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<p id="Par1" class="p p-first-last"><em>Streptococcus pyogenes</em><span> </span>Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized<span> </span><em>PCSK9</em><span> </span>mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from<span> </span><em>Parasutterella secunda</em><span> </span>(PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the<span> </span><em>PCSK9</em><span> </span>site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.</p>
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'authors' => 'Bestas B. et al.',
'description' => '<div id="Abs1" lang="en" class="tsec sec">
<div>
<p id="Par1" class="p p-first-last"><em>Streptococcus pyogenes</em><span> </span>Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized<span> </span><em>PCSK9</em><span> </span>mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from<span> </span><em>Parasutterella secunda</em><span> </span>(PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the<span> </span><em>PCSK9</em><span> </span>site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.</p>
</div>
<div class="sec"><strong class="kwd-title">Subject terms:<span> </span></strong><span class="kwd-text">Genetic engineering, Gene therapy, CRISPR-Cas9 genome editing</span></div>
</div>
<div id="Abs2" lang="en" class="tsec sec"></div>',
'date' => '2023-09-06',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10482872/',
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View::_render() - CORE/Cake/View/View.php, line 933
View::render() - CORE/Cake/View/View.php, line 473
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Dispatcher::_invoke() - CORE/Cake/Routing/Dispatcher.php, line 193
Dispatcher::dispatch() - CORE/Cake/Routing/Dispatcher.php, line 167
[main] - APP/webroot/index.php, line 118
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