CRISPR | Bio-Techne

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url('https://resources.bio-techne.com/bio-techne-assets/images/banners/immunoassay-brochure-background-1500x340-v6.png'); background-position: top center; background-repeat: no-repeat; background-size: cover;"> <div> <div class="paragraph paragraph--type--h1-2-2-text-to-media-multi-cta paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:h1_2_2_text_to_media_multi_cta:field_indv_h1"> <div><h1>CRISPR Gene Editing</h1></div> </div> <div class="field_block:paragraph:h1_2_2_text_to_media_multi_cta:field_lg_sngl_frmtd_text_global"> <div><p>CRISPR (clustered regularly interspaced short palindromic repeats) gene editing is a technique for the targeted editing and regulation of genes that can be applied to a number of biological systems; it is adapted from CRISPR-Cas9, part of the bacterial adaptive immune system.  Bio-Techne provides a collection of antibodies, small molecules and proteins to support CRISPR gene editing.</p></div> </div> <div class="field_block:paragraph:h1_2_2_text_to_media_multi_cta:field_call_to_action_global"> <a href="/search?keywords=crispr" class="btn btn-primary btn-cta mr-3 mb-3" type="link"> Shop All CRISPR-Cas9 Products </a> </div> <div class="field_block:paragraph:h1_2_2_text_to_media_multi_cta:field_multi_cta_links"> <div class="mt-3"> <a href="/research-areas/cell-and-gene-therapy" class="text-white" type="link"> <u class="text-white">Cell & Gene Therapy</u> </a> <span class="text-light"> | </span> <a href="/research-areas/stem-cells/ipsc" class="text-white" type="link"> <u class="text-white">iPSCs</u> </a> </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:h1_2_2_text_to_media_multi_cta:field_indv_media"> <div> <img 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class="teaser-valign-container" style=""> <div class="valign"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/antibody-floating-480x480.png" alt="Floating Antibody Cell" loading="lazy" > </div> </div> </div> </a> <a class="card_title_link" href="/p/antibodies/crispr-cas9-antibody-pack_nbp2-52986" > <h3 class="card_title_htag"> <div><h3>CRISPR Cas9 Antibody Pack</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Detect CRISPR-Cas proteins using our antibodies, validated for use in a range of applications.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/p/antibodies/crispr-cas9-antibody-pack_nbp2-52986" > View Antibody Pack </a> </div> </div> </div> </div> </div> <div class="col-md-4"> <div class="h-100"> <div class="d-flex flex-column h-100"> <a href="/resources/instrument-applications/crispr-cell-engineering-single-cell-dispenser" > <div class="card-img-top text-center mb-3" style="background-repeat: no-repeat; background-color: ; height:200px; width: 100%;"> <div class="teaser-valign-container" style=""> <div class="valign"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/bt-pala-side-instrument-525x300.png" alt="Bio-Techne Cell Sorter and Single Cell Dispenser side-angle" loading="lazy" > </div> </div> </div> </a> <a class="card_title_link" href="/resources/instrument-applications/crispr-cell-engineering-single-cell-dispenser" > <h3 class="card_title_htag"> <div><h3>Single Cell Isolation</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p> With Bio-Techne's single cell dispensers, Hana™ and Pala™,  fluorescently-labeled CRISPR gene-edited cells can be identified and rapidly and gently isolated. </p></div> </div> <div class="card-bottom-link"> <div> <a href="/resources/instrument-applications/crispr-cell-engineering-single-cell-dispenser" > Learn More About Bio-Techne Cell Sorter & Single Cell Dispenser </a> </div> </div> </div> </div> </div> <div class="col-md-4"> <div class="h-100"> <div class="d-flex flex-column h-100"> <a href="/reagents/small-molecules-and-peptides/lipid-nanoparticles" > <div class="card-img-top text-center mb-3" style="background-repeat: no-repeat; background-color: ; height:200px; width: 100%;"> <div class="teaser-valign-container" style=""> <div class="valign"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/products/lipids-nanoparticle-hero-480x480-v2.png" alt="lipids nanoparticle hero image" loading="lazy" > </div> </div> </div> </a> <a class="card_title_link" href="/reagents/small-molecules-and-peptides/lipid-nanoparticles" > <h3 class="card_title_htag"> <div><h3>Lipid Nanoparticles</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>We offer high quality lipids enabling you to make lipid nanoparticles for delivery of gene editing tools such as CRISPR-Cas9. </p></div> </div> <div class="card-bottom-link"> <div> <a href="/reagents/small-molecules-and-peptides/lipid-nanoparticles" > Learn About Our High Quality Lipids </a> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="workflow"></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--header"> <div> <div><h2>CRISPR-Cas9 Gene Editing Workflow</h2></div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3> </h3> <h3>i. Gene Delivery</h3> <p>There are a variety of methods for delivery of CRISPR DNA, RNA, or RNP into cells, including <a href="/reagents/small-molecules-and-peptides/lipid-nanoparticles">lipid nanoparticles</a>, viral and lentiviral vectors, and electroporation.</p> <p>Bio-Techne offers a range of reagents for use in CRISPR delivery: </p> <ul> <li><a href="/t/transfection-reagents">Small Molecule and Peptide Transfection Reagents</a> </li> <li><a href="/t/viral-transduction-enhancers">Viral Transduction Enhancers</a> </li> <li><a href="/search?keywords=lipid%20nanoparticles">Lipid Nanoparticle Components</a>.</li> </ul></div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-media-to-text mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/pei-star-transfection-reagent-application.jpg" alt="Cultures of HEK293 suspensions transfected with CMV-SEAP plasmid using either PEI STARTM or competitor PEI and expression levels readout as absorbance. " loading="lazy" > </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_lg_sngl_frmtd_text_global"> <div><p><strong>Absorbance readout of HEK293 cell suspensions transfected with a CMV-SEAP plasmid using either PEI STAR™ or competitor PEI. </strong><a href="/p/small-molecules-peptides/pei-transfection-reagent_7854">PEI STAR™ transfection reagent</a> (Catalog # <a href="/p/small-molecules-peptides/pei-transfection-reagent_7854">7854</a>) performance data comparison (HEK293): HEK 293 - 20 mL cultures containing HEK293 suspensions were transfected with a CMV-SEAP plasmid at optimized PEI/DNA ratios using either PEI STAR™ (3:1) or leading competitor PEI. SEAP expression levels were quantified 5 days post-transfection using phosphatase reporter dye and UV/Vis absorbance.</p></div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h4><strong>Efficiency of Gene Transfer</strong></h4> <p><a href="/search?keywords=crispr&category_attr=Primary%20Antibodies">CRISPR-Cas9 antibodies</a> can help verify the success of gene transfer. Comparing the expression of Cas9 in transfected versus mock transfected cells is important to assess the efficiency of transfection. With the help of CRISPR-Cas9 antibodies, one can use <a href="/applications/western-blotting">Western blot</a> and/or immunostaining to verify transfection efficiency.</p> <p>CRISPR-mediated gene editing is typically an inefficient process; the efficiency can be improved using small molecule <a href="/t/crispr-reagents">CRISPR Enhancers</a>.</p> <p> </p> <p> </p></div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3><strong>ii. Detection of CRISPR-Cas9 </strong></h3> <p>Detection of CRISPR-Cas9 involves both expression analysis as well as determination of cellular localization. Analysis of these factors can be achieved through use of CRISPR-Cas9 antibodies.</p> <h4>CRISPR-Cas9 Expression</h4> <p>The levels as well as the duration of Cas9 expression are highly critical for CRISPR-Cas9 based genome editing. In stable clones, high expression levels of Cas9 may result in non-specific activity. This may be controlled through isolating multiple clones and screening them for Cas9 expression levels through <a href="/applications/western-blotting">Western blot</a> analysis. For transient transfections, a chronic or prolonged expression of Cas9 may lead to the development of more off-target mutations. The transient nature of the expression of CRISPR-Cas9 may be checked through Western blot analysis of the transfected samples collected at various time points.</p> <h4>Subcellular Localization </h4> <p>CRISPR-Cas9 must translocate to the nuclei of transfected cells for executing its effects on DNA. Nuclear localization of Cas9 may be verified through <a href="/applications/imaging/immunocytochemistry">ICC/IF</a> or <a href="/applications/imaging/immunohistochemistry">IHC</a> staining with CRISPR-Cas9 antibodies.</p></div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-text-to-media mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p><strong>Expression of CRISPR-Cas9 in HEK293 Cells. </strong>HEK293 cells were transfected with a construct including the N-terminal 608 amino acids of <em>S. pyogenes</em> CRISPR-Cas9 fused to GFP and stained with <a href="/p/antibodies/crispr-cas9-antibody_nbp3-05548">Rabbit anti-CRISPR-Cas9 Polyclonal Antibody</a> (red; Catalog # <a href="/p/antibodies/crispr-cas9-antibody_nbp3-05548">NBP3-05548</a>). Transfected cells express the green fusion protein and bind the antibody in red, producing a yellow signal. Nuclear DNA in transfected and non-transfected cells is revealed with the blue DNA stain <a href="/search?search_type=products&keywords=dapi">DAPI</a>.</p></div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/crispr-cas9-antibody-icc.jpg" alt="CRISPR-Cas9 in HEK293 cells transfected with construct containing S. pyogenes CRISR-Cas9 fused to GFP detected by immunocytochemistry showing transfected cells (green) that bind the antibody (red) produce a yellow signal." loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>iii. Confirmation of Edits</h3> <p>Confirming successful editing within target cells is an important step in the CRISPR-Cas9 gene editing workflow is . Edits can be confirmed at the mRNA level using reverse transcriptase polymerase chain reaction (RT-PCR) or at the gene level by utilizing knockout (KO)-validated antibodies. Learn more about <a href="#background">CRISPR/Cas9 Knockout Lysates</a></p> <p><strong>Knockout Validated Antibodies by Application</strong></p> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td class="w-33"><a href="/search?search_type=products&keywords=knockout&applications=Immunocytochemistry/%20Immunofluorescence">ICC/IF KO Antibodies</a></td> <td class="w-33"><a href="/search?search_type=products&keywords=knockout&applications=Western%20Blot">Western Blot KO Antibodies</a></td> <td><a href="/search?search_type=products&keywords=knockout&applications=Simple%20Western">Simple Western KO Antibodies</a> </td> </tr> </tbody> </table></figure></div> </div> </div> <div> <div class="paragraph paragraph--type--_-3-media-to-text paragraph--view-mode--default mb-4"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--25-75"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:1_3_media_to_text:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/vimentin-affinity-purified-polyclonal-ab-knockout-validated-wb.jpg" alt="Expression of Vimentin in K562 parental cell line compared to Vimentin knockout (KO) K562 cell line in Western Blot following probing with Goat Anti-Vimentin Polyclonal Antibody followed by HRP-conjugated Secondary showing expression in the parental cell " loading="lazy" > </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:1_3_media_to_text:field_lg_sngl_frmtd_text_global"> <div><p><strong>Western blot analysis of Vimentin expression in K562 parental cell line and Vimentin knockout (KO) K562 cell line</strong>.<strong> </strong>Western blot analysis showing lysates of K562 parental cell line and Vimentin knockout (KO) K562 cell line loaded at 0.2 mg/mL. The membrane was probed with <a href="/p/antibodies/human-mouse-rat-vimentin-antibody_af2105">Goat Anti-Human/Mouse/Rat Vimentin Antigen Affinity-purified Polyclonal Antibody</a> (Catalog # <a href="/p/antibodies/human-mouse-rat-vimentin-antibody_af2105">AF2105</a>) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # <a href="https://www.bio-techne.com/p/secondary-antibodies/goat-igg-hrp-conjugated-antibody_haf017" target="_self">HAF017</a>). A specific band was detected for Vimentin at approximately 55 kDa (as indicated) in the parental K562 cell line, but is not detectable in knockout K562 cell line. GAPDH (<a href="https://www.bio-techne.com/p/antibodies/human-mouse-rat-gapdh-antibody_af5718" target="_self">AF5718</a>) is shown as a loading control. </p></div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="related"></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--header"> <div> <div><h2>Related Products</h2></div> </div> </div> </div> <div> <div class="paragraph paragraph--type--card-paragraph paragraph--view-mode--default card-para-default"> <div class="row card_row"> <div class="col-md-4"> <div class="h-100"> <div class="d-flex flex-column h-100"> <a href="/resources/instrument-applications/gene-transfer-assessment" > <div class="card-img-top text-center mb-3" style="background-repeat: no-repeat; background-color: ; height:200px; width: 100%;"> <div class="teaser-valign-container" style=""> <div class="valign"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/instruments/proteinsimple-milo-230x210.png" alt="Milo instrument by ProteinSimple a Bio-Techne brand" loading="lazy" > </div> </div> </div> </a> <a class="card_title_link" href="/resources/instrument-applications/gene-transfer-assessment" > <h3 class="card_title_htag"> <div>Milo Single Cell Westerns</div> </h3> </a> <div class="card_description flex-grow-1"> <div><p> Single-Cell Westerns with Milo enable analysis of CRISPR-based gene editing efficiency and expression uniformity with fewer cells.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/resources/instrument-applications/gene-transfer-assessment" > Learn More About Milo in Gene Transfer Assessment </a> </div> </div> </div> </div> </div> <div class="col-md-4"> <div class="h-100"> <div class="d-flex flex-column h-100"> <a href="/reagents/rnascope-ish-technology" > <div class="card-img-top text-center mb-3" style="background-repeat: no-repeat; 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background-color: ; height:200px; width: 100%;"> <div class="teaser-valign-container" style=""> <div class="valign"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/banners/bt-hero-HB-westernblot.png" alt="Western Blot Handbook Hero Image" loading="lazy" > </div> </div> </div> </a> <a class="card_title_link" href="/resources/literature/western-blot-handbook" > <h3 class="card_title_htag"> <div><h3>Western Blot eHandbook</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Our step-by-step guide to western blotting is aimed at helping you understand the principles behind this common technique and learn how to achieve reliable and reproducible results.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/resources/literature/western-blot-handbook" > Download eHandbook </a> </div> </div> </div> </div> </div> <div class="col-md-4"> <div class="h-100"> <div class="d-flex flex-column h-100"> <a href="/resources/webinars/multiplexed-single-cell-westerns-for-exploring-heterogeneity-in-pkc-signaling-and-crispr-efficiency" > <div class="card-img-top text-center mb-3" style="background-repeat: no-repeat; background-color: ; height:200px; width: 100%;"> <div class="teaser-valign-container" style=""> <div class="valign"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/webinar-icon.png" alt="Webinar on Demand Icon" loading="lazy" > </div> </div> </div> </a> <a class="card_title_link" href="/resources/webinars/multiplexed-single-cell-westerns-for-exploring-heterogeneity-in-pkc-signaling-and-crispr-efficiency" > <h3 class="card_title_htag"> <div>Single-Cell Westerns for Exploring PKC Signaling and CRISPR Efficiency</div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>In this Webinar, Dr Noemi Kedei and Dr Kelly Gardner explain how <a href="https://www.bio-techne.com/p/single-cell-western/milo_p100">Milo</a>, Bio-Techne's automated <a href="https://www.bio-techne.com/instruments/single-cell-western">single-cell western</a> (scWestern) platform is being used to explore heterogeneity in Protein Kinase C (PKC) signaling and CRISPR knockdown efficiency.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/resources/webinars/multiplexed-single-cell-westerns-for-exploring-heterogeneity-in-pkc-signaling-and-crispr-efficiency" > View Webinar </a> </div> </div> </div> </div> </div> <div class="col-md-4"> <div class="h-100"> <div class="d-flex flex-column h-100"> <a href="/resources/literature/crispr-edited-cell-lines-using-namocell-single-cell-dispensers" > <div class="card-img-top text-center mb-3" style="background-repeat: no-repeat; background-color: ; height:200px; width: 100%;"> <div class="teaser-valign-container" style=""> <div class="valign"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/literature/crispr-edited-single-cell-dispenser-thumbnail1000x1000.png" alt="Dr. Oscar Perez-Leal describes how his team leveraged a Bio-Techne Single Cell Sorter and Single Cell Dispenser to improve clonal outgrowth of CRISPR-edited cells." loading="lazy" > </div> </div> </div> </a> <a class="card_title_link" href="/resources/literature/crispr-edited-cell-lines-using-namocell-single-cell-dispensers" > <h3 class="card_title_htag"> <div>Accelerate the Development of CRISPR-edited Cell Line Models</div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Learn how the<a href="/instruments/single-cell-dispensers"> Bio-Techne's Single Cell Dispenser</a> can be used in streamlining and accelerating the development of CRISPR-edited cell line models in the whitepaper.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/resources/literature/crispr-edited-cell-lines-using-namocell-single-cell-dispensers" > Download Whitepaper </a> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="background"></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default"> <div> <div> <div class="paragraph paragraph--type--header"> <div> <div><h3>Background Information</h3></div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-what-is-crispr25" role="button" aria-expanded="false" aria-controls="expPara"> <h5>What is CRISPR?</h5> </a> </div> <div class="collapse" id="expPara-what-is-crispr25" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/figure-crispr-workflow-2222w.jpg" alt="Schematic showing the CRISPR-Cas system as a prokaryotic host defense mechanism and as a gene editing technique" loading="lazy" > </div> <div><strong>Figure 1: </strong>Schematic showing the CRISPR-Cas system.</div> <br/> <div><p><strong>CRISPR</strong> (clustered regularly interspaced short palindromic repeats) is a technology for the targeted editing and regulation of genes that can be applied to a number of biological systems.</p> <p>CRISPR-Cas9 is an RNA-guided DNA endonuclease enzyme (MW: 158.4 kDa), and is part of an acquired bacterial immune response against invading plasmids and viruses.  The CRISPR locus is composed of Cas (CRISPR associated) genes, a leader sequence and repeat spacer arrays (CRISPR repeats). The Cas genes encode nucleases that are capable of cleaving DNA. In type II CRISPR systems, invading DNA is detected by Cas1/2, which inserts a portion of the invading DNA into the cell's CRISPR array, between the CRISPR repeats, as a protospacer. The CRISPR array is then transcribed to form crRNA, which forms a complex with transactivating (tra)crRNA and Cas9. The protospacer section of the crRNA then binds to the complementary sequence on the invading DNA, which is cleaved by Cas9.</p></div> </div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-crispr-prokaryotic-host-defense-amp-gene-editing-technique25" role="button" aria-expanded="false" aria-controls="expPara"> <h5>CRISPR Prokaryotic Host Defense & Gene Editing Technique</h5> </a> </div> <div class="collapse" id="expPara-crispr-prokaryotic-host-defense-amp-gene-editing-technique25" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p>The CRISPR system has been adapted to create a technique capable of targeted gene editing at specific locations in the genome. By creating double-stranded breaks, CRISPR-Cas9 technology can be used to induce insertion or deletion mutations, specific sequence replacements or insertions, and large deletions or genomic rearrangements at any desired genomic location.</p> <p>This is made possible by designing RNA guided nucleases. In their simplest form, Cas9 is complexed with a guide RNA (gRNA), which consists of a crRNA and tracrRNA. The gRNA contains a complementary sequence to the DNA sequence of interest, and therefore guides Cas9 to the desired location. Cas9 then cleaves the DNA to create a DSB.</p> <p>DSBs can be repaired by endogenous non-homologous end-joining (NHEJ) and homology-directed repair (HDR). CRISPR-Cas9-mediated genome editing through NHEJ is imprecise but can reach efficiencies of 20-60%, whereas gene editing efficiency by HDR is more precise but has only been shown to reach 0.5-20%. As HDR is more precise than NHEJ, improving the efficiency of HDR is vital for the use of the CRISPR-Cas9 system in many applications.</p> <p>Recent research has demonstrated that small molecules can enhance the efficiency of HDR-mediated gene editing. The β<sub>3</sub> adrenergic receptor partial agonist <a href="/p/small-molecules-peptides/l-755-507_2197">L-755,507</a> has been shown to enhance the efficiency of HDR-mediated GFP insertion and point mutations by 3-fold and 9-fold, respectively in pluripotent stem cells. The small molecule <a href="/p/small-molecules-peptides/scr7-pyrazine_5342">SCR7 pyrazine</a> has also been shown to enhance HDR-efficiency, enhancing insertion of short DNA fragments by up to <strong>19-fold</strong>. Small molecules that enhance HDR efficiency could be integral to the application of the CRISPR-cas9 gene editing system.</p></div> </div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-crisprcas9-knockout-lysates23" role="button" aria-expanded="false" aria-controls="expPara"> <h5>CRISPR/Cas9 Knockout Lysates</h5> </a> </div> <div class="collapse" id="expPara-crisprcas9-knockout-lysates23" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/human-ki-67-knockout-validation.jpeg" alt="Human Ki-67 knockout validation by ICC" loading="lazy" > </div> <div>Human Ki-67/MKI67 Knockout (KO) Antibody Validation by Immunocytochemistry (ICC).</div> <br/> <div><p>The use of genetic negative controls has been proposed by the International Working Group for Antibody Validation as one of five key criteria for ensuring antibody specificity. Specificity, selectivity and fidelity of antibody-target interactions often varies based on the intricacies of specific analytical applications (e.g., immunoblot vs. immunostaining). Double knockout cell-lysates (<a href="/applications/western-blotting">Western Blot</a>) and cell-lines (Immunocytochemistry) are the best negative controls to quickly and confidently prove an antibody’s specificity for a target of interest and ensure reproducibility.</p> <p>Application of CRISPR/Cas9 gene-editing technology for the generation of knockout cell lysates and cell lines ensures the development of useful tools to validate antibody specificity and elucidate gene function. For example in In western blot  analysis, an initial indicator of antibody specificity is the presence of a single band at the expected target size, however proper antibody validation requires the use of adequate controls. Because CRISPR/Cas9 gene-editing technology enables complete removal or "knock out" of both alleles of the gene encoding the target protein, antibody specificity is confirmed by demonstrating that a protein band is only present in the wildtype and not the KO cell lysate in WB analysis.</p> <p>NOTE: To confirm differential expression, it is also important to analyze a separate target protein as a loading control (e.g., GAPDH) to demonstrate that all samples were loaded and transferred equally across wells.</p></div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </article> </div> </div> </section> </main> </div> <div id="brandsbar-footer" class="bg-bt-primary"> <div class="container-wide container-fluid text-white"> <div class="row py-5"> <div 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