{"id":6268,"date":"2023-02-17T06:48:41","date_gmt":"2023-02-17T06:48:41","guid":{"rendered":"https:\/\/beta.nanion.de\/resource_library\/2018-simplified-footprint-free-cas9-crispr-editing-of-cardiac-associated-genes-in-human-pluripotent-stem-cells\/"},"modified":"2023-02-17T06:48:42","modified_gmt":"2023-02-17T06:48:42","slug":"2018-simplified-footprint-free-cas9-crispr-editing-of-cardiac-associated-genes-in-human-pluripotent-stem-cells","status":"publish","type":"resource_library","link":"https:\/\/nanion.com\/zh-hans\/resource_library\/2018-simplified-footprint-free-cas9-crispr-editing-of-cardiac-associated-genes-in-human-pluripotent-stem-cells\/","title":{"rendered":"2018 &#8211; Simplified footprint-free Cas9\/CRISPR editing of cardiac-associated genes in human pluripotent stem cells"},"content":{"rendered":"<p>Modelling disease with hPSCs is hindered because the impact on cell phenotype from genetic variability between individuals can be greater than from the pathogenic mutation. While \u2018footprint-free\u2019 Cas9\/CRISPR editing solves this issue, existing approaches are inefficient or lengthy. Here, a simplified PiggyBac strategy shortened hPSC editing by 2 weeks and required one round of clonal expansion and genotyping rather than two, with similar efficiencies to the longer conventional process. Success was shown across 4 cardiac-associated loci (ADRB2, GRK5, RYR2, ACTC1) by genomic cleavage and editing efficiencies of 8-93% and 8-67%, respectively, including mono- and\/or bi-allelic events. Pluripotency was retained, as was differentiation into high purity cardiomyocytes (CMs; 88-99%). Using the GRK5 isogenic lines as an exemplar, chronic stimulation with the b-adrenoceptor agonist, isoprenaline, reduced beat rate in hPSC-CMs expressing GRK5-Q41 but not GRK5-L41; this was reversed by the b-blocker, propranolol. This simplified, footprint-free approach will be useful for mechanistic studies.<\/p>\n","protected":false},"featured_media":0,"template":"","products_tags":[34],"resource_type":[75],"application_areas":[50,77],"therapeutic_areas":[54],"target_categories":[335],"target_families":[336],"targets":[258],"resource_keyword":[],"class_list":["post-6268","resource_library","type-resource_library","status-publish","hentry","products_tags-cardioexcyte-96","resource_type-publication","application_areas-ion-channels","application_areas-cell-ipscs-profiling","therapeutic_areas-cardiac-safety-cipa","target_categories-intracellular-regulated-ion-channels","target_families-calcium-release-receptors","targets-ryrs"],"acf":[],"_links":{"self":[{"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/resource_library\/6268","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/resource_library"}],"about":[{"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/types\/resource_library"}],"wp:attachment":[{"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/media?parent=6268"}],"wp:term":[{"taxonomy":"products_tags","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/products_tags?post=6268"},{"taxonomy":"resource_type","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/resource_type?post=6268"},{"taxonomy":"application_areas","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/application_areas?post=6268"},{"taxonomy":"therapeutic_areas","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/therapeutic_areas?post=6268"},{"taxonomy":"target_categories","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/target_categories?post=6268"},{"taxonomy":"target_families","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/target_families?post=6268"},{"taxonomy":"targets","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/targets?post=6268"},{"taxonomy":"resource_keyword","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/resource_keyword?post=6268"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}