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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE161634 | GEO | 2022/12/31

REPOSITORIES: GEO

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Cas9 interactions with host RNAs and transposable elements impair neurodevelopment (CUT&Tag)

Project description: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.

2022-12-31 | GSE161633 | GEO

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

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Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites

Project description:CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2,500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.

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Dissolution of RNA condensates by the embryonic stem cell protein L1TD1 [RNA-seq]

Project description:L1TD1 is a cytoplasmic RNA-binding protein that is specifically expressed in pluripotent stem cells and, unlike its mouse paralogue, is essential for the maintenance of stemness in human cells. Although L1TD1 is the only known domesticated gene from a LINE-1 (L1) retroelement, the functional legacy from its ancestral protein, ORF1p of L1, and the novelty are so far unknown. Here, we determined RNAs associated with L1TD1 and found that, like ORF1p, L1TD1 binds L1 RNAs, localizes to high-density ribonucleoprotein (RNP) condensates, and increases L1 retrotransposition. Unexpectedly, L1TD1 enhanced the translation of a subset of mRNAs enriched in the condensates. In HeLa cells, ectopically expressed L1TD1 facilitated the dissolution of stress granules and granules formed by pathological mutations of TDP-43 and FUS. The glutamate-rich domain and the ORF1-homology domain facilitated dispersal of the RNPs and induced autophagy, respectively. These results provide insights into how L1TD1 regulates gene expression in pluripotent stem cells. We propose that the ability of L1TD1 to dissolve stress granules may provide novel opportunities for treatments of neurodegenerative diseases caused by disturbed stress granule dynamics.

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Haplotype-phased Callithrix jacchus embryonic stem cell line for genome editing using CRISPR/Cas9

Project description:Haplotype-phased Callithrix jacchus embryonic stem cell line for genome editing using CRISPR/Cas9

2018-07-09 | PRJEB27676 | EVA

Activation of LINE-1 antisense promoter stimulates the growth of cultured cells

Project description:Long interspersed element 1 (LINE-1, or L1) is a retrotransposon that constitutes ~17% of the human genome. Although ~6,000 full-length L1s spread throughout the human genome, their biological significance remains undetermined. L1 5’ untranslated region has a bidirectional promoter activity with a sense promoter, driving L1 mRNA, and an antisense promoter (ASP), driving L1-gene chimeric RNAs. Here, we stimulated L1 ASP activity using the CRISPR-Cas9 system to evaluate its biological impacts. Activation of L1 ASP enhances L1 retrotransposition and cell growth. Conversely, we identified epigallocatechin gallate (EGCG) as an inhibitor of L1 ASP. The inhibition of L1 ASP by EGCG decreased cell growth but did not affect L1 retrotransposition. Collectively, these results indicate that activation of L1 ASP activity fuels cell growth. To our knowledge, this is the first report demonstrating the role of L1 ASP in the biological process. Considering that L1 sequences are de-silenced in various tumor cells, activation of L1 ASP may be a cause of tumor growth and interfering with L1 ASP activity will be a potential strategy to suppress the growth.

2020-12-22 | GSE152634 | GEO

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L1 retrotransposition in neurons is mediated by MeCP2

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