当前位置: X-MOL 学术Bioeng. Transl. Med. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Highly efficient CRISPR/Cas9-mediated exon skipping for recessive dystrophic epidermolysis bullosa
Bioengineering & Translational Medicine ( IF 7.4 ) Pub Date : 2024-01-17 , DOI: 10.1002/btm2.10640
Alex du Rand 1 , John Hunt 1 , Christopher Samson 1 , Evert Loef 1 , Chloe Malhi 1 , Sarah Meidinger 1 , Chun‐Jen Jennifer Chen 1 , Ashley Nutsford 1 , John Taylor 1 , Rod Dunbar 1 , Diana Purvis 2 , Vaughan Feisst 1 , Hilary Sheppard 1
Affiliation  

Gene therapy based on the CRISPR/Cas9 system has emerged as a promising strategy for treating the monogenic fragile skin disorder recessive dystrophic epidermolysis bullosa (RDEB). With this approach problematic wounds could be grafted with gene edited, patient-specific skin equivalents. Precise gene editing using homology-directed repair (HDR) is the ultimate goal, however low efficiencies have hindered progress. Reframing strategies based on highly efficient non-homologous end joining (NHEJ) repair aimed at excising dispensable, mutation-harboring exons offer a promising alternative approach for restoring the COL7A1 open reading frame. To this end, we employed an exon skipping strategy using dual single guide RNA (sgRNA)/Cas9 ribonucleoproteins (RNPs) targeted at three novel COL7A1 exons (31, 68, and 109) containing pathogenic heterozygous mutations, and achieved exon deletion rates of up to 95%. Deletion of exon 31 in both primary human RDEB keratinocytes and fibroblasts resulted in the restoration of type VII collagen (C7), leading to increased cellular adhesion in vitro and accurate C7 deposition at the dermal-epidermal junction in a 3D skin model. Taken together, we extend the list of COL7A1 exons amenable to therapeutic deletion. As an incidental finding, we find that long-read Nanopore sequencing detected large on-target structural variants comprised of deletions up to >5 kb at a frequency of ~10%. Although this frequency may be acceptable given the high rates of intended editing outcomes, our data demonstrate that standard short-read sequencing may underestimate the full range of unexpected Cas9-mediated editing events.

中文翻译:

高效 CRISPR/Cas9 介导的外显子跳跃治疗隐性营养不良性大疱性表皮松解症

基于 CRISPR/Cas9 系统的基因治疗已成为治疗单基因脆性皮肤病隐性营养不良性大疱性表皮松解症 (RDEB) 的一种有前景的策略。通过这种方法,有问题的伤口可以用经过基因编辑的、患者特定的皮肤替代物移植。使用同源定向修复(HDR)进行精确基因编辑是最终目标,但效率低下阻碍了进展。基于高效非同源末端连接 (NHEJ) 修复的重构策略旨在切除可有可无的突变外显子,为恢复COL7A1开放阅读框提供了一种有前途的替代方法。为此,我们采用了外显子跳跃策略,使用双单向导RNA(sgRNA)/Cas9核糖核蛋白(RNP)针对含有致病性杂合突变的三个新的COL7A1外显子(31、68和109),并实现了高达至 95%。原代人 RDEB 角质形成细胞和成纤维细胞中外显子 31 的缺失导致 VII 型胶原蛋白 (C7) 的恢复,从而导致体外细胞粘附增加以及 3D 皮肤模型中真皮-表皮交界处 C7 的准确沉积。总而言之,我们扩展了适合治疗性删除的COL7A1外显子列表。作为一个偶然的发现,我们发现长读长 Nanopore 测序检测到了大的目标结构变异,其中缺失高达 > 5 kb,频率约为 10%。尽管考虑到预期编辑结果的高发生率,这个频率可能是可以接受的,但我们的数据表明,标准短读长测序可能会低估 Cas9 介导的意外编辑事件的全部范围。
更新日期:2024-01-18
down
wechat
bug