Bu raporla ilgili hiçbir çatışması vardır.

Tüm hayvan bakımı ve deneysel prosedürler Ulusal Sağlık enstitüleri ve kurumsal hayvan bakım ve kullanım Komitesi (IACUC) Michigan Üniversitesi'nde kılavuz izledi. Ve ipuçları protokol boyunca pipet RNase free çözümleri kullanın. Çalışma alanı, Pipetler, tüpler ve santrifüj ( Malzeme tabloyabakın) Üretici yönergeleri izleyerek RNase dekontaminasyon çözüm ile temiz.
1. sgRNA hedef seçimi
<ol><li>Bir web tarayıcısı kullanarak Web sayfasını açın: http://crispor.tefor.net8<ol>
<li>~ 60 baz çifti (bp) istenen düzenleme ilgi kanat sırası giriş (Yani 30 bp 5' ve 3' istenen düzenleme).</li>
		<li>Caenorhabditis elegans genom açılan menüden seçin.</li>
		<li>Protospacer bitişik Motif seçin (20 bp-NGG - SpCas9, SpCas9-HF1, eSPCas9 1.1) açılan menüsünden.</li>
		<li>Gönder'i tıklatın. Sonuç sayfası faiz düzen için en yakın üst sırada hedef sırası seçin. Kanat 60 bp Giriş dizisi içinde hiçbir uygun hedef site varsa, en fazla 100 sorgu sırası genişletin bp (Yani 50 bp istenen her iki tarafında düzenleme).</li>
	</ol>
</li>
	<li>Bir kaynaktan, örneğin synthego, aşağıdaki özelliklere sahip bir 20-mer hedef sgRNA elde etmek: 3 nmol; modifikasyon yok.</li>
	<li>Aldıktan sonra liyofilize sgRNA tüp, içeren santrifüj kapasitesi > 12.000 x g oda sıcaklığında 1 dk için. Tüp TE nükleaz Ücretsiz 60 µL ekleyin ve hafifçe yukarı ve aşağı kullanarak 15 - 20 kez P200 pipet pipetting tarafından yeniden askıya. Son 50 µM bölgedir.</li>
</ol>2. homoloji-yönetmen onarım şablon tasarımı
<ol><li>Bir ssODN HDR şablonu içeren tasarım: 1) faiz mutasyon, 2) bir benzersiz çerçeve kısıtlama endonükleaz sitesi, birini veya her ikisini NGG PAM dizisi içinde Gs sessiz 3) bir mutasyon ve ilk ve son mutasyon kanat 4) 50 bp 5' 3' homoloji silah. (Şekil 1 c) 9 , 10.<ol>
<li>Mutasyon NGG PAM sırası mümkün değilse, 5-6 sgRNA-aracılı bölünme ssODN HDR şablonunun önlemek için sgRNA hedef tanıma dizisi içinde sessiz mutasyonlar tanıtmak.</li>
	</ol>
</li>
	<li>Bir kaynaktan, örneğin idtdna, bir "Ultramer DNA Oligonükleotid" aşağıdaki parametrelerle elde etmek: Ölçek: 4 nmol; Formülasyon: None; Arıtma: Standart Desalting.</li>
	<li>Aldıktan sonra liyofilize ssODN tüp, içeren santrifüj kapasitesi > 12.000 x g oda sıcaklığında 1 dk için. Yavaşça ssODN 100 µM son bir konsantrasyon için GKD nükleaz ücretsiz2içinde O 15 - 20 kere P200 pipet kullanarak yukarı ve aşağı pipetting tarafından yeniden askıya alma.</li>
</ol>3. tasarım Genotipleme astar
<ol><li>~ 400-700 bp PCR amplicon11oluşturmak için genom değişiklik kanat ayarla bir astar tasarım.</li>
	<li>PCR bisiklet koşullarını tek ve belirli amplicon11üretmek için en iyi duruma getirme.</li>
</ol>4. enjeksiyon karışım hazırlamak
<ol><li>Tablo 1 reaktifler 4 ° C'de 2 min için maksimum hızda santrifüj kapasitesi</li>
	<li>Listelenen sırayla Tablo 1 reaktifler bir nükleaz ücretsiz PCR tüp ekleyin.</li>
	<li>Yavaşça aşağı yukarı 10 kez P20 pipet ile pipetting tarafından iyice karıştırın.</li>
	<li>Oda sıcaklığında 10 dakika için enjeksiyon karışımı kuluçkaya.</li>
</ol>5. enjeksiyon Protokolü
<ol><li>Yük enjeksiyon micropipette yaklaşık yarısı enjeksiyon ile12karıştırın. Enjeksiyon iğne takunya ya da tatili halinde enjeksiyon mix kalan kaydedin.</li>
	<li>Yavaş yavaş akan çözüm12~ 20-30 psi üretir micropipette ipucu break. Not: Büyük çap ipuçları olumsuz hayvan sağlığını etkileyen ve F1 döl verim azaltın.</li>
	<li>10-15 genç enjekte yetişkin solucanlar her iki eşey kollarında mümkünse12. Eğer değilse, bir eşey kol içine enjeksiyon yeterlidir.</li>
	<li>Oda sıcaklığında 1-2 h için kurtarmak enjekte hayvanlar (P0) 35 mm OP50 numaralı seribaşı nematodunun büyüme medya (NGM) plaka üzerinde izin. Daha sonra bireysel OP50 numaralı seribaşı 35 mm NGM tabak Platin tel solucan kullanarak tek enjekte P0 hayvanlara12seç.</li>
</ol>6. ekran P0 plakaları ve tek mCherry(+) F1s
<ol><li>İki gün sonrası enjeksiyon, F1 döl mCherry floresan stereomicroscope kullanarak yutak olarak ifade içeren P0 plakaları tanımlamak (şekil 1 d, gün 2 - 3). En mCherry(+) F1 hayvanlar içeren üç P0 plakaları seçin.</li>
	<li>Her üç seçili P0 plakalar, 8-12 mCherry(+) F1 hayvanlar 24-36 mCherry(+) F1s bir solucan kullanarak toplam tek seçin (şekil 1 d, gün 2 - 3). Not: mCherry(-) hayvan-da var saydı P0 tabakları, ancak doğru bir şekilde belirlenmesi olasılığını hayvanlar çok düzenlenmiş (şekil 2A) daha düşük.</li>
	<li>Bireysel F1ler Self-fertilize ve oda sıcaklığında 1-2 gün boyunca yumurtlamak izin verir.</li>
</ol>7. tek solucan PCR ve Genotipleme
<ol><li>1-2 gün sonra yumurta atma, F1s 7 µL solucan lizis bir solucan pick (Tablo 2, şekil 1 d, gün 4-5) kullanarak arabellek içeren bireysel PCR şerit tüp kapakları aktarın.</li>
	<li>Santrifüj PCR tüpleri tüp altına hayvan getirmek ve tüpler için 1 h-80 ° C'de dondurmak için oda sıcaklığında 1 dk. için maksimum hızda. Not: Worms-80 ° C'de süresiz olarak saklanır.</li>
	<li>Aşağıdaki programı kullanarak bir thermocycler de donmuş solucan parçalayıcı: 4 ° C 60 ° C 60 dk, 15 dk, 95 ° C için tutun.</li>
	<li>Tablo 3' te gösterilen kurulum PCR mastermix.</li>
	<li>21 µL PCR mastermix temiz PCR tüpler içine ekleyin ve sonra adım 3 4 µL solucan lizis ekleyin. İyi bir pipet kullanarak mix ve ( Malzeme tabloyabakın) üreticileri yönergeleri izleyerek PCR programını çalıştırın.</li>
	<li>Üreticinin yönergelerini izleyerek bir DNA temiz ve konsantre kit kullanarak PCR reaksiyonları arındırmak ( Malzeme tabloyabakın). DNA spin sütun 10 µL su ekleyerek elute.</li>
	<li>Tablo 4' te gösterildiği gibi set-up restriksiyon enzimi mastermix.</li>
	<li>Restriksiyon enzimi mastermix 10 µL temizlenmiş her PCR reaksiyon ekleyin ve 1-2 h 37 ° C13için kuluçkaya.</li>
	<li>Ayrı 120 V 1 x Tris-asetat-EDTA (TAE) arabellek14kullanarak çalıştırmak % 1.5 özel jel PCR ürünleri sindirilir.</li>
</ol>8. kimlik ve sıra doğrulama düzenlenen hayvan
<ol><li>Potansiyel hayvanlar14 (şekil 1 d, 4-5 gün) düzenlenmiş belirtmek bantları varlığı için sindirilmiş enzim örnekleri inceleyin. Not: Grup boyutu ve/veya desenleri bantlama beklenmedik ve karmaşık restriksiyon enzimi sindirim nöbetleşe olarak gözlenen potansiyel Indel mutasyonlar tanımlamak için bir N2 denetimini yükleyin.</li>
	<li>Potansiyel düzenlenen hayvan belirlenmesi sonra solucan lizis kalan 3 µL kullanın ve PCR reaksiyon tekrarlayın. Temiz mi veya restriksiyon enzimi sindirmek PCR reaksiyon program tamamlandıktan sonra. PCR reaksiyon % 1'özel jel, ayrı olarak yüklemek ve bir jel ekstraksiyon kiti15 kullanarak Grup ayıklamak ( Malzeme tabloyabakın).</li>
	<li>Sanger sıra16 jel doğru düzenlenmiş hayvanlar (şekil 1A) tanımlamak için F1 astar kullanarak DNA elde Not: okuma içerir Heterozygote doruklarına vahşi türü varlığı nedeniyle overlaid ve alleli tasarlanmış.</li>
	<li>Homozigoz hayvanlar doğrulanmış heterozigoz düzenlenen hayvanlardan, tek 8-12 F2 hayvanlar almak ve 7-8 adımları gerçekleştirmek için.</li>
</ol>

<ol><li>Dickinson, D. J., Goldstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Based+Methods+for+Caenorhabditis+elegans+Genome+Engineering%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering.</a> Genetics. 202, 885-901 (2016).</li>
<li>Doudna, J. A., Charpentier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+editing.+The+new+frontier+of+genome+engineering+with+CRISPR-Cas9%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Genome editing. The new frontier of genome engineering with CRISPR-Cas9.</a> Science. 346, 1258096(2014).</li>
<li>Ma, D., Liu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+Editing+and+Its+Applications+in+Model+Organisms%5BTitle%5D)+AND+%22Genomics+Proteomics+Bioinformatics%22%5BJournal%5D)">Genome Editing and Its Applications in Model Organisms.</a> Genomics Proteomics Bioinformatics. 13, 336-344 (2015).</li>
<li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Cas+systems+for+editing%2C+regulating+and+targeting+genomes%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat Biotechnol. 32, 347-355 (2014).</li>
<li>Kim, H. M., Colaiacovo, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Cas9-Guided+Genome+Engineering+in+C.+elegans%5BTitle%5D)+AND+%22Curr+Protoc+Mol+Biol%22%5BJournal%5D)">CRISPR-Cas9-Guided Genome Engineering in C. elegans.</a> Curr Protoc Mol Biol. 115, 31-31 (2016).</li>
<li>Yin, H., Kauffman, K. J., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delivery+technologies+for+genome+editing%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Delivery technologies for genome editing.</a> Nat Rev Drug Discov. 16, 387-399 (2017).</li>
<li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Res. 24, 1012-1019 (2014).</li>
<li>Haeussler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+off-target+and+on-target+scoring+algorithms+and+integration+into+the+guide+RNA+selection+tool+CRISPOR%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.</a> Genome Biol. 17, 148(2016).</li>
<li>Paix, A., Folkmann, A., Seydoux, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precision+genome+editing+using+CRISPR-Cas9+and+linear+repair+templates+in+C.+elegans%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans.</a> Methods. , (2017).</li>
<li>Prior, H., Jawad, A. K., MacConnachie, L., Beg, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+Efficient%2C+Rapid+and+Co-CRISPR-Independent+Genome+Editing+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22G3+%28Bethesda%29%22%5BJournal%5D)">Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans.</a> G3 (Bethesda). 7, 3693-3698 (2017).</li>
<li>JoVE Science Education Database. <a target="_blank" href="https://www.jove.com/science-education/5056/pcr-the-polymerase-chain-reaction">Basic Methods in Cellular and Molecular Biology. PCR: The Polymerase Chain Reaction.</a> J Vis Exp. , (2017).</li>
<li>Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+stable+transgenic+C.+elegans+using+microinjection%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Generation of stable transgenic C. elegans using microinjection.</a> J Vis Exp. , (2008).</li>
<li>JoVE Science Education Database. <a target="_blank" href="https://www.jove.com/science-education/5070/restriction-enzyme-digests"> Basic Methods in Cellular and Molecular Biology. Restriction Enzyme Digests.</a> J Vis Exp. , (2017).</li>
<li>JoVE Science Education Database. <a target="_blank" href="https://www.jove.com/science-education/5057/dna-gel-electrophoresis">Basic Methods in Cellular and Molecular Biology. DNA Gel Electrophoresis .</a> J Vis Exp. , (2017).</li>
<li>JoVE Science Education Database. <a target="_blank" href="https://www.jove.com/science-education/5063/gel-purification">Basic Methods in Cellular and Molecular Biology. Gel Purification.</a> J Vis Exp. , (2017).</li>
<li>Estrada-Rivadeneyra, D. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/29171728">Sanger sequencing.</a> FEBS J. , (2017).</li>
<li>Ludolph, A. C., Brettschneider, J., Weishaupt, J. H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22918486">Amyotrophic lateral sclerosis.</a> Curr Opin Neurol. 25, 530-535 (2012).</li>
<li>Evans, T. C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aEvans%2C+TC+%22WormBook%22">WormBook</a>. , The C. elegans Research Community, WormBook. (2006).</li>
<li>Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+transfer+in+C.elegans%3A+extrachromosomal+maintenance+and+integration+of+transforming+sequences%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO J. 10, 3959-3970 (1991).</li>
<li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337, 816-821 (2012).</li>
<li>Boyd, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Balanced+Look+at+the+Implications+of+Genomic+%28and+Other+%26%2334%3BOmics%26%2334%3B%29+Testing+for+Disease+Diagnosis+and+Clinical+Care%5BTitle%5D)+AND+%22Genes+%28Basel%29%22%5BJournal%5D)">A Balanced Look at the Implications of Genomic (and Other &#34;Omics&#34;) Testing for Disease Diagnosis and Clinical Care.</a> Genes (Basel). 5, 748-766 (2014).</li>
<li>Saccon, R. A., Bunton-Stasyshyn, R. K., Fisher, E. M., Fratta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+SOD1+loss+of+function+involved+in+amyotrophic+lateral+sclerosis%3F%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Is SOD1 loss of function involved in amyotrophic lateral sclerosis?</a> Brain. 136, 2342-2358 (2013).</li>
<li>Acevedo-Arozena, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comprehensive+assessment+of+the+SOD1G93A+low-copy+transgenic+mouse%2C+which+models+human+amyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22Dis+Model+Mech%22%5BJournal%5D)">A comprehensive assessment of the SOD1G93A low-copy transgenic mouse, which models human amyotrophic lateral sclerosis.</a> Dis Model Mech. 4, 686-700 (2011).</li>
<li>Gurney, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motor+neuron+degeneration+in+mice+that+express+a+human+Cu%2CZn+superoxide+dismutase+mutation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.</a> Science. 264, 1772-1775 (1994).</li>
</ol>

Biz üyeleri Beg laboratuvar bu el yazması eleştirel okuma için teşekkür ederiz. Suşları NIH ofisi araştırma altyapı programları (P40 OD010440) tarafından finanse edilen Caenorhabditis genetik Merkezi tarafından temin edilmiştir. Beg laboratuvar araştırma hibe tarafından NIH (R01 NS094678) ve Musküler Distrofi Derneği (MDA382300) A.A.B. için desteklenir

CRISPR-Cas9 sistemi tam olarak canlılar genom değiştirmek için güçlü ve etkili bir araçtır. Burada, biz o chimeric sgRNAs20 ssODN HDR şablonları etkinleştir ile yüksek verimli üretimi C. elegansgenomik noktası mutasyonların birleştiğinde göstermek. Önemlisi, biz floresan bir eş seçimi marker olarak kullanıldığında RNP teslim yüksek Düzenleme verimliliği üreten göstermek kolaylık ve güvenilirlik-in teknik vurgulayarak.
C. ele...

Son teknolojik gelişmeler kökten değiştirdi ve tam olarak mühendis genleri yeteneği hızlandırılmış. Özellikle, RNA güdümlü endonükleaz Çift Kişilik strand mola (DSB) faiz hedef sıra ikna etmek için Cas9 dayanan, CRISPR-Cas9 sistemi kapsamlı olarak doğru bir şekilde kullanılan model organizmalar çoğunluğu genom mühendisi kullanılmaya başlanmıştır Biyomedikal Araştırma1,2,3,4. Önemli ölçüde, CRISPR-Cas9 kullanımı bile zor türler C. elegans5gibi genom düzenleme kilidi. Türü ne olursa olsun, sistem düzenleme CRISPR-Cas9 dayalı genom ile nokta mutasyonlar üreten üç çekirdek bileşenleri üzerinde dayanır: 1) Cas9 endonükleaz, bir hedef sıra ve tasarlanmış 3) bir kullanıcı için Cas9 endonükleaz yönlendirir 2) bir tek Kılavuzu RNA (sgRNA) İstenen içeren homoloji yönetmen onarım (HDR) şablonunu faiz2/ edit(s).
Hedefleme sgRNA ve Cas9 tanıtmak için kullanabileceğiniz çeşitli yöntemler vardır nükleaz plazmid, RNA ve viral alan teslim yöntemleri6dahil olmak üzere hücre içine. Son zamanlarda, pre-complexed sgRNA-Cas9 Ribonucleoproteins (RNPs) doğrudan teslim CRISPR Cas9 esaslı genom7düzenleme güçlü ve etkili bir araç ortaya çıkmıştır. Pre-complexed CRISPR-Cas9 RNPs doğrudan teslim Yani birkaç farklı avantajı vardır: 1) RNPs bypass hücresel transkripsiyon ihtiyacını ve çeviri, 2) RNPs hızla temizlenir, hangi-ebilmek artmak özgüllük için kullanılabilir zamanı azaltarak kapalı-hedef bölünme ve 3) RNPs non-yerli dizileri rasgele tümleştirme yoluyla ana bilgisayar genom içine giriş circumvents hiçbir yabancı DNA/RNA unsurları içerir. Birlikte, bu öznitelikler muhtemel hedef kapalı etkileri en aza indirerek CRISPR hedef olarak düzenleme kısa ömürlü bir patlama sağlar.
C. eleganssiteye özgü genomik değişiklikleri tanıtmak için basit ve etkili bir iletişim kuralı tanımlar. Bu iletişim kuralı sgRNA ve tek telli Oligonükleotid (ssODN) HDR şablon tasarımı, sgRNA-Cas9 RNP kompleks ve teslim ve düzgün düzenlenmiş hayvanlar kesin tanımlaması için bir Genotipleme strateji hedefleme içerir. Bu stratejiyi kullanarak, sadece istediğiniz siteye özgü değişiklikleri elde edilebilir, ama diğer non-spesifik Indel mutasyonlar da kurtarılabilir. Böylece, bizim strateji allelic dizisi tek bir strateji kullanarak mono allelic, BI allelic nerede nesil verir ve Indel mutantlar F1 üretimi oluşturulabilir.

<table><tbody><tr><td>CRISPRevolution sgRNA&amp;nbsp; EZ Kit</td><td>Synthego Inc.</td><td></td><td>chimeric sgRNA</td></tr><tr><td>Nuclease-free TE</td><td>Synthego Inc.</td><td>provided with the sgRNA kit EZ kit</td><td></td></tr><tr><td>Nuclease-free water</td><td>Synthego Inc.</td><td>provided with the sgRNA kit EZ kit</td><td></td></tr><tr><td>4 nmole Ultramer DNA Oligo</td><td>Integrated DNA Technologies</td><td></td><td>ssODN HDR template</td></tr><tr><td>Alt-R S.p. HiFi Cas9 Nuclease 3NLS</td><td>Integrated DNA Technologies</td><td>1078728</td><td>Cas9 protein</td></tr><tr><td>pCFJ90&amp;nbsp;</td><td>Addgene</td><td>19327</td><td>Pmyo-2::mCherry Marker Plasmid</td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>DNA Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D4004</td><td></td></tr><tr><td>Zymoclean Gel DNA Recovery Kit</td><td>Zymo Research</td><td>D4002</td><td></td></tr><tr><td>Q5 Hot Start High-Fidelity 2X Master Mix</td><td>New England Biolabs</td><td>M0494L</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P2308</td><td></td></tr><tr><td>Glass Borosilicate Glass Micropipettes</td><td>Sutter Instruments</td><td>BF100-78-10</td><td>OD: 1.0mm. ID: 0.78mm</td></tr><tr><td>Trizma Hydrochloride</td><td>Sigma</td><td>T5941</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M2393</td><td></td></tr><tr><td>NP-40</td><td>Sigma</td><td>74385</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Scientific</td><td>BP337-100</td><td></td></tr><tr><td>RNaseZap Decontamination Solution</td><td>Fisher Scientific</td><td>AM9780</td><td></td></tr></tbody></table>

a rapid and facile pipeline for generating genomic point mutants in c. elegans using crispr/cas9 ribonucleoproteins

Kümelenmiş düzenli olarak serpiştirilmiş palindromik tekrarlar (CRISPR) - CRISPR - protein prokaryotik edinilmiş bağışıklık savunma sistemi kesin ökaryotik genom mühendisliği için güçlü bir araç olarak ortak tercih 9 (Cas9) ilişkili. Burada, C. elegansgenomik noktası mutasyonların verimli ve hassas nesil chimeric tek Kılavuzu RNA'ların (sgRNA) ve CRISPR-Cas9 Ribonucleoproteins (RNPs) kullanarak hızlı ve basit bir yöntem mevcut. Biz bir boru hattı sgRNA hedef seçimi, homoloji yönetmen onarım (HDR) şablonu tasarım, CRISPR-Cas9-RNP kompleks ve teslim ve doğru düzenlenmiş hayvanlar sağlam ve hızlı tanımlaması sağlar bir Genotipleme strateji tanımlamak. Yaklaşımımız sadece facile üretimi ve istenen genomik noktası tanımlaması mutant hayvanlar izin verir, ancak aynı zamanda diğer karmaşık Indel allelleri algılama yaklaşık 4-5 gün içinde yüksek verimliliği ve azaltılmış tarama iş yükü ile kolaylaştırır.

Mutasyonların insan süperoksit dismutaz 1 (SOD1) ailesel amyotrofik lateral skleroz, felç ve ölüm17' ye kaçınılmaz götüren bir yıkıcı nörodejeneratif hastalık ~ 10-%20 hesabı. İnsan SOD-%1 55 kimlik ve % 70 benzerlik C. elegans SOD-1 protein ile (şekil 1B) paylaşımı örümceklerle korunmuş bir proteindir. Basitlik, fizibilite ve CRISPR-Cas9 RNP dayalı yaklaşım verimliliğini göstermek için <...

Watch this Scientific Journal Video about A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins at JoVE.com

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Bir hızlı ve genomik noktası mutantlar C. elegans kullanarak CRISPR/Cas9 Ribonucleoproteins üreten Facile boru hattı

Burada, CRISPR-Cas9 ribonucleoproteins ve homoloji bağımlı onarım şablonları kullanarak C. elegans genom mühendisi bir yöntem mevcut.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

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Research

JoVE Journal

Genetics

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

7.5K Görüntüleme.  University of Michigan. Burada, CRISPR-Cas9 ribonucleoproteins ve homoloji bağımlı onarım şablonları kullanarak C. elegans genom mühendisi bir yöntem mevcut.

Video: A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

HiPSC Sinir Lineage Özgül Knockin Muhabirleri Verimli Nesil kullanma CRISPR / Cas9 ve Cas9 Çift Nickase Sistemi

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

Gelişim Biyolojisi

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

CRISPR genotipine bir Floresan PCR kılcal jel elektroforezi Tekniği / yüksek verimli bir Format Nakavt Mutants Cas9 aracılı

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Genetik

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

Memeli Hücrelerinde CRISPR / Cas9 aracılı Gen Knockout&#39;larının Seçime Bağlı ve Bağımsız Üretimi

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Yerinde mutagenisitesinin CRISPR/Cas9 ve SsODN insan hücrelerinde tarafından katalize nokta mutasyon onarım bir fonksiyonu olarak incelemek için standart bir metodoloji

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Verimli üretim ve Model sistem Danio rerio kimlik CRISPR/Cas9 tarafından oluşturulan Gene altını gizleme

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Gelişmiş genom Cas9 Ribonükleoprotein yılında çeşitli ile hücreleri ve organizmaların düzenleme

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

İnsan Pluripotent Kök Hücrelerinde CRISPR-CAS9 Kullanılarak Tanımlanmış Genomik Modifikasyonların Üretimi

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing&#160;in less than two weeks.

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The goal of this protocol is to enable seamless CRISPR/Cas9 editing of the C. elegans genome using assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. This protocol can be used to make a variety of genetic modifications in C. elegans including insertions, deletions, gene replacements and codon substitutions.

CRISPR/Cas9 C. elegans rbm-3.2 Geninin dpy-10 Co-CRISPR Tarama İşaretçisi ve Monte Edilmiş Ribonikleoprotein Kompleksleri kullanılarak düzenlenmesi.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been ...

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 Teknolojisi Kullanılarak Göçmen Locust'un Homozigot Mutantlarının İnşası

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 ...

Biyoloji

Single Worm PCR: A Method to Extract and Amplify Genomic DNA

Source: Prior, H., et al. <a href="https://www.jove.com/video/57518/a-rapid-facile-pipeline-for-generating-genomic-point-mutants-c">A Rapid and Facile Pipeline for Generating Genomic Point Mutants in&#160;C. elegans&#160;Using CRISPR/Cas9 Ribonucleoproteins.</a>&#160;J. Vis. Exp.&#160;(2018).
This video describes a technique to rapidly screen a single nematode for a genetic marker by PCR screening. In the example protocol, we screen for CRISPR-based genome editing.

Bir hızlı ve genomik noktası mutantlar C. elegans kullanarak CRISPR/Cas9 Ribonucleoproteins üreten Facile boru hattı

Özet

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