Name: generation of genomic deletions in mammalian cell lines via crispr/cas9
Uploaded: 2015-01-03T14:00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 at JoVE.com

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.

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We have no conflicts of interest related to this report.

CRISPR / Cas9 प्रणाली आकारों की एक श्रेणी के जीनोमिक विलोपन उत्पन्न करने के लिए इस्तेमाल किया जा सकता है। हम हटाए जाने की आवृत्ति इरादा विलोपन आकार के संबंध में विपरीत रूप से भिन्न होता है देखा है कि हालांकि, हम नियमित रूप से कई biallelic नष्ट कर दिया क्लोन उपज 100 KB अप करने के लिए अप करने के लिए 1 एमबी का विलोपन, और विलोपन ठीक करने में सक्षम किया गया है। हम एक सेल लाइन में क्रमिक रूप से शुरू करने विलोपन की दक्षता में कोई नुकसान नहीं मनाया है। इस रणनीति के कई जीन और तत्वों की मिश्रित विलोपन के निर्माण के लिए इस्तेमाल किया जा सकता है। biallelic विलोपन क्लोन प्राप्त करने की प्रक्रिया biallelic विलोपन 12 के साथ क्लोन की वांछित संख्या प्राप्त करने के लिए हटाए जाने के आकार के आधार पर जांच किए जाने की आवश्यकता क्लोन की न्यूनतम संख्या का आकलन करके तेजी लाई जा सकती है। संभाव्य वितरण में biallelic विलोपन के अभाव के साथ monoallelic विलोपन प्राप्त करने की क्षमता समारोह का पूरा नुकसान के साथ जुड़े सेल मारक का संकेत मिलता है। कम frequency या अनुपस्थित विलोपन गरीब अभिकर्मक, अक्षम sgRNAs या कारण (एक सकारात्मक नियंत्रण की कमी के कारण हटाए जाने के लिए स्क्रीन के लिए पीसीआर प्राइमरों मान्य करने के लिए) अकुशल पीसीआर स्क्रीनिंग प्राइमरों सहित परिदृश्यों की एक संख्या को प्रतिबिंबित कर सके। GFP + कोशिकाओं, अभिकर्मक दक्षता (कदम 5.2 देखें) के लिए एक किराए के रूप में इस्तेमाल किया जा सकता है ताकि GFP + कोशिकाओं में कमी होने की संभावना गरीब अभिकर्मक को दर्शाता है और जिसके परिणामस्वरूप एक विलोपन दक्षता की कमी हुई। अक्षम sgRNA के लिए नियंत्रण में मदद कर सकते हैं स्वतंत्र स्क्रीनिंग प्राइमरों के साथ दो अलग अलग sgRNA जोड़े का प्रयोग और पीसीआर प्राइमरों स्क्रीनिंग और biallelic विलोपन क्लोन प्राप्त करने की संभावना को अधिकतम। GFP + कोशिकाओं के लिए सेल छँटाई विलोपन alleles के लिए समृद्ध करती है। इस कदम से हटाया जा सकता है, चूक की संभावना monoallelic या biallelic विलोपन के साथ उन लोगों की पहचान करने के लिए और अधिक क्लोन स्क्रीनिंग की जरूरत होगी। अभिकर्मक दक्षता अनुकूलित किया जा सकता है कि डिग्री करने के लिए, हम जीनोम संपादन दक्षता बढ़ाने की उम्मीद है। <p cलड़की = &quot;jove_content&quot; लक्ष्य स्थलों पर indels की एक किस्म के साथ alleles की एक श्रृंखला में विलोपन और स्थानीय मरम्मत परिणाम आबाद है कि&gt; NHEJ घटनाओं। 10 बीपी सम्मिलन या अधिक सामान्यतः sgRNA निर्देशित दरार (चित्रा 2B) की साइट पर विलोपन - प्रमुख परिणाम छोटे ~ 1 है। अक्सर इन alleles के microhomology आधारित मरम्मत 34,35 का नतीजा हो दिखाई देते हैं। यह हम वर्णन पीसीआर आधारित पता लगाने की रणनीति बड़ा या अधिक जटिल सम्मिलन, विलोपन, व्युत्क्रमों, या rearrangements की पहचान नहीं होगा कि ध्यान दिया जाना चाहिए। इन घटनाओं में कम आम हैं, यद्यपि हम न तो विलोपन और न ही गैर-विलोपन amplicons पता लगाया जा सकता है जिसमें क्लोन मनाया, और आगे की जांच पर इन अधिक जटिल परिणामों को प्रतिबिंबित किया है। हम व्यापक CRISPR / monoallelic और गैर-विलोपन क्लोन से गैर-विलोपन alleles की &quot;scarring के&quot; Cas9 की मध्यस्थता मनाया है (चित्रा 2B देखें)। ये &quot;निशान&quot; से मिलकर बनता हैइच्छित विलोपन के बिना sgRNA दरार स्थल पर उत्पादन छोटे indels (यानी, sgRNAs ए और बी के बीच बीच के खंड का विलोपन)। ये निशान अक्सर sgRNA द्वारा लक्ष्य मान्यता में व्यवधान डाला। इसलिए हम पहले से ही sgRNAs का उपयोग कर sgRNAs के संपर्क में कोशिकाओं में alleles के retargeting में सावधानी से आग्रह करता हूं। अद्वितीय sgRNA का उपयोग होगा एक और अधिक सफल पुनर्लक्ष्यीकरण रणनीति पहले &quot;जख्म&quot; मान्यता साइटों से अलग दृश्यों। मामलों में sgRNAs की एक जोड़ी (चित्रा 1 बी, नीचे), frameshift एलील भी विलोपन के अभाव में उत्पादन किया जा सकता है exonic दृश्यों को पहचानता है। इसलिए, monoallelic विलोपन क्लोन कारण एलील 12 गैर नष्ट कर दिया पर frameshift म्यूटेशन के उच्च आवृत्ति को नुकसान के समारोह के लिए समृद्ध किया जा सकता है। 38 - CRISPR / Cas9 प्रणाली के साथ एक चिंता का विषय बंद लक्ष्य प्रभाव, यानी, अनायास ही साइटों 36 पर जीनोमिक संशोधन है। आरecent रिपोर्टों 17 के साथ छोटे गाइड RNAs सुझाव दिया है कि - 19 न्यूक्लियोटाइड CRISPR की आवृत्ति को कम कर सकते हैं / बंद लक्ष्य प्रभाव 39 Cas9 आधारित। इसके अतिरिक्त, एक nickase साथ लक्ष्य साइट प्रति दो गाइडों का उपयोग करते हुए एक डबल Nicking रणनीति बंद लक्ष्य प्रभाव सात जबकि कम से कम DSBs बनाने के लिए इस्तेमाल किया जा सकता है। वैकल्पिक रूप से, आरएनएआई के लिए इस्तेमाल किया रणनीति के अनुरूप है, हम गैर अतिव्यापी protospacer दृश्यों के साथ sgRNAs के विभिन्न जोड़े बंद लक्ष्य एक संभावित विरोध के रूप में मनाया फेनोटाइप पर लक्ष्य CRISPR / Cas9 संशोधन का परिणाम है कि प्रदर्शन करने के लिए इस्तेमाल किया जा सुझाव है कि प्रभाव। एक सुविधाजनक दृष्टिकोण स्क्रीनिंग प्राइमरों (चरण 2 देखें) का एक सेट एकाधिक sgRNA जोड़े (चित्रा 1 ए) के लिए इस्तेमाल किया जा सकता है, ताकि कम से कम दो आसन्न लेकिन गैर अतिव्यापी sgRNA जोड़े डिजाइन करने के लिए किया जाएगा। इसके अलावा, लापता अनुक्रम और / या बाधित जीन reintroducing द्वारा एक विलोपन सेल लाइन के पूरक के बीच एक कारण रिश्ते को पुष्ट कर सकते हैंएक दिया जीनोमिक विलोपन और फेनोटाइप। सेलुलर मॉडल सिस्टम के साथ काम कर रहे जीव के लिए, आरएनएआई कार्यात्मक जीनोमिक्स के लिए एक शक्तिशाली उपकरण का प्रतिनिधित्व किया है। 42 - हालांकि, इस दृष्टिकोण की सीमाओं लक्ष्य mRNA प्रतिलेख के स्तर में अधूरा कमी, एक ही जीन को लक्षित स्वतंत्र अभिकर्मकों के प्रभाव की विविधता, और बीज आधारित और गैर-बीज प्रभाव 40 सहित ज्ञात बंद लक्ष्य प्रभाव को शामिल किया है। जीनोम संपादन रणनीतियों इन चिंताओं में से कई को संबोधित करने का वादा और भावी आनुवंशिक गड़बड़ी 8,36,37 के लिए एक रोमांचक, पूरक दृष्टिकोण का प्रतिनिधित्व करते हैं। इसके अलावा, जीनोम संपादन आरएनएआई के द्वारा ही संभव है और पारंपरिक लक्ष्य-निर्धारण से चुनौतीपूर्ण नहीं एक तरह से आनुवंशिक तत्वों गैर-कोडिंग 25 दृष्टिकोण के अध्ययन के लिए अनुमति देता है। हम उत्पादन और नुकसान के समारोह एलील चिह्नित करने के लिए एक मजबूत और विशिष्ट पद्धति के रूप में CRISPR / Cas9 द्वारा जीनोमिक विलोपन की पीढ़ी के लिए प्रोत्साहित करते हैं।

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.

इस प्रयोग के लक्ष्य एमईएल कोशिकाओं में Pim1 का विलोपन था। कई गैर अतिव्यापी sgRNA जोड़े (यानी, स्वतंत्र protospacer दृश्यों) का प्रयोग बंद लक्ष्य प्रभाव (चित्रा 1 ए) के लिए नियंत्रित करने में मदद कर सकते हैं। एक सुसंगत phenotype के कई स्वतंत्र protospacer दृश्यों द्वारा साझा एक आम बंद लक्ष्य प्रभाव के लिए विरोध के रूप में एक पर लक्ष्य प्रभाव से परिणाम के लिए और अधिक होने की संभावना होगी। प्रत्येक जोड़ी एक अद्वितीय विलोपन ब्रेकपाइंट के उत्पादन के लिए नेतृत्व करेंगे। (यानी, एन ~ 150 बीपी से भी कम) करीब एक साथ, एक ही स्क्रीनिंग प्राइमरों sgRNAs के प्रत्येक सेट के द्वारा उत्पादित विलोपन का पता लगाने के लिए इस्तेमाल किया जा सकता है। जीनोमिक विलोपन जीन (चित्रा 1 बी) के संबंध में विभिन्न स्थानों में sgRNA जोड़े का उपयोग करके जीन को बाधित कर सकते हैं। उदाहरण के लिए, sgRNA जोड़ी पूरे जीन शरीर के विलोपन के लिए एक जीन पार्श्व सकता है; जोड़ी frameshift indels बनाने की क्षमता के साथ, दो एक्सॉनों के भीतर स्थित हो सकता है, भले ही एक या दोनों allelतों हटाया नहीं गया था; या जोड़ी एक विशेष isoform के विघटन की अनुमति के लिए एक विशिष्ट एक्सॉन पार्श्व सकता है। Pim1 के लिए इस्तेमाल किया हटाए जाने की रणनीति पूरे जीन शरीर, एक 8 केबी विलोपन (चित्रा 2) को हटाने के लिए sgRNAs flanking डिजाइन करने के लिए किया गया था। इस रणनीति के कारण Pim1 जीन की अपेक्षाकृत छोटे आकार के हिस्से में चुना गया था। इस उदाहरण नीचे (क्रिक) कतरा पर शीर्ष (वाटसन) कतरा और एक पीएएम पर एक पीएएम (हरा) से पता चलता है; हालांकि, डीएसबी ऊपर या नीचे किनारा करने के लिए पीएएम अनुक्रम स्थानीयकरण से स्वतंत्र है। sgRNA जोड़े नीचे किनारा पर, शीर्ष किनारा पर दोनों दोनों पीएएम दृश्यों है, या प्रत्येक का एक कर सकते हैं। ऊपर वर्णित प्रोटोकॉल का उपयोग करना, दो sgRNA, डिजाइन pX330 अभिव्यक्ति वेक्टर में क्लोन है, और एक GFP रिपोर्टर (2A चित्रा) के साथ-साथ electroporation द्वारा एमईएल कोशिकाओं को दिया गया। GFP + कोशिकाओं के शीर्ष 3% दो दिनों के बाद electroporation हल और कमजोर पड़ने सीमित पर clonally चढ़ाया गया। स्क्रीनचित्रा 2 में दिखाया गया है आईएनजी प्राइमरों चरण 2 में वर्णित के रूप में बनाया गया है और कर रहे थे। पीसीआर स्थितियों पैतृक एमईएल कोशिकाओं से और &quot;थोक&quot; हल कोशिकाओं से अलग gDNA का उपयोग कर अनुकूलित किया गया। 10 दिनों के चढ़ाना के बाद, gDNA सभी क्लोन से अलग किया गया था और गैर-विलोपन (एनडी) और विलोपन (डी) amplicons के पैटर्न के अनुसार गैर-विलोपन, monoallelic और biallelic विलोपन क्लोन की पहचान की है, जो पीसीआर के माध्यम से हटाए जाने, के लिए जांच (चित्रा 3) । गैर-विलोपन क्लोन गैर-विलोपन amplicon की उपस्थिति और विलोपन amplicon के अभाव होने के रूप में पहचान की गई। Monoallelic क्लोन गैर-विलोपन और विलोपन amplicons दोनों की उपस्थिति होने के रूप में पहचान की गई। Biallelic क्लोन गैर-विलोपन amplicon के अभाव और विलोपन amplicon की उपस्थिति होने के रूप में पहचान की गई। इस विलोपन के लिए, 400 क्लोन जांच की गई 126 monoallelic विलोपन क्लोन और 32 biallelic delet जो पहचान<html> आयन क्लोन (यह विलोपन आवृत्ति विलोपन आकार 12 के साथ बदलता रहता है कि नोट करना महत्वपूर्ण है)। Biallelic विलोपन क्लोन चयनित और मीडिया के 8 मिलीलीटर के साथ बोतल के लिए ले जाया गया। विस्तार के लिए 5 दिन की इजाजत देने के बाद, प्रत्येक क्लोन biallelic विलोपन और विलोपन amplicons सटीक विलोपन (चित्रा 2B) की पहचान करने के लिए सेंगर अनुक्रमण के अधीन थे पुष्टि करने के लिए gDNA की पीसीआर द्वारा पुनर्परीक्षण किया गया था। विलोपन amplicons के भीतर विविधता अपूर्ण INDEL के गठन NHEJ मरम्मत को दर्शाता है। मामलों के बहुमत में indels खुला monoallelelic विलोपन क्लोन में गैर-विलोपन एलील की अनुक्रमण, monoallelic और biallelic विलोपन क्लोन दोनों आवश्यक हैं जहां अनुप्रयोगों के लिए महत्वपूर्ण हो सकता है, जो भी गैर-हटाए एलील अक्सर CRISPR / Cas9 द्वारा संपादित किया है कि प्रदर्शन । शाही सेना biallelic विलोपन क्लोन से अलग और (चित्रा 4) Pim1 अभिव्यक्ति की हानि पुष्टि करने के लिए आर टी-qPCR के द्वारा विश्लेषण किया गया था। तरीके &quot;&gt; <img alt="चित्रा 1" src="/files/ftp_upload/52118/52118fig1highres.jpg" /> संभव विलोपन रणनीतियों के चित्रा 1. योजनाबद्ध। (ए) जीनोमिक विलोपन के लिए दो उदाहरण sgRNA जोड़े (क्रमशः, काले और नारंगी में दिखाया गया है)। नीले तीर गैर-विलोपन amplicon पता लगाने के लिए प्राइमरों से संकेत मिलता है और लाल तीर विलोपन amplicon पता लगाने के लिए प्राइमरों संकेत मिलता है। sgRNA 17 पदों और 18 पर लाल और नीले रंग में डाला जाता है sgRNA साइटों-एक 1 / ए 2 और पदों पर 17 के बीच की भविष्यवाणी की Cas9 दरार का संकेत एक लाल रेखा के साथ sgRNA साइटों बी 1 / बी 2 पर बैंगनी और नारंगी में डाला जाता है और 18 (बी) के CRISPR निर्देशित चोली खड़ी काले रंग की लाइनों के रूप में दिखाया जाता है। नीले तीर गैर-विलोपन amplicon पता लगाने के लिए प्राइमरों से संकेत मिलता है और लाल तीर विलोपन amplicon पता लगाने के लिए प्राइमरों संकेत मिलता है। <a href="https://www.jove.com/files/ftp_upload/52118/52118fig1large.jpg" target="_blank">देखने के लिए यहां क्लिक करें</a>इस आंकड़े का एक बड़ा संस्करण। <img alt="चित्रा 2" src="/files/ftp_upload/52118/52118fig2highres.jpg" /> चित्रा उत्पादन और अनुक्रमण विलोपन और गैर-विलोपन एलील सहित Pim1 का विलोपन, पता लगाने के लिए 2. रणनीति। पीसीआर आधारित स्क्रीनिंग रणनीति की (ए) योजनाबद्ध Pim1 विलोपन क्लोन की पहचान करने के लिए। एक प्राइमर जोड़ी विलोपन (नीले तीर) और एक प्राइमर जोड़ी के लिए आंतरिक स्थित है विलोपन (लाल तीर) के लिए बाहरी स्थानीय है। sgRNA दृश्यों (protospacer दृश्यों) बैंगनी रंग में दिखाया गया है। खड़ी लाल लाइनों के पदों पर 17 और sgRNA अनुक्रम में से 18 के बीच की भविष्यवाणी की Cas9 दरार का संकेत मिलता है। (बी) सेंगर अनुक्रमण sgRNA मान्यता स्थल पर INDEL गठन का पता चलता है। sgRNA दृश्यों हरे रंग में बैंगनी और PAM दृश्यों में दिखाया गया है। विलोपन घटनाओं श कर रहे हैंपानी का छींटा के निशान और सम्मिलन के एक बराबर संख्या से खुद को नीले रंग में डाला जाता है। कार्यक्षेत्र लाल लाइनों के पदों पर 17 और sgRNA के 18 के बीच।, दरार साइट की भविष्यवाणी संकेत मिलता है <a href="https://www.jove.com/files/ftp_upload/52118/52118fig2large.jpg" target="_blank">यह आंकड़ा का एक बड़ा संस्करण देखने के लिए यहां क्लिक करें।</a> <img alt="चित्रा 3" src="/files/ftp_upload/52118/52118fig3highres.jpg" /> चित्रा 3। गैर-विलोपन, monoallelic, और biallelic क्लोन की पहचान के प्रतिनिधि जेल। प्रत्येक क्लोन के लिए, बाईं लेन गैर-विलोपन amplicon (नीले रंग में &quot;एन डी&quot;) और सही लेन का प्रतिनिधित्व करता है लाल रंग में विलोपन amplicon (&quot;डी&quot; का प्रतिनिधित्व करता है )। व्यक्तिगत क्लोन बिंदीदार रेखा से अलग हो रहे हैं। तीन monoallelic विलोपन क्लोन क्लोन 5, 6 हैं, और अनुक्रमण डेटा के रूप में देखा 2. तीन biallelic विलोपन क्लोन, ग के लिए विलोपन बैंड क्लोन 15, 17, और 13 हैं<html> लोन 13 विलोपन जंक्शन पर बड़ा विलोपन की वजह से एक छोटे आकार की है। <img alt="चित्रा 4" src="/files/ftp_upload/52118/52118fig4highres.jpg" /> Biallelic विलोपन क्लोन में Pim1 अभिव्यक्ति की चित्रा 4 में कमी। Pim1 अभिव्यक्ति आर टी-qPCR के द्वारा दो biallelic विलोपन क्लोन के लिए गणना की गई। ΔCt विधि - डेटा 2 का उपयोग कर GAPDH के लिए सामान्यीकृत किया गया था। <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> Protospacer अनुक्रम </td></tr><tr><td> sgRNA-ए </td><td> 5&#39;-TAGGAAAATGACTCGTCACT-3 &#39; </td></tr><tr><td> sgRNA-बी </td><td> 5&#39;-GTCTATCCTATCTCGATAAA-3 &#39; </td></tr></tbody></table> टेबल Pim1 का विलोपन के लिए दो sgRNA के लिए 1. 20 मेर protospacer दृश्यों। 543 &quot;&gt; <tbody><tr height="23"><td height="23" style="height:23px;width:101px;"></td><td style="width:443px;"> Protospacer अनुक्रम के रिवर्स पूरक </td></tr><tr height="23"><td height="23" style="height:23px;"> sgRNA-ए-आर.सी. </td><td> 5&#39;-AGTGACGAGTCATTTTCCTA-3 &#39; </td></tr><tr height="23"><td height="23" style="height:23px;"> sgRNA-बी आर सी </td><td> 5&#39;-TTTATCGAGATAGGATAGAC-3 &#39; </td></tr></tbody></table> तालिका 1 से दो sgRNA के लिए protospacer दृश्यों की तालिका 2 रिवर्स पूरक। <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> दृश्यों </td></tr><tr><td> sgRNA-ए </td><td> 5&#39;-CACCTAGGAAAATGACTCGTCACT-3 &#39; </td></tr><tr><td> sgRNA-ए-आर.सी. </td><td> 5&#39;-AAACAGTGACGAGTCATTTTCCTA-3 &#39; </td></tr><tr><td> sgRNA-बी </td><td> 5&#39;-CACCGTCTATCCTATCTCGATAAA-3 &#39; </td></tr><tr><td> sgRNA-बी आर सी </td><td> 5&#39;-AAACTTTATCGAGATAGGATAGAC-3 &#39; </td></tr></tbody></table> तालिका 3. Protospacer दृश्यों और उनके रिवर्स &quot;CACC&quot; और &quot;AAAC&quot; के साथ पूरक BbsI प्रतिबंध एंजाइम का उपयोग कर pX330 वेक्टर में क्लोनिंग के लिए जोड़ा गया। <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> दृश्यों </td></tr><tr><td> sgRNA-ए </td><td> 5&#39;- CACCGTAGGAAAATGACTCGTCACT-3 &#39; </td></tr><tr><td> sgRNA-ए-आर.सी. </td><td> 5&#39;- AAACAGTGACGAGTCATTTTCCTAC-3 &#39; </td></tr><tr><td> sgRNA-बी </td><td> 5&#39;- CACCGTCTATCCTATCTCGATAAA-3 &#39; </td></tr><tr><td> sgRNA-बी आर सी </td><td> 5&#39;- AAACTTTATCGAGATAGGATAGAC-3 &#39; </td></tr></tbody></table> PX330 वेक्टर के U6 प्रमोटर से तालिका 4 sgRNA अभिव्यक्ति CACC अनुक्रम के बाद और पहले एक जी न्यूक्लियोटाइड के अलावा द्वारा बढ़ाया है20-मेर। एक अतिरिक्त जी न्यूक्लियोटाइड के अलावा रिवर्स पूरक oligo के 3 &#39;के अंत (जैसे, sgRNA-ए) में एक सी न्यूक्लियोटाइड के अलावा आवश्यकता है। 20-मेर (protospacer अनुक्रम) के पहले की स्थिति में पहले से ही एक जी न्यूक्लियोटाइड है हालांकि, अगर एक और जी जोड़ने के लिए कोई जरूरत नहीं है (उदाहरण के लिए, sgRNA-बी) और रिवर्स पूरक की अंतिम स्थिति के लिए सी जोड़ने के लिए कोई ज़रूरत नहीं oligo।

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.

CRISPR / Cas9 के माध्यम से स्तनधारी सेल लाइनों में जीनोमिक विलोपन का सृजन

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

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Unit 2

Analyzing Gene Expression and Function

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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.

आरएनएआई स्क्रीनिंग के लिए स्तनधारी कोशिकाओं में मिशन esiRNA

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 ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

जीनोमिक डीएनए / आरएनए और डीएनए संकर की शाही सेना युक्त परिवर्तन का उपयोग कर oligonucleotides द्वारा जनरेशन

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

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.

खमीर एकाधिक जीन विलोपन ले उपभेदों की पीढ़ी के लिए ग्रीन दानव प्रक्रिया

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.

टेट्रासाइक्लिन-inducible (Tet पर) shRNA या सीडीएनए अभिव्यक्ति के साथ स्थिर मानव कोशिका लाइनों का सृजन

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.

Anaerobic विकास और स्तनधारी सेल लाइनों के रखरखाव

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.

स्पार्क को सिलिंग: कमजोर इलेक्ट्रिक मछली में CRISPR/Cas9 जीनोम संपादन

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.

CRISPR/Cas9 का उपयोग कर प्राथमिक मानव बी कोशिकाओं के जीनोम इंजीनियरिंग

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.

टीजीएफ-β-मध्यस्थता एंडोथेलियल से मेसेंचिमल ट्रांजिशन (एंडएमटी) और CRISPR/Cas9 जीन संपादन का उपयोग करके एंडएमटी प्रभावकर्ताओं का कार्यात्मक मूल्यांकन

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.

CRISPR/Cas9 प्रौद्योगिकी का उपयोग करके प्रवासी टिड्डियों के होमोजीगस उत्परिवर्ती का निर्माण

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.