TALEN和CRISPER基因靶向技术

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2013/12/22,#,2021,11,13,划时代,的靶向,基因操作技术,-,TALEN,和,CRISPR/Cas9,Outline,Background,Transcription activator-like (TAL) effector,CRISPR/Cas9,我们研究一个作物的性状，最终都需要做一件事：改变基因，从必要性和充分性两方面说明基因功能。,1Loss function：降低基因或删除基因,Give function: 提高基因的表达,Background,生物学家的梦想：,随心所欲地,操作基因,划时代的基因靶向操作技术,：,TALE,与,CRISPR/Cas9,使靶向操作不再是梦,我们不再大海捞针！,经典基因操作：,Gene,trap:,化学诱变；,转座子,-,海量的筛选,+good Luck,同源重组：,一般物种几率低,10,-6,；,ES cell,10,-2,难！,3.,突破性的发现：,RNAi-Knockdown;,不完全敲除，临时性。,4.,充满梦想却幻灭的,ZFN,：,难以完全找到匹配的,3,连子,锌指,；,Off-target,严重。,美国加州大学Dave Segal教授说： “ With zinc fingers, we have to let the DNA tell us where we target,5.,完美基因靶向操作： 识别特异,DNA,序列结构,+,操作,DNA,的酶,TALEN,技术形成的关键研究,Sugisaki Hiroyuki Jens Boch,发现FokI核酸酶 破译TALE序列 1981 2021,Daniel F. Voytas Adam J.Bogdanove,将TALE DNA结合motif与FokI核酸酶融,合，推出与ZFN同类型的双链DNA剪刀，,称之为TALEN。(2021),TALE,的发现迅速成为科技最前沿,TALE domain: DNA 识别域,TALE，植物病原体黄单胞菌 Xantomonas 用于调控宿主基因,由34 个氨基酸长度的重复单位构成,第12，13位点氨基酸 repeat-variable di-residue，RVD不同,RVD决定识别位点不同,Nuclease domain: DNA剪切域,FokI，发现于海床黄杆菌 Flavobacterium okeanokoites,采取其非特异性DNA剪切结构域,形成二聚体时发生剪切,N,C,TALE,FokI,LTPDAVVAIAS,NI,GGKQALETVQRLLPVLCQDHG,NI,A,HD,C,NG,T,NN,G,A,T,T,C,T,G,C,T,A A,C,T,C,A,T,A,C,TALEN,核酸酶,DNA,识别域,DNA,剪切域,FastTALE,TM,连接,TALEN,N,C,N,C,TALEN modules at 9 positions,TALEN backbone vectors,TALEN assembled vectors,Promoter,FokI,Promoter,FokI,x4,x16,x4,x16,x4,x16,x4,x16,x4,x16,x4,x16,x16,x16,x4,x16,Position 1,Position 2,Position 3,Position 4,Position 5,Position 6,Position 7,Position 8,Position 9,1/2,9 x 16 dimers, 7 x 4 monomers,Complete library: 172,CMV, EF1a, Ubi, 35S, T7, SP6,Maximum RVDs: 19,Minimum RVDs: 12,一步连接,只要,4-5,小时，这些片段就全部连接,亚克隆到植物表达载体上,通过Ti载体介导转染插入植物基因组中，然后表达目标蛋白-TALEN，TALEN剪切DNA，造成突变体。最后通过交配将TALEN去除，到达无痕基因敲除的目的。,We targeted the rice bacterial blight susceptibility,gene,Os11N3,(also called,OsSWEET14,) for TALEN-based disruption. This rice gene encodes a member of the,SWEET sucrose-efflux transporter family,and is hijacked by,X. oryzae,pv.,oryzae, using its endogenous TAL effectors AvrXa7 or PthXo3, to activate the gene and thus divert sugars from the plant cell so as to satisfy the pathogens nutritional needs and enhance its persistence.,The,Os11N3,promoter contains an effector-binding element (EBE) for AvrXa7, overlapping with another EBE for PthXo3 and with the TATA box.,Os-11N3,插入突变或者被,RNA,介导沉默后，对那些依赖,AvrXa7,和,PthXo3,的致病小种的特异感病性丧失。,24bp,17bp,24bp,We deployed two pairs of designer TALENs (pair 1 and pair 2) independently to induce mutations in these overlapping EBEs of the,Os11N3,promoter and thus to interfere with the virulence function of AvrXa7 and PthXo3, but not the developmental function of,Os11N3.,T2,代表型,The loss of induction was specific to Os11N3, as the induction of Os04g19960, a transposon coding gene collaterally targeted by AvrXa7, was not prevented.,All mutant plants were morphologically normal compared to wild-type plants, indicating that the,developmental function of Os11N3 was not disrupted,.,Removal of T-DNA sequences,containing TALEN genes from TALEN-modified rice plants using,genetic crossing,.,将识别特异,DNA,序列的,TALE,与,转录激活因子,VP64,融合，,表达的融合蛋白结合启动子附近的特异,DNA,序列， 并通过,VP64,激活区域与,Pol II,结合,，激活,基因的转录，提高了内源目标基因的表达。,TALEA-,人工转录因子,-,提高内源基因的表达,TALE,应用展望,-TALEX,将识别特定,DNA,序列的,TALE,蛋白与不同的蛋白酶融合，实现不同的靶向基因操作的应用：,调控控表达的,TALEN,或,TALEA-TALEL,抑制基因表达,- TALER,敲除基因,- TALEN,启动子附近的甲基化,- TALEM,特定序列的重组酶,-TALER,。,TALE,蛋白新功能,清华大学生命学院施一公教授研究组Cell Reports,2021报道转录激活因子样效应蛋白TALE能够特异识别DNA-RNA杂合链，并且能够保护DNA-RNA杂合链不被核酸酶降解。,他们针对HIV病毒逆转录过程中的一段DNA-RNA杂合序列设计了特异的含有23个重复单元的TALE蛋白TALEHIV，该蛋白可以有效地阻止RNase H 对这段DNA-RNA杂合链中RNA链降解。如果HIV在逆转录过程中，不能有效降解RNA，那么不能完成其基因组的扩增。因此，这一发现为抑制HIV病毒提供了新思路。,RNase H是一种核糖核酸内切酶，它能够特异性地水解杂交到DNA链上的RNA磷酸二酯键，故能分解RNA/DNA杂交体系中的RNA链。该酶不能消化单链或双链DNA。,RNase A是一种被详细研究和具有广泛应用的核酸内切酶。RNase A 对RNA有水解作用，但对DNA那么不起作用。,CRISPR/Cas9 System,1987年，日本大阪大学Osaka University在对一种细菌编码的碱性磷酸酶alkaline phosphatase基因进行研究时，发现在这个基因编码区域的附近存在一小段不同寻常的DNA片段，这些片段是由简单的重复序列组成的，而且在片段的两端还存在一段不太长的特有的序列，正是这个特有的序列以后被证明发挥了DNA的定向识别功能。,2021以后，研究者们在包括?science?和?nature biotechnology?等著名杂志上发表多篇文章介绍CRISPR-Cas系统，并且已成功在人类、小鼠、斑马鱼等物种上实现精确的基因修饰。,CRISPR-Cas主要由两局部组成：,识别,切割,CasCRISPR associated：,存在于CRISPR位点附近，是一种双链DNA核酸酶。它与folk酶功能类似，但是它并不需要形成二聚体就能发挥作用。,CRISPR-Cas是很多细菌和大局部古生菌的天然免疫系统，通过对入侵的病毒和核酸进行特异性的识别，利用Cas蛋白进行切割，从而到达对自身的免疫。,CRISPR：clustered regularly interspaced short palindromic repeats,CRISPR 是一个特殊的DNA重复序列家族, CRISPR 位点通常由短的高度保守的重复序列(repeats) 组成, 重复序列的长度通常 2148 bp, 重复序列之间被 2672 bp 间隔序列(spacer)隔开。CRISPR就是通过这些间隔序列space与靶基因进行识别。,CRISPR-Cas系统赋予原核细胞针对外源DNA特异性免疫，而这种特异性是由间隔序列spacer决定的。这种免疫功能的有无是由CRISPR 间隔序列的动态性变化决定的，即通过增加或删除间隔序列spacer来实现的。,如果改造成我们的目的基因，就可以定向的进行基因修饰,2021年1月29日在?nature biotechnology?上发表的?RNA-guided editing of bacterial genomes using,CRISPR-Cas systems?一文中，作者利用CRISPR-Cas系统用设计好的DNA模板替换的相应基因来到达基因的定向修饰。,42nt,NGG,20-25nt,40nt,化脓性链球菌终止子,The sgRNA is a 102-nt-long chimeric noncoding RNA, consisting of a 20-25nt target-specific complementary region, a 42-nt Cas9-binding RNA structure and a 40-nt transcription terminator derived from S.,pyogenes,. The PAM sequence, consisting of a 2- to 5-bp recognition site that varies depending on the CRISPR system and the host organism, is a key motif that is recognized by the CRISPR machinery for spacer acquisition and subsequent tar -get interference.,The choice of the target sites also affects the level of transcrip -tion repression. By using fluorescent reporter genes in E. coli, we have observed that the repression is inversely correlated with the distance of the target site from the transcription start site. Thus,to achieve better repression in bacteria, target sites within the 5 end of the gene should be selected. In human cells, we recom -mend selecting multiple target sites within the promoter-proximal region (targeting either the template or nontemplate strand) or,within the coding region (targeting the nontemplate strand), as epigenetic modifications and local chromatin structures might impede CRISPRi binding.,Design of sgRNAs,The PAM site is essential for dCas9 binding to the DNA, limiting the number of targetable sites within a genome. To avoid off-target effects, we recommend searching the genome for the 14-nt specificity region consisting of the 12-nt seed region of the sgRNA and 2 of the 3-nt (NGG) PAM in the genome, in order to rule out additional potential binding sites .Any sgRNA designed with more than one binding site should be discarded.,转化原生质体，检测突变活性：酶切,+,测序,26.0%,36.5%,The frequency of mutations induced by sgRNA:Cas9 in Os02g23823 was lower (26.0%) than that induced by TALENs (36.5%) whereas, in OsBADH2, it was considerably higher(26.5% versus 8.0%).,Wt+pst1,Wt+pst1,Wt-,Wt,-,Mutations in OsPDS -SP1 were identified in 9 of 96 independent transgenic plants (9.4%) In addition, biallelic mutations were identified in 3 of the 9 plants mutated in OsPDS -SP1. Two of them were homozygous for the same one-nucleotide insertion. And mutations in OsBADH2 in 7 of 98 transgenic plants (7.1%),Phenotypes of the,pds,mutants. (1) Nontransgenic wild-type rice plant. (2) Monoallelic mutant. (3) Biallelic homozygous mutant. (4) Biallelic heterozygous mutant. Mutants 3 and 4 have the albino and dwarf phenotype.,To examine homology-directed repair (HDR), we designed a single-stranded oligo with a KpnI + EcoRI site to be introduced into,OsPDS,. Two of 29 single colonies had the expected insertion of the KpnI + EcoRI site into,OsPDS,.,We next evaluated potential off-target effects of two sgRNA:Cas9 constructs targeting the OsMPK2 or OsPDS genes. Three nearly identical sequences, PDS_NI-1, MPK2_NI-1 and MPK2_NI-2, with one-base or three-base mismatches to the PDS-SP1 and MPK2 target sites were identified from the rice genome,The system described can in principle target any sequences, such as 5-A-N(20)-GG-3 and 5 -G-N(20)-GG-3 in rice and wheat, respectively. Use of the rice U3 promoter and the wheat U6 promoter constrains the first positions in the corresponding RNA transcripts to be A and G, respectively.,Limitations of the CRISPRi method,First, the requirement for an NGG PAM sequence for S.,pyogenes,Cas9 limits the availability of target sites,in the genome. And recent studies have suggested that the S. pyogenes Cas9 protein could partially recognize an NAG PAM, which might increase both the number of targeta -ble genome sites and that of potential off-target sites.,Second, the targeting specificity is determined only by a 14-nt-long region (the 12 nt of the sgRNA and the 2 nt of the PAM), which might confer,off-target,effects in organisms with large genomes. The theoretical sequence length for unique targeting with a 14-nt recognition sequence is 268 Mb (4,14,).,谢谢大家！,