Name: using a fluorescent pcr-capillary gel electrophoresis technique to genotype crispr/cas9-mediated knockout mutants in a high-throughput format
Uploaded: 2017-04-08T14:00:00
Description: Watch this Scientific Journal Video about Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format at JoVE.com

Forfatterne takker Ms Tan Shi Min, Ms Helen Ong, og Dr. Zhao Yi for at hjælpe med kapillargelelektroforese eksperimenter. Dette arbejde blev støttet af NMRC / IRG give NMRC / 1314/2011 og MOE AcRF Tier 2 Fondens tilskud MOE2011-T2-1-051.

1. Opnåelse CRISPR / Cas9 målrettede Single-cellekloner <ol><li> Frø HEPG2-celler på en plade med 6 brønde ved 500.000 celler per brønd i 2 ml af antibiotikum-frit Dulbeccos Modified Eagle Medium (DMEM) suppleret med 10% føtalt bovint serum (FBS). Inkuber i 24 timer ved 37 ° C og 5% CO2. </li><li> Transficere celler med plasmid coudtrykker Cas9 og specifik sgRNA mod genet af interesse ved anvendelse af en passende transfektionsreagens i henhold til fabrikantens anvisninger. BEMÆRK: For eksem...

<ol>
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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=Knockout+mice+created+by+TALEN-mediated+gene+targeting.">Knockout mice created by TALEN-mediated gene targeting.</a> Nat. Biotechnol. 31, 23-24 (2013).</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, 84-87 (2014).</li><li>Zhou, 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-throughput+screening+of+a+CRISPR/Cas9+library+for+functional+genomics+in+human+cells.">High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.</a> Nature. 509, 487-491 (2014).</li><li>Guschin, D. 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=A+rapid+and+general+assay+for+monitoring+endogenous+gene+modification.">A rapid and general assay for monitoring endogenous gene modification.</a> Methods Mol. Biol. 649, 247-256 (2010).</li><li>Kim, H. J., Lee, H. J., Kim, H., Cho, S. W., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+genome+editing+in+human+cells+with+zinc+finger+nucleases+constructed+via+modular+assembly.">Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly.</a> Genome Res. 19, 1279-1288 (2009).</li><li>Ramlee, M. K., Yan, T., Cheung, A. M., Chuah, C. T., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+genotyping+of+CRISPR/Cas9-mediated+mutants+using+fluorescent+PCR-capillary+gel+electrophoresis.">High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis.</a> Sci. Rep. 5, 15587 (2015).</li><li>Liu, C. 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=Distinct+Responses+of+Stem+Cells+to+Telomere+Uncapping-A+Potential+Strategy+to+Improve+the+Safety+of+Cell+Therapy.">Distinct Responses of Stem Cells to Telomere Uncapping-A Potential Strategy to Improve the Safety of Cell Therapy.</a> Stem cells. 34, 2471-2484 (2016).</li><li>Doench, J. 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=Rational+design+of+highly+active+sgRNAs+for+CRISPR-Cas9-mediated+gene+inactivation.">Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.</a> Nat. Biotechnol. 32, 1262-1267 (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=Identification+and+characterization+of+essential+genes+in+the+human+genome.">Identification and characterization of essential genes in the human genome.</a> Science. 350, 1096-1101 (2015).</li><li>Sanjana, N. E., Shalem, O., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vectors+and+genome-wide+libraries+for+CRISPR+screening.">Improved vectors and genome-wide libraries for CRISPR screening.</a> Nat. Methods. 11, 783-784 (2014).</li><li>Urnov, F. 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=Highly+efficient+endogenous+human+gene+correction+using+designed+zinc-finger+nucleases.">Highly efficient endogenous human gene correction using designed zinc-finger nucleases.</a> Nature. 435, 646-651 (2005).</li><li>Kim, H., Kim, J. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+genome+editing+and+phenotyping+facilitated+by+high+resolution+melting+curve+analysis.">High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis.</a> PLoS One. 9, 114632 (2014).</li><li>Kim, J. M., Kim, D., Kim, S., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+with+CRISPR-Cas-derived+RNA-guided+endonucleases.">Genotyping with CRISPR-Cas-derived RNA-guided endonucleases.</a> Nat. Commun. 5, 3157 (2014).</li></ol>

Den banke ud af et specifikt gen i en model udvalgt cellelinje er blevet rutine til at belyse den rolle, som genet spiller i denne særlige cellulære kontekst. Faktisk flere genom-dækkende skærme er i øjeblikket tilgængelige, der bruger CRISPR / Cas9 system til at målrette næsten alle kendte humane gener i genomet 14, 15, 16. Med disse store skærme (eller endog mindre målestok målretning af individuelle gener), er det...

Reverse genetiske tilgange har tilladt forskerne at belyse virkningerne af specifikke ændringer i genomet på cellen eller hele organismen. For eksempel kan ekspressionen af et bestemt gen svækkes ved gen knockdown 1, 2 (delvis reduktion) eller gen-knockout 3, 4 (komplet ablation) for at bestemme den virkning, dette har på funktionen af cellen eller på udvikling af organismen. Gen-knockout eksperimenter er blevet lettere siden indførelsen af ​​sekvensspecifikke programmerbare nukleaser, såsom zink-finger nukleaser (ZFNs) og transkriptions- aktivator-lignende effektor nukleaser (Talens). Men den relativt nylige karakterisering af de grupperede regelmæssigt afbrudt kort palindrom gentagelse (CRISPR) / Cas9 system har gjort det ekstremt nemt for alle laboratorier rundt om i verden for at udføre gene knockout eksperimenter. I det væsentlige, CRISPR / Cas9 systemet består af to væsentlige komponenter-en enkelt guide RNA (sgRNA), som genkender og binder via basen komplementaritet til en specifik sekvens i genomet, og en endonuklease kaldet Cas9. Eftervirkningerne af specifik binding og virkningen af ​​sgRNA-Cas9 kompleks på genomisk DNA er dobbeltstrenget spaltning af DNA. Dette igen, udløser DNA beskadigelse respons mekanisme i cellen, som efterfølgende repareret via ikke-homologe endesammenslutning (NHEJ) eller homolog rekombination (HR) veje. Da NHEJ reparationsmekanisme (men ikke HR-mekanismen) resulterer ofte i vilkårlig insertion eller deletion af nukleotider på stedet for reparation, hvilket resulterer i insertion / deletion (InDel) mutationer, kan det forårsage læserammen for et exon at flytte. Dette kan resultere i knockout af genet grund af for tidlig terminering af translation og nonsense-medieret henfald 5, 6,7. Trods den bekvemmelighed gav ved indføring af CRISPR / Cas9 system udskære et gen, genotypebestemmelse af kloner af målrettede celler er stadig en flaskehals, især i en høj kapacitet indstilling 8, 9. Eksisterende teknikker enten lider store iboende begrænsninger eller er økonomisk bekostelige. For eksempel SURVEYOR eller T7E1 assay, som er et enzymatisk assay, der påviser fejlparringer i DNA-duplekser 10, er ikke i stand til at skelne mellem vildtype kloner og homozygote mutanter (kloner, hvis alleler er muterede identisk), da disse kloner har identiske alleler og dermed ikke udgør fejlparringer i deres DNA-sekvens 11. Hertil kommer, at anvendelsen af ​​Sanger-sekventering, hvilket betragtes som den gyldne standard i genotypebestemmelse af mutante kloner, i en high-throughput setup er uønsket på grund af dets høje omkostninger. Her præsenteres en itailed protokol af det fluorescerende PCR-kapillargelelektroforese teknik, som kan omgå begrænsningerne ved de øvrige eksisterende genotypebestemmelsesteknikker og er særlig nyttig ved udførelse af en high-throughput screen for nuklease-medieret knockout kloner. Denne metode er teknisk enkel at udføre og sparer tid og omkostninger.

<table border="0" cellpadding="0" cellspacing="0" width="1548">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:401px;">HEPG2 cells</td>
			<td style="width:196px;">ATCC</td>
			<td style="width:135px;">HB-8065</td>
			<td style="width:816px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Dulbecco&#39;s Modified Eagles Medium (DMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pSpCas9(BB)-2A-GFP plasmid</td>
			<td>Addgene</td>
			<td>PX458</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.5%), no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Water, Molecular Biology Grade</td>
			<td>GE Healthcare</td>
			<td>SH30538.02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CRISPR sgRNA insert oligonucleotide (sense)</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-CACCGCTAACCTTTCAGCCTGCCTA-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CRISPR sgRNA insert oligonucleotide (anti-sense)</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-AAACTAGGCAGGCTGAAAGGTTAGC-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Unlabeled PCR amplification forward primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-CACTAACTCCAATGCTTCAGTTTC-3&#39;; this primer is also used to sequence PCR amplified alleles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-FAM-labeled fluorescent PCR forward primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-6-FAM-CACTAACTCCAATGCTTCAGTTTC-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEX-labeled fluorescent PCR forward primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-HEX-CACTAACTCCAATGCTTCAGTTTC-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Unlabeled PCR reverse primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-CCTCTTCCAAGTCTGCTTATGT-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq PCR Core Kit</td>
			<td>QIAGEN</td>
			<td>201223</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hi-Di Formamide</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311320</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneScan 500 LIZ Dye Size Standard</td>
			<td>Thermo Fisher Scientific</td>
			<td>4322682</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Optical 96-Well Reaction Plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>4306737</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3500xL Genetic Analyzer</td>
			<td>Thermo Fisher Scientific</td>
			<td>4405633</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3500 Series 2 program</td>
			<td>Thermo Fisher Scientific</td>
			<td>4476988</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gene Mapper 5 program</td>
			<td>Thermo Fisher Scientific</td>
			<td>4475073</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentra Puregene Cell Kit</td>
			<td>QIAGEN</td>
			<td>1045696</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wizard SV Gel and PCR Clean-Up System</td>
			<td>Promega</td>
			<td>A9282</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NAP1L1 Antibody (N-term)</td>
			<td>Abgent</td>
			<td>AP1920b</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclear Matrix Protein p84 antibody [5E10]</td>
			<td>GeneTex</td>
			<td>GTX70220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase AffiniPure Goat Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-035-144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase AffiniPure Sheep Anti-Mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>515-035-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a fluorescent pcr-capillary gel electrophoresis technique to genotype crispr/cas9-mediated knockout mutants in a high-throughput format

Udviklingen af ​​programmerbare genom-redigeringsværktøjer har lettet anvendelsen af ​​revers genetik til at forstå de roller specifikke genomiske sekvenser spiller i cellernes funktion og hele organismer. Denne årsag er blevet voldsomt hjulpet på vej af den nylige indførelse af CRISPR / Cas9 systemet et alsidigt værktøj, der giver forskerne at manipulere genomet og transkriptom for at blandt andet, knock out, banke ned, eller banke i gener i en målrettet måde. Med henblik på at udskære et gen, CRISPR / Cas9-medieret dobbeltstrengede brud rekruttere det ikke-homolog endesammenbinding DNA-reparationsvej at indføre rammeforskydningssignalsekvensen-forårsager insertion eller deletion af nukleotider ved pausen site. Dog kan en privat guide RNA forårsage uønskede off-target effekter, og at udelukke disse ud, anvendelsen af ​​flere guide RNA&#39;er er nødvendig. Denne mangfoldighed af mål betyder også, at en screening af kloner med høj volumen er påkrævet, hvilket igen giver anledning til at anvende en efstrækkelig højt gennemløb teknik til genotype knockout kloner. Aktuelle genotypebestemmelsesteknikker enten lider af iboende begrænsninger eller pådrage sig høje omkostninger, dermed gør dem uegnede til high-throughput formål. Her, vi detaljeret protokol for anvendelse af fluorescerende PCR, som anvender genomisk DNA fra råt cellelysat som skabelon, og derefter løse PCR-fragmenterne via kapillær gelelektroforese. Denne teknik er nøjagtig nok til at skelne et basepar forskel mellem fragmenter og dermed er tilstrækkelig i indikerer tilstedeværelse eller fravær af et rammeskift i den kodende sekvens af det målrettede gen. Denne præcise viden effektivt udelukker behovet for en bekræftende sekventering skridt og giver brugerne mulighed for at spare tid og omkostninger i processen. Endvidere er denne teknik vist sig at være alsidig i genotypebestemmelse forskellige pattedyrceller af forskellige vævsoprindeise målrettet ved hjælp af styrestænger RNA&#39;er mod talrige gener, som vist her og andre steder.

Det fluorescerende PCR-kapillargelelektroforese teknikken beskrevet her forventes at kunne anvendes på enhver målrettes region i genomet i stort set enhver cellelinie, som er modtagelig for fremmed DNA levering. Vi har tidligere vist sin ansøgning ved at målrette tre gener i en kolorektal cancer cellelinje 12. Her, viser vi dens virkningsfuldhed i genotypebestemmelse af en hepatocellulært carcinom cellelinie, HEPG2, målrettet med en CRISPR / Cas9 konstruktio...

Watch this Scientific Journal Video about Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format at JoVE.com

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

Genotypebestemmelse teknikken beskrevet her, som kobler fluorescerende polymerasekædereaktion (PCR) til kapillær gelelektroforese muliggør high-throughput genotyping af nuklease-medieret knockout kloner. Den omgår begrænsninger af andre genotypebestemmelsesteknikker står og er mere omkostningseffektive end sekventeringsfremgangsmåder.

Under anvendelse af et fluorescerende PCR-kapillær gelelektroforese Teknik til genotype CRISPR / Cas9-medieret Knockout Mutanter i et format med høj kapacitet

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

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High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

High-Throughput robotersvejsede Assisted Isolering af temperaturfølsomme Lethal mutanter i<em&gt; Chlamydomonas reinhardtii</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

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

Udvælgelsesafhængig og uafhængig generering af CRISPR / Cas9-medierede Gene Knockouts i Pattedyrceller

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

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

Generation af indfødte kromatin Immunoprecipitation sekventering biblioteker for Nucleosome tæthed analyse

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: En High Throughput sekventering-baseret teknik til identifikation af sent replikerende regioner i genomet

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Robust DNA isoleret og høj overførselshastighed sekventering bibliotek konstruktion til Herbarium prøver

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-medieret målrettede Integration In Vivo ved hjælp af en homologi-medieret End at deltage-baseret strategi

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

En protokol til produktion af Integrase-mangelfuld Lentiviral vektorer for CRISPR/Cas9-medieret gen Knockout i dividere celler

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Høj overførselshastighed identifikation af synergistiske Drug kombinationer af overlapning2 metode

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

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

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

En hurtig og letkøbt Pipeline for generering af genomisk punkt mutanter i C. elegans bruger CRISPR/Cas9 Ribonucleoproteins

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