这项工作是由武田科学基金会（TF）的支持;旭硝子基金会;上原纪念基金会（HF）;在仓田纪念日立科学技术基金会（TF和HF）;格兰特提供的援助青年科学家（B）（＃25830131），格兰特 - 在急救科学研究（C）（＃15K06895）（TF）;和格兰特 - 在急救科学研究的创新领域"转录周期"（＃25118512＆＃15H01354），格兰特 - 在急救科学研究（B）（＃15H04329），格兰特提供的援助的探索性研究（ ＃26650059）和"基因组支持"（＃221S0002）（HF）由教育部，文化，体育，科学和技术的日本。

工程DNA结合分子认识到目标轨迹的1.设计<ol><li>对于使用CRISPR复合enChIP，使用CRISPRdirect网络工具（http://crispr.dbcls.jp），以确定所关注形容为先前18的基因组区域的候选gRNA靶序列。此Web工具将返回表格23 bp的基因组位点5&#39;-N 20 NGG-3&#39;的目标区域内。 </li><li>合成一个gBlock包括U6启动子序列和5&#39;- N 20中5&#39;-N 20 NGG-3&#39;使用商业服务的序列（ 见图 2）19,20。用于亚克隆的目的，包括gBlock以外适当的限制性酶位点。 </li><li>插入gBlock到适当的载体如先前所述16。对于gBlocks几个逆转录病毒载体（请参阅材料 ）。 </li><li>对于使用TAL蛋白enChIP，设计TAL蛋白识别TARG等位点。生成质粒编码使用商业服务TAL蛋白。产生含有稠合有一个或多个标记，如3×FLAG如前所述15的TAL蛋白质的表达载体。 </li></ol> 2.建立细胞的enChIP分析<ol><li>表达3×FLAG-dCas9和gRNA（CRISPR基于程序）或3×FLAG-TAL（TAL蛋白质基过程）在细胞中被如先前所描述的14-17进行分析。 <ol><li>使用瞬时转染，如果转染效率是高的。可含有3×FLAG-dCas9和CMV启动子的表达载体（ 见材料）。 </li><li>考虑建立稳定的转化使用常规方法，如果瞬时转染效率是低的。除了 ​​为gBlock上述逆转录病毒载体，是3×FLAG-dCas9的反转录病毒载体可用（ 参见材料）。 </li></ol></li><li&gt;通过使用如先前14-17描述的抗FLAG抗体（抗体）免疫印迹分析确认3XFLAG-dCas9或3XFLAG-TAL蛋白在转染细胞中的表达。 注意:这些标记的蛋白质的表达，也可以通过细​​胞内染色用抗FLAG-FITC和随后的FACS分析证实。一种协议，用于细胞内染色可以从我们的主页（http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/iChIP_protocols/english.html）下载。 </li><li>确认gRNA通过标准的RT-PCR法表达。 </li><li>蛋白质与感兴趣的基因组区域相互作用的定量识别，可以考虑使用稳定同位素标记的通过在细胞培养物（SILAC）分析结合enChIP（enChIP-SILAC）氨基酸。 注:媒体的SILAC分析，可以从商业供应商处购买。 SILAC是在检测特异性相互作用强大。 <ol><li>培养细胞SILAC重介质。准备细胞文化前进红色在SILAC光介质作为阴性对照。使用至少5×10 7个细胞的每个SILAC介质（重或轻）。为有效的标记，同时添加L-赖氨酸2HCl的，13 C 6和L-精氨酸盐酸，在SILAC重媒体13 C 6，15 N 4。注:至少有五细胞分裂所必需的标签蛋白。 <ol><li>例如，培养人纤维肉瘤HT1080细胞稳定表达3XFLAG-dCas9和gRNA在37℃和5％的CO 2在DMEM培养基SILAC和透析的胎牛血清与赖氨酸2HCl的加L-精氨酸盐酸（光介质）或13 C 6 L-赖氨酸二盐酸盐加13 C 6 15 N 4 L-精氨酸盐酸（重介质）（ 见材料）16。添加50毫克轻或重的L-赖氨酸-2HCl的和L-精氨酸盐酸在500毫升培养基中。 </li><li> Trypsinize和replate细胞才汇合，使细胞保持指数growtH。 </li></ol></li></ol></li></ol> 3.交联细胞与甲醛<ol><li>对细胞悬浮培养，转移2×10 7细胞表达3XFLAG-TAL蛋白质或3XFLAG-dCas9加gRNA和暂停在30ml常规培养基在50ml离心管中。当使用更多的细胞，提高试剂的数量和金额比例。对于SILAC实验，混合在重介质或光介质（各5×10 7个细胞）培养的细胞和1×10 8个细胞的总分为5试管含有2×10 7细胞。 </li><li>添加810微升37％的甲醛的30毫升细胞悬浮液（最终浓度1％），并在37℃下5分钟。对于贴壁细胞，直接通过加入810微升37％的甲醛至30ml培养基中固定的细胞在培养皿中。 </li><li>停止交联通过加入3.1毫升1.25M的甘氨酸溶液（最终浓度127毫摩尔），和在室温下孵育10分钟。 </li><li>通过离心（300×g下5分钟，4℃）收集细胞。小心弃去上清液，包括甲醛，并将其存储在适当的废液瓶。 <ol><li>对于贴壁细胞，分离细胞用细胞刮刀和收获在50ml管中，并作为本步骤中所述收集细胞。 </li><li>对于SILAC实验，混合在重介质或光介质（5×10 7个细胞每）培养分离的细胞，1×10 8个细胞的总分为5试管含有2×10 7细胞，并如在本描述的收集细胞步骤。 </li></ol></li><li>每管洗细胞沉淀用30ml的PBS两次。小心弃去上清液，包括甲醛，并根据化学品的安全指导方针将其存储在bottle.Handle甲醛废适当的废物。固定的细胞可以冷冻并储存于-80℃。 </li></ol> 4.制备染色质（每2×107细胞 ） <ol><li>暂停固定的细胞在10毫升细胞裂解缓冲液（10mM的Tris（pH 8.0）中，1毫摩尔EDTA，0.5％IGEPAL CA-630，和1×蛋白酶抑制剂），并在冰上孵育10分钟。 </li><li>离心样品（830×g下在4℃，8分钟），小心弃去上清液。 </li><li>暂停沉淀在10ml核裂解缓冲液（10毫摩尔Tris（pH值8.0），1mM EDTA中，0.5M氯化钠，1％的Triton X-100，0.5％脱氧胆酸钠，0.5％月桂酰肌氨酸，和1x蛋白酶抑制剂）。冰上孵育10分钟，并涡旋振荡每2-3分钟。 </li><li>离心样品（830× 在4℃，8分钟g），并小心地弃去上清液。 </li><li>在10ml PBS中洗涤沉淀。将沉淀（染色质级分）可以储存在-80℃之后在液态氮立即冻结。 </li></ol> 5.超声处理的染色质（每2×10 7细胞） <ol><li>暂停在800微升的染色质部分改进的裂解缓冲液3（10毫摩尔Tris（pH值8.0），1mM EDTA中，150 mM氯化钠，0.1％脱氧胆酸钠，0.1％SDS中，和1x蛋白酶抑制剂），并转移到1.5ml试管。 </li><li>超声处理使用超声波仪（参见材料）的染色质和条件如下:输出:3;税:100％（连续）;时间:自由。执行超声处理10-18个循环持续10秒和在冰上冷却20秒。为了避免过热，孵育样品在冰上2分钟，每5-6个周期。为了避免起泡，保持声处理探针0.5厘米管子的底部上方的尖端的位置。 </li><li>离心样品（16,000×g下在4℃下进行10分钟），将上清转移到1.5ml试管。将超声处理的染色质可以储存在-80℃之后在液态氮立即冻结。 </li></ol> 6.评估染色质碎片的<ol><li>混合10微升片段化的染色质与85微升蒸馏水。 </li><li>加入4微升和的5 M氯化钠孵育在65℃CO / N。 </li><li>加入1微升10毫克/毫升RNA酶A和孵育在37℃下45分钟。 </li><li>加入2微升的0.5M EDTA（pH 8.0）中，4微升的1M Tris（pH6.8）中，和1微升20mg / ml的蛋白酶K，再孵育在45℃下1.5小时。 </li><li>单独的10微升样品通过电泳中不包含染色染料，如SYBR绿1％琼脂糖凝胶。 </li><li>染色凝胶来评价零散染色质的长度的分布。产生0.5-2 kbp的（范围0.2-4千碱基对）的平均长度条件，推荐。 </li><li>纯化通过苯酚-氯仿提取或通过使用DNA提取试剂盒（ 参见材料）从其余样品的DNA。将纯化的DNA可以作为输入的DNA来估计enChIP分析的产率（见8.11）。 </li></ol> 7.制备磁珠共轭与抗体（每2×10 7细胞） <ol><li>预削2 1.5 ml的管，一个用于使用正常小鼠IgG预澄清，另一个用于孵育抗FLAG抗体。添加150微升蛋白G偶联的磁珠（参见材料），以每管。 </li><li>将管上的磁体支架，等待3分钟。弃去上清液吹打。 </li><li>暂停在1ml PBS中含有0.01％Tween-20（PBS-T）的珠。将管上的磁性底座，等待2分钟。弃去上清液吹打，然后重复步骤。 </li><li>暂停在1ml含有0.1％BSA的PBS-T中的珠子。 </li><li>加入15微克正常小鼠IgG或抗FLAG抗体和旋转4°CO / N。 </li><li>后短暂离心，然后将管上的磁体支架，等待3分钟。弃去上清液吹打。 </li><li>暂停在1ml的PBS-T中的珠子。反转样品几次后短暂离心。将管上的磁体支架，等待3分钟，然后通过移液丢弃上清液。重复洗两次用PBS-T（总共三个洗涤步骤）。 </li></ol> 8.染色质免疫沉淀（每2×10 7细胞） <ol><li>混合在5.3中制备的分散的染色质）（约800微升）与五分之一体积的（大约200微升含5％的Triton X-100（1％的Triton X-100的最终浓度）改进的裂解缓冲液3）。 </li><li>添加染色质溶液，以在其中结合有正常小鼠IgG蛋白G偶联的磁珠制备的管。旋转，在4℃下进行1小时。 </li><li>将管上的磁体支架，等待3分钟。 </li><li>将上清液转移到在其中结合有抗FLAG抗体蛋白G偶联的磁珠制备的管。旋转，在4°CO / N。 </li><li>将管上的磁体支架，等待3分钟。弃去上清液吹打。 </li><li>暂停在1ml的低盐缓冲液（20mM的Tris（pH 8.0）中，2毫摩尔EDTA，150毫摩尔NaCl，1％三珠吨X-100，0.1％SDS中，和1x蛋白酶抑制剂），并旋转，在4℃下5分钟。将管上的磁体支架，等待3分钟。弃上清，吹打，重复清洗步骤。 </li><li>洗用高盐缓冲液（20mM的Tris（pH 8.0）中，2毫摩尔EDTA，500毫摩尔NaCl，1％的Triton X-100，0.1％SDS中，和1x蛋白酶抑制剂）的两倍珠。 </li><li>洗用的LiCl缓冲液（10mM的Tris（pH 8.0）中，1毫摩尔EDTA，250毫的LiCl，0.5％IGEPAL CA-630，0.5％脱氧胆酸钠，和1x蛋白酶抑制剂）的两倍珠。 </li><li>洗用TBS-IGEPAL CA-630（50毫摩尔Tris（pH值7.5），150 mM氯化钠，0.1％IGEPAL CA-630，和1x蛋白酶抑制剂）一旦珠。 </li><li>暂停在200μl洗脱缓冲液的珠（50毫摩尔Tris（pH值7.5），150 mM氯化钠，0.1％IGEPAL CA-630，1×蛋白酶抑制剂，和500微克/毫升3×FLAG肽）和在37℃下20分钟。将管上的磁体支架，等待3分钟。将上清液转移到一个新的1.5毫升管，并重复ELUT离子一步。 </li><li>净化洗脱液通过苯酚-氯仿提取或通过使用DNA提取试剂盒的DNA小部分（例如，5％）（见材料）。将纯化的DNA可用于PCR用特定引物集来估计产量enChIP分析由与如先前14,16所述制备步骤6.7输入的DNA进行比较。 </li></ol> 9. SDS-PAGE，染色，质谱分析<ol><li>混合洗脱物（400微升）用1毫升2-丙醇，加入50μl3M乙酸钠（pH 5.2）和5微升20毫克/毫升糖原。沉淀的染色质在-20℃CO / N。 </li><li>离心样品（16,000×g下在4℃下30分钟）并弃去上清液。冲洗用1ml 70％乙醇，离心再次的粒料（16,000×g下在4℃下进行10分钟）。吹打完全弃上清。 </li><li>暂停在40微升2×样品缓冲液（125毫摩尔Tris（pH6.8）中，10％的2-MERC沉淀aptoethanol，4％SDS，10％蔗糖和0.004％溴酚蓝）。涡旋5分钟，以完全溶解沉淀，然后孵育在100℃下30分钟以变性蛋白和逆转交联。 </li><li>对于SDS-PAGE凝胶上的运行40微升样品直至染料达到1厘米以下的井。注意:我们通常使用4-20％梯度凝胶，但其他％凝胶可以使用。 </li><li>染色用考马斯亮蓝或银染的凝胶。 </li><li>切胶成五片（2 mm高）。 </li><li>如先前13-16说明进行凝胶消化和质谱分析。 </li><li>对于SILAC实验用5×10 7个细胞每（总共1×10 8个细胞），从最初的5管在40微升2×样品缓冲液（这是没有必要按比例增加）暂停粒料。 </li></ol> 10.纯化RNA和RNA测序分析<ol><li>为了净化enChIP后的RNA，加酶抑制剂的5单位/毫升（SEë材料），以所有的缓冲溶液，除改进的裂解缓冲液3和洗脱缓冲液中，向其中添加RNA酶抑制剂的40单位/毫升。 </li><li>混合洗脱物（400微升），16微升的5M NaCl和孵育在65℃下2小时。 </li><li>加入1毫升酸 - 胍 - 酚系试剂（参见材料）到样品中。涡旋15秒，然后在室温下孵育5-15分钟。离心样品15分钟，12000×g下和室温。 </li><li>上清转移到一个新的1.5毫升管，加入5微升对溴苯甲醚的。涡旋15秒，然后在室温下孵育3-5分钟。离心样品以12,000×克和RT 10分钟。 </li><li>转移上清液（约1毫升）到新的2ml管中，加入1毫升2-丙醇。倒置该管并在室温下孵育10分钟。装载到混合物中的RNA纯化试剂盒的柱（ 参见材料）。离心列在12000×1分钟 G和RT。 </li><li>洗用400μl的RNA洗涤缓冲液中的RNA纯化试剂盒（参见材料）的列。 </li><li>的5微升DNA酶I（1U /微升），8微升10×DNA酶I反应缓冲液，3微升DNA酶/无RNase水，和64微升的RNA洗涤缓冲液中添加80μl的DNA酶I的鸡尾酒（混合物的RNA纯化试剂盒（参见材料））到柱上。孵育样品在37℃下进行15分钟，然后在12000×g下和RT离心30秒。 </li><li>用400微升RNA预洗缓冲液中的RNA纯化试剂盒（见材料）的两倍洗列。 </li><li>洗脱用50μlDNA酶/无RNase水的核糖核酸。将洗脱的RNA可用于RNA测序。 </li></ol>

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Biol. 15, 26 (2014).</li><li>Fujita, T., Kitaura, F., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+of+the+Thy28-MYH9+axis+in+B+cell-specific+expression+of+the+Pax5+gene+in+chicken+B+cells.">A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells.</a> PLoS One. 10, (2015).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+and+identification+of+associated+proteins+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP)+using+CRISPR.">Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.</a> Biochem. 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Rep. 3, 3171 (2013).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+proteins+associated+with+an+IFN&#947;-responsive+promoter+by+a+retroviral+expression+system+for+enChIP+using+CRISPR.">Identification of proteins associated with an IFN&#947;-responsive promoter by a retroviral expression system for enChIP using CRISPR.</a> PLoS One. 9, e103084 (2014).</li><li>Fujita, T., Yuno, M., Okuzaki, D., Ohki, R., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+non-coding+RNAs+associated+with+telomeres+using+a+combination+of+enChIP+and+RNA+sequencing.">Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing.</a> PLoS One. 10, e0123387 (2015).</li><li>Naito, Y., Hino, K., Bono, H., Ui-Tei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPRdirect:+software+for+designing+CRISPR/Cas+guide+RNA+with+reduced+off-target+sites.">CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites.</a> Bioinformatics. , (2014).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</li><li>Esvelt, K. 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The authors have filed a patent on enChIP (Patent name: 'Method for isolating specific genomic regions using DNA-binding molecules recognizing endogenous DNA sequences'; Patent number: PCT/JP2013/74107). H.F. is a member of the Advisory Board of Addgene.

Here, we describe the purification of specific genomic regions using engineered DNA-binding molecules such as the CRISPR system and TAL proteins, and the identification of proteins and RNAs bound to these genomic regions. Binding of engineered DNA-binding molecules to the genome may affect chromatin structure, including nucleosome positioning, and may abrogate genomic functions, as described in CRISPR interference experiments21. To avoid these potential aberrant effects, we propose specific guidelines for choosing target genomic regions. First, to avoid potential inhibition of the recruitment of RNA polymerases and transcription factors, as well as disruption of nucleosome positioning around the transcription start site, the target regions for analyses of promoter regions should be several hundred base pairs upstream of (5' to) the transcription start site. By contrast, when analyzing genomic regions with distinct boundaries, such as enhancers and silencers, genomic regions that are directly juxtaposed to these regions can be targeted because it is less likely that the binding of engineered DNA-binding molecules will affect their functions. Furthermore, it is best to avoid using target regions that are conserved among different species, because important DNA-binding molecules often bind to evolutionarily conserved regions and inhibition of their binding might disrupt the functions of the target genomic regions. In this regard, it is always necessary to check that the function of the target genomic region is maintained in the established cells used for enChIP analyses. Because multiple gRNAs can be tested easily and it is tedious and expensive to generate multiple versions of TAL or zinc-finger proteins recognizing different target genomic regions, enChIP using CRISPR is more advantageous than enChIP using other proteins.
It has been shown that dCas9 binds to off-target sites although affinity to those sites might be weaker than that to the target sites22-25. There are several ways to manage contamination of molecules bound to those off-target sites. First, the use of several, at least two, different gRNAs would be recommended. Those molecules commonly observed in enChIP using distinct gRNAs would be true positives. Second, comparison of different conditions for enChIP would be effective in cancelling contamination of non-specific molecules and molecules bound to off-target sites. Examples of those comparison sets would be (i) stimulation (-) and (+), or (ii) different cell types such as T cells vs. B cells. Finally, quantitative analysis of binding of candidate molecules should be performed to confirm their specific binding to the target sites. It is preferable to prepare cells expressing only dCas9 but not gRNA as a negative control.
Using enChIP analyses, we were able to successfully identify a number of known and novel molecules interacting with specific genomic regions (Tables 1-3)14-17. However, this technique failed to detect some other known proteins interacting with these regions. For example, STAT1 reportedly associates with the IRF-1 promoter upon IFN&#947; stimulation8, but our enChIP-SILAC analysis did not detect STAT1 as a protein induced to interact with this genomic region16. In addition, in the enChIP-MS analysis of telomeres, we did not detect shelterin proteins consisting of TRF-1 and TRF-215, which have been shown to interact with telomeres26. There are a few potential reasons for these discrepancies. First, the stoichiometry of binding of Stat1 to the IRF-1 promoter might be very low. It is reasonable that enChIP-MS, including enChIP-SILAC, detects proteins that are more abundantly associated with target genomic regions; hence, the analysis of more cells might be necessary to detect these proteins. Increases in the sensitivities of MS instruments would also contribute to the efficient detection of proteins with low stoichiometric binding. Second, some proteins, possibly including Stat1 and shelterins, might be difficult targets for MS analyses. Third, in our analysis of telomere-binding proteins15, the 3&#215;FLAG-TAL proteins recognizing telomeres (3&#215;FN-Tel-TAL) might have blocked the binding of shelterins to telomeres in a competitive fashion.
In contrast to the relative difficulty of detecting transcription factors binding to specific genomic regions using enChIP, we successfully identified epigenetic regulators such as histone modification enzymes using enChIP analyses. The success of this technique may be due to the fact that epigenetic regulators bind to a broad range of genomic regions; hence, more proteins per genomic region are available for MS. Because epigenetic regulators are increasingly recognized as important targets for drugs against intractable diseases such as cancer, enChIP would be a useful tool for the identification of epigenetic drug targets.

与感兴趣的特定基因组区域相关联的分子的识别，需要理解的基因组功能，例如转录和表观遗传调控调节的机制。虽然几种技术已经开发了用于特定的基因组区域1-7中的生化分析，它们没有被广泛使用，因为其固有的问题，如有限的应用程序（例如，只对于高拷贝数或基因座基因座与重复），并在此阶段太多的时间和努力需要。 为了容易地执行特定的基因组区域的生化分析，我们已经开发了两种基因座特异性染色质免疫沉淀技术，即插入的ChIP（Internet控制器）8-13和工程化DNA结合分子介导的ChIP（enChIP）14-17 。在Internet控制器，感兴趣的基因座是由标记的外源DNA结合蛋白如莱克斯插入识别序列答轨迹然后通过使用标记的DNA结合蛋白的亲和纯化分离。在enChIP，工程化DNA结合分子，如锌指蛋白，转录激活物状（TAL）的蛋白质，和群集定期相互间隔短回文重复序列（CRISPR）配合物，用于标记的感兴趣位点（ 图1）。随后，将基因组区域被标记的DNA结合分子的亲和纯化分离。 之一的enChIP过Internet控制器的一个优点是，插入的外源DNA结合蛋白的识别序列是没有必要的。靶向使用CRISPR络合物选自Cas9（dCas9）的无催化活性的形式的和导向的RNA（gRNA）位点比这些区域通过Internet控制器或enChIP使用TAL和锌指蛋白靶向容易得多。这里，我们描述了一步一步的协议enChIP与质谱联用和RNA测序（RNA片段）识别位点Associa大泰德蛋白质和RNA的分别。

<table><tbody><tr><td>gBlock synthesis service</td><td>Life Technologies</td><td>Gene Synthesis by GeneArt</td><td></td></tr><tr><td>gBlock synthesis service</td><td>IDT (Integrated DNA Technologies)</td><td>gBlocks Gene Fragments</td><td></td></tr><tr><td>pSIR-neo</td><td>Addgene</td><td>51128</td><td></td></tr><tr><td>pSIR-GFP</td><td>Addgene</td><td>51134</td><td></td></tr><tr><td>pSIR-DsRed-Express2</td><td>Addgene</td><td>51135</td><td></td></tr><tr><td>pSIR-hCD2</td><td>Addgene</td><td>51143</td><td></td></tr><tr><td>TAL synthesis service</td><td>Life Technologies</td><td>GeneArt Precision TALs</td><td></td></tr><tr><td>3xFLAG-dCas9/pCMV-7.1</td><td>Addgene</td><td>47948</td><td></td></tr><tr><td>3&amp;times;FLAG-dCas9/pMXs-puro</td><td>Addgene</td><td>51240</td><td></td></tr><tr><td>3&amp;times;FLAG-dCas9/pMXs-IG</td><td>Addgene</td><td>51258</td><td></td></tr><tr><td>3&amp;times;FLAG-dCas9/pMXs-I2</td><td>Addgene</td><td>51259</td><td></td></tr><tr><td>3&amp;times;FLAG-dCas9/pMXs-neo</td><td>Addgene</td><td>51260</td><td></td></tr><tr><td>anti-FLAG M2 Ab</td><td>Sigma-Aldrich</td><td>F1804</td><td></td></tr><tr><td>FITC-conjugated anti-FLAG M2</td><td>Sigma-Aldrich</td><td>F4049</td><td></td></tr><tr><td>DMEM medium for SILAC</td><td>Life Technologies</td><td>89985</td><td>Other medium can be purchased from Life Technologies</td></tr><tr><td>Dialyzed FBS for SILAC</td><td>Life Technologies</td><td>89986</td><td></td></tr><tr><td>L-Lysine-2HCl for SILAC</td><td>Life Technologies</td><td>89987</td><td>For Light medium</td></tr><tr><td>L-Arginine-HCl for SILAC</td><td>Life Technologies</td><td>89989</td><td>For Light medium</td></tr><tr><td>L-Lysine-2HCl, 13C6 for SILAC</td><td>Life Technologies</td><td>89988</td><td>For Heavy medium</td></tr><tr><td>L-Arginine-HCl, 13C6, 15N4 for SILAC</td><td>Life Technologies</td><td>89990</td><td>For Heavy medium</td></tr><tr><td>Complete, mini, EDTA-free</td><td>Roche Diagnostics</td><td>4693159</td><td></td></tr><tr><td>Ultrasonic Disruptor UD-201</td><td>Tomy Seiko</td><td></td><td></td></tr><tr><td>ChIP DNA Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D5205</td><td></td></tr><tr><td>Dynabeads-Protein G</td><td>Life Technologies</td><td>DB10004</td><td></td></tr><tr><td>RNasin Plus RNase Inhibitor</td><td>Promega</td><td>N2611</td><td></td></tr><tr><td>Isogen II</td><td>Nippon Gene</td><td>311-07361</td><td></td></tr><tr><td>Direct-zol RNA Miniprep kit</td><td>Zymo Research</td><td>R2050</td><td></td></tr><tr><td>LTQ Orbitrap Velos</td><td>Thermo Fisher Scientific</td><td></td><td>A component of a nanoLC-MS/MS system for MS analysis</td></tr><tr><td>nanoLC</td><td>Advance, Michrom Bioresources</td><td></td><td>A component of a nanoLC-MS/MS system for MS analysis</td></tr><tr><td>HTC-PAL autosampler</td><td>CTC Analytics</td><td></td><td>A component of a nanoLC-MS/MS system for MS analysis</td></tr></tbody></table>

isolation of specific genomic regions and identification of associated molecules by enchip

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

总体而言，靶基因组区域的1-30％可使用enChIP纯化。 图3包括代表数据表示enChIP的产率分析靶向端粒和干扰素（IFN）调节因子1（IRF-1）基因的启动子。作为典型的结果，表1列出实施例与在IRF-1启动子中鉴定由enChIP-SILAC， 表一的IFNγ特异性方式相关的蛋白2列出确定enChIP与质谱联用端粒结合蛋白（enChIP-MS） ，和表3列出用确定enChIP-RNA测序端粒相关联的的RNA。 <img alt="图1" src="/files/ftp_upload/53478/53478fig1.jpg" /> 图 enChIP 1.概述 。 enChIP使用CRISPR（A，B）和 TAL（C，D，E </STRONG&gt;）显示。感兴趣的轨迹被打上了设计DNA结合分子，如蛋白质TAL或由Cas9（DCAS）的催化活性形式的CRISPR系统和引导RNA（gRNA）。的分子间的相互作用是用甲醛固定或其它交联剂，如果需要的话。随后，固定的染色质通过超声处理或酶促消化分段。加标签的基因组区域是由亲和纯化来纯化。最后，交联逆转，相互作用的分子（基因组区域，RNA的，和蛋白质）均采用新一代测序和质谱鉴定。 <a href="https://www.jove.com/files/ftp_upload/53478/53478fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53478/53478fig2.jpg" /> 图2.序列gBlock的。U6启动子（黑色），对G核苷酸b安伏的导向序列（蓝色），引导序列（gRNA间隔物）（红色），所述支架序列（绿色），及终止子序列（橙色）被示出。 <img alt="图3" src="/files/ftp_upload/53478/53478fig3.jpg" /> 图3.产量的代表enChIP分析 。 （A）中的百分比的输入为目标的IRF-1基因座和非靶Sox2的基因座。 （B）的百分比的输入为目标端 ​​粒和非目标γ-卫星。该数字已调整，并从以前的出版物15,16修改。 <table fo:keep-together.within-page="1"><tbody><tr><td> 分类 </td><td> 蛋白质 </td></tr><tr><td>转录</td><td> DDX1，PARP1，CKAP4，Pescadillo同源，PURβ， 活化的RNA聚合酶II转录激活p15基因， BTF3，Myb的结合蛋白1A </td></tr><tr><td>组蛋白脱乙酰化， 辅阻遏物成分</td><td> RBBP4，PA2G4，TBL3 </td></tr><tr><td>乙酰转移酶</td><td>蛋白质精氨酸N-甲基转移酶1 </td></tr><tr><td> DNA拓扑异构酶</td><td> DNA拓扑异构酶2α </td></tr><tr><td>组蛋白</td><td>组蛋白H2A.Z，组蛋白H3.2 </td></tr></tbody></table> 表1:用 在确定enChIP-SILAC一个IFNγ特异性方式的人IRF-1的启动子区相关的蛋白质的例子的表已被改编从先前的出版物16。 <table fo:keep-together.within-page="1"><tbody><tr><td> 分类 </td><td> 蛋白质 </td></tr><tr><td>哺乳动物端粒结合蛋白</td><td> PML，RPA，CDK1，PARP1，PCBP1 </td></tr><tr><td>端粒碧在酵母nding蛋白质 或其他生物</td><td> IMP4 </td></tr><tr><td>与蛋白质相互作用的端粒结合 蛋白质[相关端粒结合蛋白] </td><td> DNA聚合酶α（POLA1）Cdc13p] ARMC6 [TRF2]，CTBP1 [FOXP2-POT1] exportin-5 [叔]，GNL3L [TRF1] exportin-1 [叔]，14-3-3 [叔] </td></tr><tr><td>蛋白定位于异</td><td> BEND3 </td></tr><tr><td>蛋白质调控的表观遗传标记</td><td> KDM5C </td></tr><tr><td>蛋白质的突变影响端粒功能</td><td> DNA聚合酶α（POLA1），HAT1，Nup133， CDK7，DPOE1，PRDX1，TYSY， 谷氨酸半胱氨酸连接酶，谷，SMRC1 </td></tr></tbody></table> 表2:用 确定enChIP-MS鼠标端粒相关蛋白的例子表已adapted从以前的出版物15。 <table fo:keep-together.within-page="1"><tbody><tr><td> 分类 </td><td> RNA的 </td></tr><tr><td>端粒酶组件</td><td> TERC，RMRP </td></tr><tr><td>端粒的RNA </td><td> TERRAS </td></tr><tr><td> scaRNAs </td><td> Scarna6，Scarna10，Scarna13，Scarna2 </td></tr><tr><td> H / ACA 52]形成</td><td> Snora23，Snora74a，Snora73b，Snora73a </td></tr><tr><td> C / D 52]形成</td><td> Snord17，Snord15a，Snord118 </td></tr><tr><td> lncRNA </td><td> Neat1 </td></tr></tbody></table> 表3: 具有确定enChIP-RNA的序列表已被改编自先前公布17 鼠标端粒相关的RNA的例子 。

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Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

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JoVE Journal

Biology

10.4K 次观看.  Osaka University. The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

视频: Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

科研

遗传学

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

使用色素免疫沉淀-外核酶（ChIP-Exo）对小鼠干细胞衍生神经元中的蛋白质-DNA相互作用进行高分辨率映射

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

生物学

<meta charset="utf8"></head><body>推进新型微生物培养的 iChip 方法

Isolation of Soil Microorganisms Using iChip Technology

The iChip isolation technique uses an in-situ isolation device that increases the cultivability of previously unculturable microorganisms. Microorganisms are an important source of novel chemistries and potentially bioactive molecules. However, only 1% of environmental microorganisms can be cultured using conventional laboratory methods. With the rise in antimicrobial resistance, finding new drugs to combat infections and diseases is of foremost importance, and a critical method to finding new molecules is the discovery of new microorganisms. By incubating colonies of soil microorganisms in the wells of a 96-well plate, sealed with a semipermeable membrane and incubated on top of soil, the microbes are in contact with water and growth factors from the soil, allowing for the isolation of novel microbes in a laboratory setting. After a period of domestication in an iChip, microorganisms can potentially be subcultured onto conventional media and used for further study. This device is valuable to bioactive molecule discovery and soil microbiome research and has been used previously in both applications.

<meta charset="utf8"></head><body>使用 iChip 技术分离土壤微生物

The iChip technique makes use of an inexpensive and simple in-situ isolation device that increases the rates of novel microorganism discovery from the soil. Novel microorganisms can be used for further study pertaining to the soil microbiome or natural product discovery, amongst other applications.

使用 iChip 技术分离土壤微生物

The iChip isolation technique uses an in-situ isolation device that increases the cultivability of previously unculturable microorganisms. Microorganisms ...

生物化学

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

配对末端标签测序（嘉PET）测绘染色质相互作用和了解转录调控染色质相互作用分析

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

具体的基因组区域及相关分子鉴定enChIP隔离

摘要

探索更多视频

此视频中的章节

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射

使用色素免疫沉淀-外核酶（ChIP-Exo）对小鼠干细胞衍生神经元中的蛋白质-DNA相互作用进行高分辨率映射

采用芯片识别亚型特定的靶基因的全基因组分析

使用 iChip 技术分离土壤微生物

使用 iChip 技术分离土壤微生物

使用 iChip 技术分离土壤微生物

配对末端标签测序（嘉PET）测绘染色质相互作用和了解转录调控染色质相互作用分析

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择

具体的基因组区域及相关分子鉴定enChIP隔离

摘要

探索更多视频

此视频中的章节

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射

使用色素免疫沉淀-外核酶（ChIP-Exo）对小鼠干细胞衍生神经元中的蛋白质-DNA相互作用进行高分辨率映射

采用芯片识别亚型特定的靶基因的全基因组分析

使用 iChip 技术分离土壤微生物

使用 iChip 技术分离土壤微生物

使用 iChip 技术分离土壤微生物

配对末端标签测序（嘉PET）测绘染色质相互作用和了解转录调控染色质相互作用分析

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择