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href="https://missionbio.com/company/blog/">Blog</a></span> <span class="bread-arrow"></span> <span class="breadcrumb_last" aria-current="page">CRISPR-edited gene pools: Exploring variation in gene-edited cells</span></span></div></div>  <br /> <div class="resources-wrap detail"> <div class="_1300-container inner-content resource"> <div class="resource-category detail"> <div class="text-block">blog</div> </div> <div class="resource-detail-title"> <div class="resource-detail-image" style="background-image: url(https://missionbio.com/wp-content/uploads/2024/02/CRISPR-illustration.png);"></div> <div class="resource-detail-title-copy"> <div class="blog-post-category"><a href="https://missionbio.com/company/blog/category/single-cell-applications/" rel="category tag">Single-Cell Applications</a> | <a href="https://missionbio.com/company/blog/category/special-topics/" rel="category tag">Special Topics</a></div> <div class="resource-detail-author">February 29, 2024 by Brittany Enzmann, PhD <span class="span-reading-time rt-reading-time"><span class="rt-label rt-prefix">•</span> <span class="rt-time"> 5</span> <span class="rt-label rt-postfix">min read</span></span></div> <h1 class="resource-detail-main-title">CRISPR-edited gene pools: Exploring variation in gene-edited cells</h1> </div> </div> <div class="resource-content"> <div class="entry-content clrfix"> <p><span style="font-weight: 400;">Genome engineering techniques, like CRISPR, have revolutionized how scientists approach research and precision therapeutics. While a powerful technique, especially for engineering novel therapeutics, CRISPR introduces some inherent challenges related to heterogeneous editing outcomes that include variations in zygosity, off-target effects, and co-occurring multiplex edits. Here we explore these variations, including how they occur and how single-cell technology can help identify and quantitate these outcomes.</span></p> <p> </p> <h2><span style="font-weight: 400;">How do CRISPR-edited pools become heterogeneous?</span></h2> <p><span style="font-weight: 400;">Despite the promise of precision of advanced genome editors, editing outcomes remain largely unpredictable. Different cells subjected to the same editing regimen can yield distinct combinations of edits, varying not only across multiple on-target sites but also between on-target and off-target locations. The result is that genome editing of pools is often very heterogeneous.</span></p> <p><img fetchpriority="high" decoding="async" class="aligncenter wp-image-11923" src="https://missionbio.com/wp-content/uploads/2024/02/CRISPR-heterogeneity.png" alt="" width="400" height="201" srcset="https://missionbio.com/wp-content/uploads/2024/02/CRISPR-heterogeneity.png 1022w, https://missionbio.com/wp-content/uploads/2024/02/CRISPR-heterogeneity-300x151.png 300w, https://missionbio.com/wp-content/uploads/2024/02/CRISPR-heterogeneity-768x386.png 768w, https://missionbio.com/wp-content/uploads/2024/02/CRISPR-heterogeneity-480x241.png 480w, https://missionbio.com/wp-content/uploads/2024/02/CRISPR-heterogeneity-240x120.png 240w" sizes="(max-width: 400px) 100vw, 400px" /></p> <p style="text-align: center;"><em><i><span style="font-weight: 400;">Figure 1. CRISPR editing of two intended targets (A and B) leads to heterogeneous outcomes, in which the edit genotypes, zygosity status, and co-occurrence vary across cells (illustrated by gradation in color)</span></i></em></p> <p> </p> <p><span style="font-weight: 400;">Figure 1 shows an example of an editing experiment where there are wild type cells that are genome-edited using CRISPR at two intended targets, Target A in yellow and Target B in blue. After editing, the result is a mixture of cells, some of which are edited with the editing shown as a gradation of the blue and yellow colors to represent the variability in the pool.</span></p> <h2></h2> <h2><span style="font-weight: 400;">What are the types of variation in CRISPR-edited cells?</span></h2> <h3></h3> <h3><span style="font-weight: 400;">Variation in zygosity</span></h3> <h2><img decoding="async" class="aligncenter wp-image-11924" src="https://missionbio.com/wp-content/uploads/2024/02/variation-in-zygostiy.png" alt="" width="400" height="223" srcset="https://missionbio.com/wp-content/uploads/2024/02/variation-in-zygostiy.png 935w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-zygostiy-300x167.png 300w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-zygostiy-768x429.png 768w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-zygostiy-480x268.png 480w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-zygostiy-240x134.png 240w" sizes="(max-width: 400px) 100vw, 400px" /></h2> <p style="text-align: center;"><em><i><span style="font-weight: 400;">Figure 2. Edits at an intended target may be bi-allelic (present on both homologous chromosomes of a diploid cell) or mono-allelic (present on just one chromosome).</span></i></em></p> <p> </p> <p><span style="font-weight: 400;">One source of variation is at the level of zygosity status (Fig 2). In our example, let鈥檚 assume the cells are diploid and that two homologous chromosomes can be edited for each target site. You could have a bi-allelic edit where both chromosomes are edited, or you could have a mono-allelic edit where one chromosome is edited and one chromosome remains wild type.</span></p> <h3></h3> <h3><span style="font-weight: 400;">Variation in variant genotype</span></h3> <p><img decoding="async" class="aligncenter wp-image-11926" src="https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype-1024x333.png" alt="" width="800" height="260" srcset="https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype-1024x333.png 1024w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype-300x98.png 300w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype-768x250.png 768w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype-1536x499.png 1536w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype-1800x585.png 1800w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype-1440x468.png 1440w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype-1280x416.png 1280w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype-480x156.png 480w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype-240x78.png 240w, https://missionbio.com/wp-content/uploads/2024/02/variation-in-genotype.png 2030w" sizes="(max-width: 800px) 100vw, 800px" /></p> <p style="text-align: center;"><em><i><span style="font-weight: 400;">Figure 3. Cells that are edited with the same guide RNA at the same target site may have different variant genotypes. Cells may be homozygous, compound heterozygous, or heterozygous at the target locus.</span></i></em></p> <p> </p> <p><span style="font-weight: 400;">If we dig into this further, more variation is possible. In the example above (Fig. 3), for a bi-allelic edit (left), one option is a homozygous edit, where the same edit occurs on both alleles (in this case, an insertion of two G nucleotides).</span></p> <p><span style="font-weight: 400;">Another possibility is a compound heterozygote, where both chromosomes are still edited, but there are different indels on each of those chromosomes (in this example, the insertion of two G nucleotides in the first, and the insertion of a T nucleotide in the second).</span></p> <p><span style="font-weight: 400;">Finally, you can have a heterozygous edit, where one chromosome is wild type and one is edited (insertion of two G nucleotides).</span></p> <h3></h3> <h3><span style="font-weight: 400;">Variation in co-occurrence of edits</span></h3> <p><img loading="lazy" decoding="async" class="aligncenter wp-image-11927" src="https://missionbio.com/wp-content/uploads/2024/02/variation-of-cooccurrence-1024x489.png" alt="" width="500" height="239" srcset="https://missionbio.com/wp-content/uploads/2024/02/variation-of-cooccurrence-1024x489.png 1024w, https://missionbio.com/wp-content/uploads/2024/02/variation-of-cooccurrence-300x143.png 300w, https://missionbio.com/wp-content/uploads/2024/02/variation-of-cooccurrence-768x367.png 768w, https://missionbio.com/wp-content/uploads/2024/02/variation-of-cooccurrence-480x229.png 480w, https://missionbio.com/wp-content/uploads/2024/02/variation-of-cooccurrence-240x115.png 240w, https://missionbio.com/wp-content/uploads/2024/02/variation-of-cooccurrence.png 1063w" sizes="auto, (max-width: 500px) 100vw, 500px" /></p> <p style="text-align: center;"><i><span style="font-weight: 400;">Figure 4.聽</span></i><i><span style="font-weight: 400;">When editing multiple intended target loci at the same time (illustrated as A and B on the left), not all cells will have both A and B targets edited. </span></i></p> <p> </p> <p><span style="font-weight: 400;">Because our experiment involves editing multiple targets (A and B), variation can also exist on how many targets are actually edited across cells. </span><span style="font-weight: 400;">Some cells might have both targets edited at the single cell level, but some cells might just have one target edited (Fig 4, left)聽 </span><span style="font-weight: 400;">If off-target edits are present, there might be different combinations of on- and off-target edits that also vary across cells (Fig 4, right).</span></p> <h3></h3> <h3><span style="font-weight: 400;">Variation in cell types or states that are edited</span></h3> <p><img loading="lazy" decoding="async" class="aligncenter size-full wp-image-11928" src="https://missionbio.com/wp-content/uploads/2024/02/11.png" alt="" width="226" height="205" /></p> <p style="text-align: center;"><i><span style="font-weight: 400;">Figure 5. Cell types/states can be identified by the signature of cell-surface proteins. Gene editing may differentially edit some cell types/states more readily than others, leading to cell-to-cell variation in editing outcomes.</span></i></p> <p> </p> <p><span style="font-weight: 400;">Finally, if we assume the cells in our experiment differ in cell type or cell state, editing may differ across these categories. So this is an additional axis of variation that must be considered.</span></p> <p><span style="font-weight: 400;">It鈥檚 important to note that while bulk sequencing is appropriate for some questions, bulk assays do not preserve the information of individual cells and can鈥檛 answer these questions in variation outlined above. Only single-cell聽 analysis can provide insight into these levels of variation.</span></p> <p> </p> <h2><span style="font-weight: 400;">The Tapestri Genome Editing Solution offers comprehensive analysis of gene edits</span></h2> <p><span style="font-weight: 400;">So how do we address all these questions in variation? The </span><a href="https://missionbio.com/products/platform/"><span style="font-weight: 400;">Tapestri Platform </span></a><span style="font-weight: 400;">can be used for a diverse array of applications ranging from assessing tumor heterogeneity, all the way to cell characterization for genome editing and cell and gene therapies, as well as disease modeling.</span></p> <p> </p> <p><img loading="lazy" decoding="async" class="aligncenter size-large wp-image-11930" src="https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-1024x438.png" alt="" width="1024" height="438" srcset="https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-1024x438.png 1024w, https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-300x128.png 300w, https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-768x329.png 768w, https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-1536x658.png 1536w, https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-2048x877.png 2048w, https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-1800x771.png 1800w, https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-1440x617.png 1440w, https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-1280x548.png 1280w, https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-480x206.png 480w, https://missionbio.com/wp-content/uploads/2024/02/Tapestri-gene-editing-240x103.png 240w" sizes="auto, (max-width: 1024px) 100vw, 1024px" /></p> <p> </p> <p><span style="font-weight: 400;">The</span><a href="https://missionbio.com/applications/genome-editing/"><span style="font-weight: 400;"> Tapestri Genome Editing Solution</span></a><span style="font-weight: 400;"> is an end-to-end solution that includes reagents, the Tapestri instrument, and a robust software solution for analysis. This assay is high throughput and can analyze 1000s of cells at a time, unlike plate-based assays which are restricted to fewer cells. The assay is also targeted, meaning the whole genome does not need to be sequenced.</span></p> <p>The Tapestri Genome Editing Solution addresses:</p> <ul> <li style="font-weight: 400;" aria-level="1"><b>On-target single & multiplexed editing.</b><span style="font-weight: 400;"> If you are editing one or multiple sites simultaneously, the solution can analyze all these targets together.</span></li> <li style="font-weight: 400;" aria-level="1"><b>Predicted off-target editing</b><span style="font-weight: 400;"><strong>.</strong> If you know the predicted genomic location of the off-target edits, the solution can confirm if off-target editing is occurring. There are multiple tools to predict off-target edits prior to a Tapestri assay.</span></li> <li style="font-weight: 400;" aria-level="1"><b>Edit zygosity</b><span style="font-weight: 400;"><strong>.</strong> The analysis will tell you whether one or both chromosomes are edited at each target site and the identity of the variants.聽</span></li> <li style="font-weight: 400;" aria-level="1"><b>Edit co-occurrence</b><span style="font-weight: 400;"><strong>.</strong> The analysis will report the co-occurrence of edits at multiple intended targets (on-targets) and the co-occurrence of on- and off-targets in individual cells.</span></li> <li style="font-weight: 400;" aria-level="1"><b>Multi-omic analysis.</b> Cell surface proteins can be co-measured with genome edits to provide insights into the immunophenotype of edited cells or to validate the knockout of a target.</li> </ul> <p> </p> <p><span style="font-weight: 400;">To learn more about how to analyze heterogeneous outcomes in genome-edited pools, check out our </span><a href="https://missionbio.com/resources/presentations/crispr-genome-editing-synthego/"><span style="font-weight: 400;">joint webinar</span></a><span style="font-weight: 400;"> with Synthego or download our </span><a href="https://missionbio.com/resources/technical-notes/unprecedented-resolution-of-genome-editing-outcomes-with-single-cell-analysis/"><span style="font-weight: 400;">Genome Editing Solution Application Note</span></a><span style="font-weight: 400;"> for more information.</span></p> </div> <div class="blog-author"> <h4>About the Author</h4> <div class="saboxplugin-wrap" itemtype="http://schema.org/Person" itemscope itemprop="author"><div class="saboxplugin-tab saboxplugin-tab-about"><div class="saboxplugin-gravatar"><a href='https://missionbio.com/author/enzmann/'><img src="https://missionbio.com/wp-content/uploads/2021/03/Author-Brittany-Enzmann.jpg" width="100" height="100" alt="" itemprop="image"></a></div><div class="saboxplugin-authorname"><a href="https://missionbio.com/author/enzmann/" class="vcard author" rel="author" itemprop="url"><span class="fn" itemprop="name">Brittany Enzmann, PhD</span></a></div><div class="saboxplugin-desc"><div itemprop="description"><p>Brittany is a Bay Area native who is passionate about biology and enjoys conveying complex topics in creative ways. She loves travel and photography, and has a particular weakness for cute dogs.</p> </div></div><div class="clearfix"></div><div class="saboxplugin-socials "><a target="_blank" href="https://www.linkedin.com/in/brittanyenzmann/" area-label="Linkedin" rel="nofollow" class="saboxplugin-icon-grey"><svg aria-hidden="true" class="sab-linkedin" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 448 512"><path fill="currentColor" d="M100.3 480H7.4V180.9h92.9V480zM53.8 140.1C24.1 140.1 0 115.5 0 85.8 0 56.1 24.1 32 53.8 32c29.7 0 53.8 24.1 53.8 53.8 0 29.7-24.1 54.3-53.8 54.3zM448 480h-92.7V334.4c0-34.7-.7-79.2-48.3-79.2-48.3 0-55.7 37.7-55.7 76.7V480h-92.8V180.9h89.1v40.8h1.3c12.4-23.5 42.7-48.3 87.9-48.3 94 0 111.3 61.9 111.3 142.3V480z"></path></svg></span></a></div></div></div> </div> <div class="blog-back"> <a href="https://missionbio.com/company/blog/" class="back-link" />Back to blog</a> </div> </div>   <br /> <div class="share-block"> <h3 class="heading">SHARE THIS PAGE</h3> 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