
in-frame integrations into account, with 10.3%
The rate of functional targeting of the locus was still higher.
執行敲入
HR，祖衝之等人。 （2013年）顯示，在1.5％的速率種系傳遞在斑馬魚中，採用高效TALEN對（高達98％插入缺失率）。
因組綠色熒光蛋白1 / Cas9，有66％的插入缺失突變率。
生殖傳輸速率為NEUROD：綠色熒光蛋白基因高達31％。
即使只是走在框架集成進去，有10.3％
軌跡的功能定位率仍然較高。

32. Advantage of CRISPR/Cas9 knock-in in homology-independent
knock-in >5.7-kb-long DNA cassettes
Site specific knock-in
eGFPbait-E2A-KalTA4 construct easy to used any eGFP into a KalTA4 transgenic line
Allows the simultaneous targeting of several sequences and may also be used for gene replacement.
通過同源性無關的CRISPR/ Cas9介導敲入在斑馬魚
敲入>5.7 kb的長DNA盒
網站具體敲入
eGFPbait-E2A-KalTA4構建易於使用的任何綠色熒光蛋白成KalTA4轉基因株系
允許多個序列的同時靶向，並且也可以用於基因置換。

33. Discussion

34. Discussion and Conclusions
Targeting the kif5aa locus
integration efficiency was considerably increased by using a combination of the kif5aa-specific sgRNA kif5aa 1 and sgRNA eGFP 1, with its corresponding eGFP DNA donor (Fig. 4)
Can be easily replaced with reporter genes such as GFP to generate fluorescent fusion proteins, or other heterologous transcription factors such as TetR or LexA.
The simplicity of the DNA target vector
offer an easier alternative to BAC transgenesis.
sequences are of small size, they can be generated easily by PCR or oligonucleotide cloning, and no long homology stretches between donor and target site are required.
DNA靶載體的簡單
提供給BAC轉基因更容易的選擇。
序列是小尺寸的，它們可以很容易地通過PCR或寡克隆產生，並且供體和目標部位之間沒有長同源拉伸是必需的。

35. IRES VS 2A peptide Action WHY ?  Test efficiency
IRES  IN FRAM or NO IN FRAM
2A peptide  IN FRAM
Promoter in insert Direct expression
WHY ?  Test efficiency
因為是第一個用這個方法的人
所以想幫大家測試IN FRAM 的效率
時間軸

36. Thank you for listening ~

37. TALEN vs Zinc fingers vs CRISPR
(B)
(C)
Guide
Artificial Protein
RNA
Effector
single stranded cut
Double stranded cut
Experimential Design
Complicated
Simple

38. A diagram of CRISPR/Cas9-mediated conversion of an eGFP-transgenic line into an eGFPbait-E2A-KalTA4 transgenic line
CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish
VOL.9 NO.12 | 2014 | nature protocols

39. sgRNA eGFP 1 VS sgRNA eGFP 2
設計了 sgRNA eGFP 1 和 sgRNA eGFP 2 兩種 sgRNA序列在 embryos 上表現進行比較，從螢光表現上可以發現sgRNA eGFP 1 表現的較亮。
兩者The number of successfully converted embryos  sgRNA eGFP 2 15% 、sgRNA eGFP 1 76%

40. UAS/RFP Tracing Transgene Expression in Living Zebrafish Embryos
Developmental Biology 233, 329–346 (2001)

41. 1