Cas9 interactions with host RNAs and transposable elements impair neurodevelopment (irCLIP-seq)

The CRISPR (clustered regulatory interspaced short palindromic repeats)/ Cas9 (CRISPR-associated protein 9) system-based precise genome editing has revolutionized biomedical studies. As the CRISPR/Cas9 system has been also recently considered to permanently cure genetic diseases via human germline genome editing, a careful risk assessment of this genome engineering tool is required in human early development. Here we perform comprehensive analysis to evaluate the potential impact of Cas9 in human early embryogenesis. We find that even in the absence of synthetic guide RNA, Cas9 can be still guided by endogenous RNAs, and cut the genome of human embryonic cells at low frequency, and the resulting DNA damage induces the intrinsic immune response. Moreover, Cas9 interferes with the spliceosome, and with the suppressor machinery (e.g. L1TD1, APOBEC3G and PIWIL4) of LINE-1 (L1) retrotransposition. Even the transient presence of Cas9 in human embryonic cells induces robust de novo L1 retrotransposition that exacerbates DNA damage, and results in compromised neurodevelopment. Besides the ethical issues, these inevitable and detrimental Cas9-associated impacts on human embryonic development raise such serious safety concerns as to question the clinical use of human germline genome editing. We first established a hESC line that expressed Cas9 (fused with FLAG) under doxycycline treatment and then performed irCLIP-seq experiments of Cas9. Two replicate Cas9 irCLIP libraries were sequenced.

基于成簇规律间隔短回文重复序列（clustered regulatory interspaced short palindromic repeats，CRISPR）/CRISPR相关蛋白9（CRISPR-associated protein 9，Cas9）系统的精准基因组编辑技术，已彻底革新了生物医学研究领域。鉴于CRISPR/Cas9系统近期被认为可通过人类生殖系基因组编辑永久治愈遗传疾病，因此有必要针对该基因组工程工具在人类早期发育过程中的应用开展严谨的风险评估。本研究通过全面分析，评估Cas9在人类早期胚胎发生过程中的潜在影响。研究发现，即便不存在合成向导RNA（guide RNA，gRNA），Cas9仍可被内源性RNA引导，并以低频率切割人类胚胎细胞的基因组，由此产生的DNA损伤会激活固有免疫应答。此外，Cas9会干扰剪接体功能，并影响LINE-1（L1）逆转录转座的抑制机制（如L1TD1、APOBEC3G及PIWIL4）。即便是Cas9在人类胚胎细胞中短暂存在，也会诱导显著的新生L1逆转录转座，进而加剧DNA损伤，并导致神经发育受损。除伦理问题外，这些与Cas9相关且不可避免的有害影响会干扰人类胚胎发育，由此引发的严重安全顾虑也对人类生殖系基因组编辑的临床应用提出了质疑。本研究首先构建了可在强力霉素（doxycycline）诱导下表达FLAG标签融合Cas9的人类胚胎干细胞（human embryonic stem cell，hESC）系，随后开展了Cas9的irCLIP-seq实验，共构建并测序了两份重复的Cas9 irCLIP文库。