Thanks to Jason Wright for suggesting the Golden Gate Assembly cloning strategy and Katherine Helming and members of Orkin lab, particularly Jian Xu, Guoji Guo, Elenoe Smith, and Partha Das for helpful discussions. This work was supported by NIH R01HL032259 and P30DK049216 (Center of Excellence in Molecular Hematology) to S.H.O. and NIDDK K08DK093705 to D.E.B.

<ol>
	<li>Jinek, M., 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=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li><li>Cong, L., 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</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 (6121), 823-826 (2013).</li><li>Gaj, T., Gersbach, C. A., Barbas, C. F. Z. F. N., TALEN, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas-based+methods+for+genome+engineering.">CRISPR/Cas-based methods for genome engineering.</a> Trends in Biotechnology. 31 (7), 397-405 (2013).</li><li>Barrangou, R., 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=CRISPR+provides+acquired+resistance+against+viruses+in+prokaryotes.">CRISPR provides acquired resistance against viruses in prokaryotes.</a> Science. 315, 1709-1712 (2007).</li><li>Ding, Q., 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=Enhanced+efficiency+of+human+pluripotent+stem+cell+genome+editing+through+replacing+TALENs+with+CRISPRs.">Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.</a> Cell Stem Cell. 12 (4), 393-394 (2013).</li><li>Ran, F. A., 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=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li><li>Ran, F. A., 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=Double+nicking+by+RNA-guided+CRISPR+Cas9+for+enhanced+genome+editing+specificity.">Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.</a> Cell. 154 (6), 1380-1389 (2013).</li><li>Larson, M. H., 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=CRISPR+interference+(CRISPRi)+for+sequence-specific+control+of+gene+expression.">CRISPR interference (CRISPRi) for sequence-specific control of gene expression.</a> Nature Protocols. 8 (11), 2180-2196 (2013).</li><li>Yang, H., 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=One-step+generation+of+mice+carrying+reporter+and+conditional+alleles+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.</a> Cell. 154 (6), 1370-1379 (2013).</li><li>Wang, H., 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=One-step+generation+of+mice+carrying+mutations+in+multiple+genes+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.</a> Cell. 153 (4), 910-918 (2013).</li><li>Canver, M. C., 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=Characterization+of+Genomic+Deletion+Efficiency+Mediated+by+CRISPR/Cas9+in+Mammalian+Cells.">Characterization of Genomic Deletion Efficiency Mediated by CRISPR/Cas9 in Mammalian Cells.</a> Journal of Biological Chemistry. 289 (31), 21312-21324 (2014).</li><li>Xiao, A., 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=Chromosomal+deletions+and+inversions+mediated+by+TALENs+and+CRISPR/Cas+in+zebrafish.">Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish.</a> Nucleic Acids Research. 41 (14), e141 (2013).</li><li>Zhou, J., 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=Dual+sgRNAs+facilitate+CRISPR/Cas9+mediated+mouse+genome+targeting.">Dual sgRNAs facilitate CRISPR/Cas9 mediated mouse genome targeting.</a> The FEBS Journal. , (2014).</li><li>Wang, T., 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=Genetic+screens+in+human+cells+using+the+CRISPR-Cas9+system.">Genetic screens in human cells using the CRISPR-Cas9 system.</a> Science. 343 (6166), 80-84 (2014).</li><li>Shalem, O., 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=Genome-scale+CRISPR-Cas9+knockout+screening+in+human+cells.">Genome-scale CRISPR-Cas9 knockout screening in human cells.</a> Science. 343 (6166), 84-87 (2014).</li><li>Niu, Y., 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=Generation+of+Gene-Modified+Cynomolgus+Monkey+via+Cas9/RNA-Mediated+Gene+Targeting+in+One-Cell+Embryos.">Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos.</a> Cell. 156 (4), 836-843 (2014).</li><li>Guo, X., 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=Efficient+RNA/Cas9-mediated+genome+editing+in+Xenopus+tropicalis.">Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.</a> Development. 141 (3), 707-714 (2014).</li><li>Xie, K., Yang, Y. <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+genome+editing+in+plants+using+a+CRISPR-Cas+system.">RNA-guided genome editing in plants using a CRISPR-Cas system.</a> Molecular Plant. 6 (6), 1975-1983 (2013).</li><li>Waaijers, S., 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=CRISPR/Cas9-targeted+mutagenesis+in+Caenorhabditis+elegans.">CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans.</a> Genetics. 195 (3), 1187-1191 (2013).</li><li>Bassett, A. R., Liu, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+and+Genome+Editing+in+Drosophila.">CRISPR/Cas9 and Genome Editing in Drosophila.</a> Journal of Genetics and Genomics. 41 (1), 7-19 (2014).</li><li>Schwank, G., 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=Functional+Repair+of+CFTR+by+CRISPR/Cas9+in+Intestinal+Stem+Cell+Organoids+of+Cystic+Fibrosis+Patients.">Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients.</a> Cell Stem Cell. 13 (6), 653-658 (2013).</li><li>Wu, Y., 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=Correction+of+a+Genetic+Disease+in+Mouse+via+Use+of+CRISPR-Cas9.">Correction of a Genetic Disease in Mouse via Use of CRISPR-Cas9.</a> Cell Stem Cell. 13 (6), 659-662 (2013).</li><li>Lappalainen, T. M., 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=Transcriptome+and+genome+sequencing+uncovers+functional+variation+in+humans.">Transcriptome and genome sequencing uncovers functional variation in humans.</a> Nature. 501 (7468), 506-511 (2013).</li><li>Bauer, D. E., 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=An+Erythroid+Enhancer+of+BCL11A+Subject+to+Genetic+Variation+Determines+Fetal+Hemoglobin+Level.">An Erythroid Enhancer of BCL11A Subject to Genetic Variation Determines Fetal Hemoglobin Level.</a> Science. 342 (6155), 253-257 (2013).</li><li>Engler, C., Kandzia, R., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one+pot,+one+step,+precision+cloning+method+with+high+throughput+capability.">A one pot, one step, precision cloning method with high throughput capability.</a> PloS One. 3, e3647 (2008).</li><li>Gehl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation:+theory+and+methods,+perspectives+for+drug+delivery,+gene+therapy+and+research.">Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research.</a> Acta Physiologica Scandinavica. 177, 437-447 (2003).</li><li>Zhang, S., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purifying+Plasmid+DNA+from+Bacterial+Colonies+Using+the+Qiagen+Miniprep+Kit.">Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit.</a> J. Vis. Exp. (6), e247 (2007).</li><li>Froger, A., Hall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Plasmid+DNA+into+E.+coli+Using+the+Heat+Shock+Method.">Transformation of Plasmid DNA into E. coli Using the Heat Shock Method.</a> J. Vis. Exp. (6), e253 (2007).</li><li>Finney, M., Nisson, P. E., Rashtchian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+PCR+products.">Molecular cloning of PCR products.</a> Current Protocols in Molecular Biology. Chapter 15. (Unit 15.4), (2001).</li><li>Gordanpour, A., Nam, R. K., Sugar, L., Bacopulos, S., Seth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+Detection+in+Prostate+Tumors+by+Quantitative+Real-time+PCR+(qPCR).">MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR).</a> J. Vis. Exp. (63), e3874 (2012).</li><li>Schularick, N. M., Clark, J. J., Hansen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Culture+of+Human+Vestibular+Schwannomas.">Primary Culture of Human Vestibular Schwannomas.</a> J. Vis. Exp. (89), e51093 (2014).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Blotting:+Sample+Preparation+to+Detection.">Western Blotting: Sample Preparation to Detection.</a> J. Vis. Exp. (44), e2359 (2010).</li><li>McVey, M., Lee, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMEJ+repair+of+double-strand+breaks+(director&#8217;s+cut):+deleted+sequences+and+alternative+endings.">MMEJ repair of double-strand breaks (director&#8217;s cut): deleted sequences and alternative endings.</a> Trends in Genetics. 24 (11), 529-538 (2008).</li><li>Symington, L. S., Gautier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+break+end+resection+and+repair+pathway+choice.">Double-strand break end resection and repair pathway choice.</a> Annual Review of Genetics. 45, 247-271 (2011).</li><li>Fu, Y., 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=High-frequency+off-target+mutagenesis+induced+by+CRISPR-Cas+nucleases+in+human+cells.">High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.</a> Nature Biotechnology. 31 (9), 822-826 (2013).</li><li>Wu, X., 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=Genome-wide+binding+of+the+CRISPR+endonuclease+Cas9+in+mammalian+cells.">Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.</a> Nature Biotechnology. , (2014).</li><li>Hsu, P. D., 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=DNA+targeting+specificity+of+RNA-guided+Cas9+nucleases.">DNA targeting specificity of RNA-guided Cas9 nucleases.</a> Nature Biotechnology. 31 (9), 827-832 (2013).</li><li>Fu, Y., 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=Improving+CRISPR-Cas+nuclease+specificity+using+truncated+guide+RNAs.">Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.</a> Nature Biotechnology. 32, 279-284 (2014).</li><li>Alem&#225;n, L. M., Doench, J., Sharp, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Comparison+of+siRNA-induced+off-target+RNA+and+protein+effects.">a Comparison of siRNA-induced off-target RNA and protein effects.</a> RNA. 13 (3), 385-395 (2007).</li><li>Anderson, E. M., 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=Experimental+validation+of+the+importance+of+seed+complement+frequency+to+siRNA+specificity+Experimental+validation+of+the+importance+of+seed+complement+frequency+to+siRNA+specificity.">Experimental validation of the importance of seed complement frequency to siRNA specificity Experimental validation of the importance of seed complement frequency to siRNA specificity.</a> RNA. 14, 853-861 (2008).</li><li>Marine, S., 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=Common+seed+analysis+to+identify+off-target+effects+in+siRNA+screens.">Common seed analysis to identify off-target effects in siRNA screens.</a> Journal of Biomolecular Screening. 17 (3), 370-378 (2012).</li></ol>

We have no conflicts of interest related to this report.

وكريسبر / نظام Cas9 يمكن استخدامها لتوليد الحذف الجينومية من مجموعة واسعة من الأحجام. على الرغم من أننا لاحظنا أن وتيرة حذف يختلف عكسيا فيما يتعلق يقصد حجم الحذف، وكنا قادرين على استعادة الحذف تصل إلى 1 ميغابايت، والحذف تصل إلى 100 كيلو بايت تسفر بشكل روتيني متعددة biallelic حذف الحيوانات المستنسخة. وقد لاحظنا أي خسارة في كفاءة الحذف إدخال بالتتابع في خط الخلية. هذه الاستراتيجية يمكن استخدامها لإنشاء حذف اندماجي العديد من الجينات والعناصر. يمكن المعجل عملية الحصول على استنساخ حذف biallelic عن طريق تقدير الحد الأدنى لعدد من الحيوانات المستنسخة اللازمة ليتم عرضه على أساس حجم الحذف للحصول على العدد المطلوب من الحيوانات المستنسخة مع حذف biallelic 12. القدرة على الحصول على حذف monoallelic مع غياب حذف biallelic في توزيع احتمالي يمكن أن تشير إلى الفتك الخلية المرتبطة خسارة كاملة وظيفة. منخفض الاب<html> equency أو الحذف غائبة يمكن أن يعكس عددا من السيناريوهات بما في ذلك الفقراء ترنسفكأيشن، sgRNAs غير فعالة أو غير فعالة الاشعال فحص PCR (بسبب عدم وجود مراقبة إيجابية للتحقق من صحة الاشعال PCR للكشف عن الحذف). خلايا GFP + يمكن استخدام كبديل للكفاءة ترنسفكأيشن (راجع الخطوة 5.2)، لذلك من المرجح يعكس انخفاضا في GFP + الخلايا الفقراء ترنسفكأيشن وانخفض والناتجة كفاءة الحذف. باستخدام اثنين من أزواج sgRNA مختلفة مع الاشعال فحص مستقلة يمكن أن تساعد في السيطرة على sgRNA غير فعال وفحص PCR الاشعال وتعظيم فرص الحصول على استنساخ حذف biallelic. خلية الفرز لGFP + الخلايا يثري للالأليلات الحذف. بينما هذه الخطوة قد يتم حذف أو إغفال من المرجح أن تستلزم فحص أكثر الحيوانات المستنسخة لتحديد تلك مع الحذف monoallelic أو biallelic. إلى درجة أن كفاءة ترنسفكأيشن قد يكون الأمثل، فإننا نتوقع كفاءة تحرير الجينوم ليتم تعزيزه. معشوقة = &quot;jove_content&quot;&gt; الأحداث NHEJ التي تكمن وراء الحذف ونتيجة إصلاح المحلية في سلسلة من الأليلات مع مجموعة متنوعة من indels في المواقع المستهدفة. نتائج السائدة هي ~ 1 صغيرة - 10 بي بي الإدراج أو الحذف أكثر شيوعا في موقع انشقاق sgRNA الموجهة (الشكل 2B). وغالبا ما تظهر هذه الأليلات أن تكون نتيجة القائم على microhomology إصلاح 34،35. وتجدر الإشارة إلى أن الاستراتيجية القائمة على الكشف PCR-وصفنا لن يحدد الإدراج أكبر أو أكثر تعقيدا، والحذف، العكس، أو إعادة ترتيب. على الرغم من أن هذه الأحداث هي أقل شيوعا، لاحظنا الحيوانات المستنسخة التي يمكن من خلالها الكشف عن لا حذف ولا amplicons غير الحذف، وعند إجراء مزيد من التحقيق تعكس هذه النتائج أكثر تعقيدا. وقد لاحظنا كريسبر واسعة / Cas9 بوساطة &quot;تندب&quot; من الأليلات غير الحذف من monoallelic وعدم الحذف استنساخ (انظر الشكل 2B). هذه &quot;ندوب&quot; تتكون منindels صغيرة تنتج في موقع sgRNA الانقسام دون حذف المقصود (أي حذف الجزء التدخل بين sgRNAs ألف وباء). هذه الندوب في كثير من الأحيان يقطع الاعتراف المستهدفة من قبل sgRNA. ولذا فإننا نحث الحذر في إعادة توجيه الأليلات في الخلايا التي سبق تعرضها للsgRNAs باستخدام نفس sgRNAs. استراتيجية إعادة توجيه أكثر نجاحا من شأنه الاستفادة sgRNA فريدة من نوعها تتابعات متميزة من قبل &quot;ندوب&quot; المواقع الاعتراف. في الحالات عندما يتعرف على زوج من sgRNAs تسلسل exonic (الشكل 1B، أسفل)، التي يمكن أن تنتج الأليلات انزياح الإطار حتى في حالة عدم وجود الحذف. ولذلك، استنساخ حذف monoallelic يمكن المخصب لالخسارة من وظيفة بسبب وتيرة عالية من الطفرات انزياح الإطار على غير حذف-أليل 12. واحد قلق مع كريسبر / نظام Cas9 هو خارج الهدف آثار، أي تعديل الجيني في مواقع غير مقصودة 36-38. Rوأشارت تقارير ecent أن الرنا دليل أقصر مع 17-19 النيوكليوتيدات يمكن أن تقلل من تكرار كريسبر / خارج الهدف آثار 39 Cas9 المستندة. بالإضافة إلى ذلك، استراتيجية مزدوجة تخصر باستخدام اثنين من أدلة في الموقع المستهدف مع nickase يمكن استخدامها لإنشاء DSBs مع التقليل من خارج الهدف آثار 7. بدلا من ذلك، مماثلة لالاستراتيجيات المستخدمة لرني، نقترح أن أزواج مختلفة من sgRNAs مع غير متداخلة متواليات protospacer أن تستخدم لإظهار أن النمط الظاهري لوحظ هو نتيجة للكريسبر / Cas9 تعديل على هدف مقابل لإمكانات بعيدا عن الهدف تأثير. ومن شأن اتباع نهج مناسب يكون لتصميم اثنين على الأقل المجاورة ولكن غير متداخلة أزواج sgRNA بحيث مجموعة واحدة من الاشعال الفرز (راجع الخطوة 2) يمكن أن تستخدم للأزواج sgRNA متعددة (الشكل 1A). وعلاوة على ذلك، واستكمال خط الخلية حذف من إعادة تقديم تسلسل في عداد المفقودين و / أو الجينات تعطلت يمكن إثبات وجود علاقة سببية بينحذف الجينومية معين، والنمط الظاهري. لعلماء الأحياء العمل مع أنظمة نموذج الخلوي، ورني يمثل أداة قوية لعلم الجينوم وظيفية. ومع ذلك، وشملت القيود المفروضة على هذا النهج غير مكتمل تخفيض في مستويات النص مرنا الهدف، عدم تجانس تأثير الكواشف مستقلة تستهدف نفس الجين، والمعروف خارج الهدف آثار بما في ذلك الآثار القائمة على البذور وغير المصنفة 40-42. استراتيجيات تحرير الجينوم وعد لمعالجة العديد من هذه الشواغل وتمثل توجها مثيرا، مكملة لاضطراب وراثي المحتملين 8،36،37. وعلاوة على ذلك، والتحرير الجينوم يسمح لدراسة غير الترميز العناصر الجينية بطريقة غير ممكن بواسطة رني والتي تمثل تحديا استهداف التقليدية نهج 25. ونحن نشجع الجيل الحذف الجينومية من قبل كريسبر / Cas9 كوسيلة من وسائل قوية ومحددة لإنتاج وتوصيف الخسارة من وظيفة الأليلات.

Recent advances in genome engineering technology have allowed for unprecedented opportunities for site-specific modification of the genome. This technology may be utilized to investigate the function of genes and regulatory elements via prospective genetic perturbation. Zinc finger nucleases (ZFNs), transcription-activator like (TAL) effector nucleases (TALENs), and CRISPR/Cas9 RNA-guided nucleases each leverage customizable DNA specificity to localize a nuclease for the introduction of DSBs1&#8211;3. The resulting DSBs can be repaired by indel-forming NHEJ or by homology-directed repair (HDR) using a donor template4.
The CRISPR/Cas9 nuclease pathway, an adaptive immune system in prokaryotic cells5, has been recently adapted for mammalian genome engineering2,3. This tool has been demonstrated to be an inexpensive, efficient, and reliable genome engineering technique6. Briefly, a complex of Streptococcus pyogenes-derived Cas9 nuclease and a sgRNA achieve target recognition via Watson-Crick base-pairing with cognate genomic DNA sequences. sgRNAs include 20-mer sequences complementary to genomic sequences adjacent to an obligate protospacer adjacent motif (PAM) NGG. Cas9 induces a DSB at a predictable position within the target site. Additionally, variants of Cas9 with single-strand cleavage capacity or catalytic inactivity may be used to facilitate &#8220;nicking&#8221; or transcriptional regulation respectively7&#8211;9. CRISPR/Cas9 has been used for a wide range of applications including both knock-in and knockout10,11, large-scale genomic deletions12&#8211;14, pooled library screening for gene discovery15,16, genetic engineering of numerous model organisms10,11,17&#8211;21, as well as gene therapy22,23.
Here we describe a protocol for efficient deletion of desired genomic regions. The protocol includes CRISPR design, cloning, and delivery, as well as deletion, identification, and characterization. Genomic deletions can be generated by the introduction of two CRISPR sgRNAs with Cas9 to induce repair of the resultant two DSBs by NHEJ with deletion of the intervening segment. This strategy has been used to create deletions ranging from one kilobase to over one megabase12. Deletions can be informative for the study of genes and other genetic elements, either in isolation or in combination. There are several potential advantages of genomic deletions as compared to HDR or single-site small indel production. First, this method capitalizes on the high efficiency of NHEJ in many cellular contexts7. The high frequency of deletion limits the number of clones needed to be screened to identify informative clones. Deletion frequency is inversely related to deletion size. Biallelic deletion clones may be retrieved at frequencies at least as great as of probabilistic expectation12. Second, both monoallelic and biallelic deletions may be easily identified and distinguished by conventional PCR, simplifying the screening process. Strategies relying on small indels or point mutations may require RFLP, allele-specific PCR, T7EN1 cleavage assay, Sanger sequencing, RT-qPCR, or immunoblotting, which may be more laborious. Third, by removing a substantial portion of a gene or element of interest, a reliable loss-of-function allele may be obtained. In contrast, frameshift mutations in protein-coding sequences may not always induce nonsense-mediated decay, may produce a hypomorphic or neomorphic allele, or target an exon excluded from an alternate isoform24. Finally, deletions may be particularly revealing for the study of non-coding DNA such as regulatory elements since frameshift mutations as produced by single-site indels would not be relevant25.

<table border="0" cellpadding="0" cellspacing="0" width="842">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:388px;">Name of Material/ Equipment</td>
			<td style="width:168px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 Polynucleotide Kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase (with associated ligation buffer)</td>
			<td>New England Biolabs</td>
			<td>M0202T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5&#39;-Triphosphate (ATP)</td>
			<td>New England Biolabs</td>
			<td>P0756S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA, Molecular Biology Grade</td>
			<td>New England Biolabs</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BbsI Restriction Enzyme (with associated NEB Buffer 2.1)</td>
			<td>New England Biolabs</td>
			<td>R0539S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pSpCas9(BB) (pX330)</td>
			<td>Addgene</td>
			<td>42230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S.O.C. Medium</td>
			<td>Life Technologies</td>
			<td>15544-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BTX ECM 830</td>
			<td>Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BTX Solution and 2mm Cuvettes</td>
			<td>Harvard Apparatus</td>
			<td>45-0803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pmaxGFP Plasmid</td>
			<td>Lonza</td>
			<td>VPA-1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickExtract DNA Extraction Solution</td>
			<td>Epicentre</td>
			<td>QE09050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HotStarTaq PCR Master Mix Kit</td>
			<td>Qiagen</td>
			<td>203443</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zero Blunt PCR Cloning Kit</td>
			<td>Life Technologies</td>
			<td>K2700-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of genomic deletions in mammalian cell lines via crispr/cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

وكان الهدف من هذه التجربة حذف Pim1 في الخلايا MEL. استخدام عدة أزواج غير متداخلة sgRNA (أي تسلسل protospacer المستقلة) قد يساعد في السيطرة على الآثار خارج الهدف (الشكل 1A). ومن شأن النمط الظاهري يتفق أن يكون أكثر من المرجح أن تنتج عن تأثير على الهدف بدلا من تأثير المشترك بعيدا عن الهدف تتقاسمها عدة سلاسل protospacer مستقلة. أن كل زوج أن يؤدي إلى إنتاج حذف نقطة فريدة من نوعها. إذا قريبة من بعضها البعض (أي، ن أقل من 150 ~ بي بي)، ويمكن استخدام نفس الاشعال فحص للكشف عن عمليات الحذف التي تنتجها كل مجموعة من sgRNAs. الحذف الجينومية قد يعطل الجينات باستخدام أزواج sgRNA في مواقع مختلفة فيما يتعلق الجين (الشكل 1B). على سبيل المثال، فإن الزوج قد sgRNA الجناح جين للحذف من الجسم الجين بأكمله؛ يمكن أن يكون موجودا الزوج خلال يومين الإكسونات، مع القدرة على خلق indels انزياح الإطار حتى لو واحد أو كلا علاللم يتم حذف العناوين. أو هذا الزوج قد تكتنف اكسون محدد للسماح تعطل لشكل الإسوي معين. كانت استراتيجية الحذف تستخدم لPim1 لتصميم المرافقة sgRNAs لحذف الجسم الجين بأكمله، وهو حذف 8 كيلو بايت (الشكل 2). وقد تم اختيار هذه الاستراتيجية ويرجع ذلك جزئيا إلى الحجم الصغير نسبيا من الجين Pim1. يوضح هذا المثال PAM واحد (الأخضر) على رأس (واتسون) حبلا وPAM واحد على الجزء السفلي (كريك) حبلا. ومع ذلك، DSB مستقلة للتوطين تسلسل PAM إلى حبلا العلوي أو السفلي. يمكن أزواج sgRNA على حد سواء متواليات PAM على حبلا العلوي، سواء على حبلا أسفل، أو واحدة من كل. باستخدام بروتوكول المذكورة أعلاه، وقد صممت اثنين sgRNA، المستنسخة في pX330 التعبير ناقلات، وتسليمها إلى خلايا MEL التي كتبها Electroporation للجنبا إلى جنب مع مراسل GFP (الشكل 2A). تم فرز أعلى 3٪ من الخلايا GFP + يومين بعد electroporation ومطلي clonally في الحد من التخفيف. شاشةوقد صممت الاشعال جي كما هو موضح في الخطوة 2 وكما هو مبين في الشكل (2). وقد الأمثل الظروف PCR باستخدام gDNA معزولة من خلايا MEL الأبوية ومن &quot;الأكبر&quot; خلايا فرزها. بعد 10 أيام والطلاء، وتم عزل gDNA من جميع الحيوانات المستنسخة وفرزهم للحذف عن طريق PCR، والتي حددت عدم الحذف، monoallelic واستنساخ حذف biallelic وفقا لأنماط من غير حذف (ND) وحذف (D) amplicons (الشكل 3) . وتم تحديد الحيوانات المستنسخة غير الحذف وجود وجود amplicon غير الحذف وغياب amplicon الحذف. تم تحديد استنساخ Monoallelic وجود وجود كل من غير حذف وamplicons الحذف. تم تحديد استنساخ Biallelic وجود غياب amplicon غير الحذف وبحضور amplicon الحذف. لهذا الحذف، تم فحص 400 الحيوانات المستنسخة والتي حددت 126 استنساخ حذف monoallelic و 32 DELET biallelic<html> استنساخ أيون (من المهم أن نلاحظ أن التردد الحذف يختلف مع حجم حذف 12). تم اختيار الحيوانات المستنسخة حذف Biallelic وانتقل الى قوارير مع 8 مل من وسائل الإعلام. بعد السماح 5 أيام للتوسع، تم اختبارها من قبل كل استنساخ PCR من gDNA لتأكيد تعرضوا biallelic الحذف وحذف amplicons لسانجر تسلسل لتحديد حذف الدقيق (الشكل 2B). عدم التجانس داخل amplicons الحذف يعكس ناقص تشكيل INDEL NHEJ إصلاح. تسلسل أليل غير الحذف في استنساخ حذف monoallelelic كشف indels في معظم الحالات، مما يدل على أنه حتى أليل غير حذف وكثيرا ما تم بواسطة كريسبر / Cas9، والتي قد تكون مهمة للتطبيقات حيث كلاهما مطلوب monoallelic وbiallelic استنساخ الحذف . تم عزل الحمض النووي الريبي من استنساخ حذف biallelic وتحليلها بواسطة RT-QPCR لتأكيد فقدان Pim1 التعبير (الشكل 4). طرق &quot;&gt; <img alt="الشكل (1)" src="/files/ftp_upload/52118/52118fig1highres.jpg" /> الشكل 1. تخطيطي لاستراتيجيات الحذف ممكنة. (A) اثنين من أزواج سبيل المثال sgRNA لحذف الجينومية (كما هو موضح باللون الأسود والبرتقالي، على التوالي). الأسهم الزرقاء تشير الاشعال للكشف عن amplicon غير الحذف والسهام الحمراء تشير الاشعال للكشف عن amplicon الحذف. sgRNA المواقف 17 و 18 ويسلط الضوء باللون الأحمر والأزرق في sgRNA-مواقع-A 1 / A 2 ثم يتم تمييز في اللون الأرجواني والبرتقالي في sgRNA-مواقع-B 1 / B 2 مع خط أحمر يشير إلى انشقاق Cas9 توقع بين المواقف 17 وترد 18. (B) الانشقاقات الموجهة كريسبر كخطوط سوداء العمودية. الأسهم الزرقاء تشير الاشعال للكشف عن amplicon غير الحذف والسهام الحمراء تشير الاشعال للكشف عن amplicon الحذف. <a href="https://www.jove.com/files/ftp_upload/52118/52118fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض</a>نسخة أكبر من هذا الرقم. <img alt="الشكل 2" src="/files/ftp_upload/52118/52118fig2highres.jpg" /> الشكل 2. استراتيجية لإنتاج وكشف عن حذف Pim1، بما في ذلك حذف التسلسل والأليلات غير الحذف. (A) تخطيطي للاستراتيجية فحص PCR القائم على تحديد Pim1 استنساخ الحذف. يقع زوج واحد التمهيدي الداخلية لحذف (الأسهم الزرقاء) وزوج التمهيدي واحد المترجمة خارجي لحذف (الأسهم الحمراء). وتظهر تسلسل sgRNA (تسلسل protospacer) باللون الأرجواني. الخطوط الحمراء العمودية تشير إلى انشقاق Cas9 توقع بين المواقف 17 و 18 من تسلسل sgRNA. (B) سانجر تسلسل يكشف تشكيل INDEL في موقع الاعتراف sgRNA. وتظهر تسلسل sgRNA في تسلسل الأرجواني وPAM باللون الأخضر. أحداث الحذف هي SHالخاصة من قبل عدد مساو من علامات اندفاعة والإدراج ويسلط الضوء في الزرقاء. خطوط حمراء عمودية تشير إلى توقع الموقع الانقسام، بين المواقف 17 و 18 من sgRNA. <a href="https://www.jove.com/files/ftp_upload/52118/52118fig2large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> <img alt="الشكل (3)" src="/files/ftp_upload/52118/52118fig3highres.jpg" /> الشكل (3). هلام الممثل تحديد عدم الحذف، monoallelic، واستنساخ biallelic للحصول على كل استنساخ، الممر الأيسر يمثل عدم الحذف-amplicon (&quot;ND&quot; باللون الأزرق) والممر الأيمن يمثل amplicon حذف (&quot;D&quot; في الحمراء ). يتم فصل الحيوانات المستنسخة الفردية عن طريق الخطوط المنقطة. استنساخ حذف monoallelic الثلاثة هي استنساخ 5 و 6 و 2. استنساخ حذف biallelic الثلاثة هي استنساخ 15، 17، و 13. كما رأينا في تسلسل البيانات، والفرقة الحذف لج<html> وحيد 13 لديها حجم أصغر من المقرر أن الحذف أكبر عند تقاطع الحذف. <img alt="الشكل (4)" src="/files/ftp_upload/52118/52118fig4highres.jpg" /> تم احتساب الرقم 4. فقدان Pim1 التعبير في استنساخ حذف biallelic. Pim1 التعبير عن اثنين من الحيوانات المستنسخة حذف biallelic التي كتبها RT-QPCR. تم تطبيع البيانات لGAPDH باستخدام 2 - طريقة ΔCt. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> Protospacer تسلسل </td></tr><tr><td> sgRNA-A </td><td> 5&#39;-TAGGAAAATGACTCGTCACT-3 &quot; </td></tr><tr><td> sgRNA-B </td><td> 5&#39;-GTCTATCCTATCTCGATAAA-3 &quot; </td></tr></tbody></table> الجدول 1. 20 مير تسلسل protospacer لمدة sgRNA لحذف Pim1. 543 &quot;&gt; <tbody><tr height="23"><td height="23" style="height:23px;width:101px;"></td><td style="width:443px;"> تكملة العكسية للProtospacer تسلسل </td></tr><tr height="23"><td height="23" style="height:23px;"> sgRNA-A-RC </td><td> 5&#39;-AGTGACGAGTCATTTTCCTA-3 &quot; </td></tr><tr height="23"><td height="23" style="height:23px;"> sgRNA-B-RC </td><td> 5&#39;-TTTATCGAGATAGGATAGAC-3 &quot; </td></tr></tbody></table> الجدول 2. تكملة عكسي من متواليات protospacer لsgRNA اثنين من الجدول 1. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> متواليات </td></tr><tr><td> sgRNA-A </td><td> 5&#39;-CACCTAGGAAAATGACTCGTCACT-3 &quot; </td></tr><tr><td> sgRNA-A-RC </td><td> 5&#39;-AAACAGTGACGAGTCATTTTCCTA-3 &quot; </td></tr><tr><td> sgRNA-B </td><td> 5&#39;-CACCGTCTATCCTATCTCGATAAA-3 &quot; </td></tr><tr><td> sgRNA-B-RC </td><td> 5&#39;-AAACTTTATCGAGATAGGATAGAC-3 &quot; </td></tr></tbody></table> الجدول 3. تسلسل Protospacer والعكس بهم يكمل مع &quot;CACC&quot; و &quot;AAAC&quot; وأضافت للاستنساخ في ناقلات pX330 باستخدام BBSI انزيم قيود. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> متواليات </td></tr><tr><td> sgRNA-A </td><td> 5&#39;- CACCGTAGGAAAATGACTCGTCACT-3 &quot; </td></tr><tr><td> sgRNA-A-RC </td><td> 5&#39;- AAACAGTGACGAGTCATTTTCCTAC-3 &quot; </td></tr><tr><td> sgRNA-B </td><td> 5&#39;- CACCGTCTATCCTATCTCGATAAA-3 &quot; </td></tr><tr><td> sgRNA-B-RC </td><td> 5&#39;- AAACTTTATCGAGATAGGATAGAC-3 &quot; </td></tr></tbody></table> ومما يعزز الجدول التعبير 4. sgRNA من المروج U6 من متجه pX330 من خلال إضافة النوكليوتيدات G بعد تسلسل CACC وقبل20-مير. إضافة إلى G النوكليوتيدات إضافية يتطلب إضافة النوكليوتيدات C في نهاية 3 &#39;من تكملة بنسبة ضئيلة العكسي (على سبيل المثال، sgRNA-A). ومع ذلك، إذا كان المركز الأول من بين 20 مير (تسلسل protospacer) هو بالفعل النوكليوتيدات G، ليست هناك حاجة لإضافة G آخر (على سبيل المثال، sgRNA-B)، وهناك حاجة لإضافة C إلى الموقف النهائي للمكملا عكسي بنسبة ضئيلة.

Watch this Scientific Journal Video about Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 at JoVE.com

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

جيل من الجينوم الحذف في خطوط خلايا الثدييات عبر كريسبر / Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

generation-of-genomic-deletions-in-mammalian-cell-lines-via-crisprcas9

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 2

Analyzing Gene Expression and Function

SCIENTISTS IN ACTION

94.9K Views.  Harvard Medical School. CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

Video: Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

esiRNA بعثة لفحص رني في خلايا الثدييات

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

عملية الوحش الأخضر لتوليد سلالات الخميرة حمل حذوفات متعددة الجينات

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

جيل من خطوط الخلايا البشرية مستقرة مع shRNA (تيت-on) التتراسيكلين أو محرض التعبير [كدنا]

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

نمو اللاهوائية وصيانة خطوط خلايا الثدييات

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

إسكات شراره: CRISPR/كاس 9 الجينوم التحرير في الأسماك الكهربائية ضعيف

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

هندسة الجينوم للخلايا B البشرية الأولية باستخدام CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&amp;#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

TGF-β بوساطة البطانية إلى الانتقال المتوسط (EndMT) والتقييم الوظيفي لمؤثري EndMT باستخدام CRISPR/Cas9 تحرير الجينات

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

بناء طفرات متماثلة الزيجوت من الجراد المهاجر باستخدام تقنية كريسبر / كاس 9

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

كريسبر / كاس 9 التحرير الجيني للخلايا الجذعية المكونة للدم والخلايا السلفية لتطبيقات العلاج الجيني

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

تعديلات دقيقة بوساطة CRISPR-Cas9 في قلوب الزرد

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...