AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity
AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity
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DOI：
10.1016/j.omtm.2020.07.007
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发表时间：
2020-09-11
影响因子：
4.7
通讯作者：
Tai, Phillip W. L.
中科院分区：
文献类型：
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作者：
Ngoc Tam Tran;Heiner, Cheryl;Tai, Phillip W. L.
基因治疗领域受到两种技术的启发，这两种技术彻底改变了遗传疾病的治疗：基于腺相关病毒（AAV）的载体和成簇的规则间隔短回文重复序列（CRISPR）-Cas基因编辑工具。当结合到一个平台上时，这些安全和广泛的热带生物疗法可以被设计成靶向人类基因组中的任何区域，以纠正遗传缺陷。不幸的是，对AAV载体中CRISPR组分的设计相容性的研究很少。使用AAV-基因组群体测序（AAV-GPseq），我们先前发现具有强DNA二级结构的自身互补AAV载体设计可引起高度的截短事件，从而影响生产和载体功效。我们假设含有几个环区域的单向导RNA（sgRNA）支架也可能损害载体完整性。因此，我们已经推进了AAV-GPseq方法，以也询问单链AAV载体，以研究携带Cas9-sgRNA盒的载体基因组是否可以引起截短事件。我们发现，sgRNA序列本身不会产生高度的截短事件。然而，我们证明了以尾对尾构型携带双sgRNA表达盒的载体基因组设计导致截短。此外，我们发现，在反向末端重复序列的区域缺失的形式固有的某些AAV载体质粒的异质性可以询问。
The gene therapy field has been galvanized by two technologies that have revolutionized treating genetic diseases: vectors based on adeno-associated viruses (AAVs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-editing tools. When combined into one platform, these safe and broadly tropic biotherapies can be engineered to target any region in the human genome to correct genetic flaws. Unfortunately, few investigations into the design compatibility of CRISPR components in AAV vectors exist. Using AAV-genome population sequencing (AAV-GPseq), we previously found that self-complementary AAV vector designs with strong DNA secondary structures can cause a high degree of truncation events, impacting production and vector efficacy. We hypothesized that the single-guide RNA (sgRNA) scaffold, which contains several loop regions, may also compromise vector integrity. We have therefore advanced the AAV-GPseq method to also interrogate single-strand AAV vectors to investigate whether vector genomes carrying Cas9-sgRNA cassettes can cause truncation events. We found that on their own, sgRNA sequences do not produce a high degree of truncation events. However, we demonstrate that vector genome designs that carry dual sgRNA expression cassettes in tail-to-tail configurations lead to truncations. In addition, we revealed that heterogeneity in inverted terminal repeat sequences in the form of regional deletions inherent to certain AAV vector plasmids can be interrogated.