Robust genome editing via modRNA-based Cas9 or base editor in human pluripotent stem cells.
Robust genome editing via modRNA-based Cas9 or base editor in human pluripotent stem cells.
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DOI：
10.1016/j.crmeth.2022.100290
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发表时间：
2022-09-19
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CRISPR系统为生物医学研究带来了革命性的变化，因为它们为基因组编辑提供了前所未有的机会。然而，将CRISPR系统应用于人多能干细胞(HPSCs)的一个瓶颈是如何方便有效地递送CRISPR效应器。在这里，我们开发了基于修饰mRNA(ModRNA)的CRIPSR系统，该系统利用Cas9和p53DD或碱基编辑程序(ABE8e)modRNA，通过简单的基于脂质的转染来敲除hPSCs中的基因。ABE8E modRNA被用来破坏剪接供体位点，导致目标转录本的剪接缺陷，最终导致基因敲除。使用我们的modRNA CRISPR系统，Cas9+p53DD modRNA和ABE8e modRNA的敲除效率分别达到73.3%±11.2%和69.6±3.8%，显著高于基于质粒的系统。综上所述，我们证明了我们的基于非整合modRNA的CRISPR方法作为更有效和更容易获得的hPSC基因组编辑技术具有很大的前景。通过Cas9/p53DD modRNA对hPSCs进行基因编辑，获得了高达84%的基因敲除效率。Cas9/p53DD modRNA方法优于基于质粒和RNP的方法ABE8E modRNA获得了5倍于质粒ABE8E的基因敲除效率ABE8E稳健而有效的敲除干细胞中的基因的方法对于理解基因在干细胞分化过程中的功能是必不可少的。以质粒为基础的CRISPR系统可用于产生基因敲除，但效率较低，而且质粒DNA可能整合到基因组中，从而影响基因组的完整性。我们试图开发基于非整合和高效的修饰mRNA(ModRNA)的CRISPR系统(Cas9或碱基编辑者)，该系统可用于在人类胚胎干细胞和诱导多能干细胞中实现强大的基因敲除，以规避基于质粒的CRISPR系统所面临的挑战。Haideri等人。开发基于modRNA的非整合CRISPR系统，以实现hPSC中强大而高效的基因敲除。他们进一步建立了ABE8e碱基编辑者modRNA协议来扰乱剪接供体位点。这些非整合的方法可以保持基因组的完整性，并显著提高基因敲除效率。
CRISPR systems have revolutionized biomedical research because they offer an unprecedented opportunity for genome editing. However, a bottleneck of applying CRISPR systems in human pluripotent stem cells (hPSCs) is how to deliver CRISPR effectors easily and efficiently. Here, we developed modified mRNA (modRNA)-based CRIPSR systems that utilized Cas9 and p53DD or a base editor (ABE8e) modRNA for the purposes of knocking out genes in hPSCs via simple lipid-based transfection. ABE8e modRNA was employed to disrupt the splice donor site, resulting in defective splicing of the target transcript and ultimately leading to gene knockout. Using our modRNA CRISPR systems, we achieved 73.3% ± 11.2% and 69.6 ± 3.8% knockout efficiency with Cas9 plus p53DD modRNA and ABE8e modRNA, respectively, which was significantly higher than the plasmid-based systems. In summary, we demonstrate that our non-integrating modRNA-based CRISPR methods hold great promise as more efficient and accessible techniques for genome editing of hPSCs. Transfection of hPSCs with Cas9 modRNA yielded 90% transfection efficiency Gene editing of hPSCs via Cas9/p53DD modRNA yielded up to 84% knockout efficiency Cas9/p53DD modRNA method was better than plasmid- and RNP-based methods ABE8e modRNA achieved 5-fold knockout efficiency relative to the plasmid ABE8e Robust and efficient methods for knocking out genes in stem cells are indispensable in understanding the function of a gene during stem cell differentiation. Plasmid-based CRISPR systems can be used to generate gene knockouts, but the efficiency is low, and plasmid DNA may integrate into the genome and thus compromise genome integrity. We sought to develop non-integrating and efficient modified mRNA (modRNA)-based CRISPR systems (Cas9 or base editor) that can be used to achieve robust gene knockouts in both human embryonic stem cells and induced pluripotent stem cells to circumvent the challenges faced by plasmid-based CRISPR systems. Haideri et al. develop non-integrating modRNA-based CRISPR systems for achieving robust and efficient gene knockouts in hPSCs. They further establish an ABE8e base editor modRNA protocol to disrupt the splice donor site. These non-integrating approaches can preserve genome integrity and significantly enhance knockout efficiency.