CRISPR/Cas9 Gene Editing System Can Alter Gene Expression and Induce DNA Damage Accumulation

Clustered regularly interspaced short palindromic repeats (CRISPR) and the associated protein (Cas) gene editing can induce P53 activation, large genome fragment deletions, and chromosomal structural variations. Here, gene expression was detected in host cells using transcriptome sequencing following CRISPR/Cas9 gene editing. We found that the gene editing reshaped the gene expression, and the number of differentially expressed genes was correlated with the gene editing efficiency. Moreover, we found that alternative splicing occurred at random sites and that targeting a single site for gene editing may not result in the formation of fusion genes. Further, gene ontology and KEGG enrichment analysis showed that gene editing altered the fundamental biological processes and pathways associated with diseases. Finally, we found that cell growth was not affected; however, the DNA damage response protein—γH2AX—was activated. This study revealed that CRISPR/Cas9 gene editing may induce cancer-related changes and provided basic data for research on the safety risks associated with the use of the CRISPR/Cas9 system.

[1]  P. Veys,et al.  Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia , 2022, Science Translational Medicine.

[2]  Jiazhi Hu,et al.  CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo , 2022, Nucleic acids research.

[3]  K. Burns,et al.  Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites , 2022, Nature Communications.

[4]  A. Madi,et al.  Frequent Aneuploidy in Primary Human T Cells after CRISPR-Cas9 cleavage , 2022, Nature biotechnology.

[5]  OUP accepted manuscript , 2022, Nucleic Acids Research.

[6]  L. Feuk,et al.  CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations , 2021, bioRxiv.

[7]  D. Cappellen,et al.  CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells , 2021, Nature Communications.

[8]  Weiwei Zhang,et al.  In-depth assessment of the PAM compatibility and editing activities of Cas9 variants , 2021, Nucleic acids research.

[9]  Mark D. M. Leiserson,et al.  A systematic genome-wide mapping of oncogenic mutation selection during CRISPR-Cas9 genome editing , 2021, Nature Communications.

[10]  Cheng-Zhong Zhang,et al.  Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing , 2020, Nature Genetics.

[11]  Amanda M Li,et al.  CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. , 2020, The New England journal of medicine.

[12]  Ashish Ranjan Sharma,et al.  CRISPR-Cas9: A Preclinical and Clinical Perspective for the Treatment of Human Diseases. , 2020, Molecular therapy : the journal of the American Society of Gene Therapy.

[13]  Yao Fu,et al.  CRISPR-Cas9 gene editing causes alternative splicing of the targeting mRNA. , 2020, Biochemical and biophysical research communications.

[14]  Oana M. Enache,et al.  Cas9 activates the p53 pathway and selects for p53-inactivating mutations , 2020, Nature Genetics.

[15]  Tao Tao,et al.  mTOR signaling pathway and mTOR inhibitors in cancer: progress and challenges , 2020, Cell & Bioscience.

[16]  Howard Y. Chang,et al.  CRISPR-engineered T cells in patients with refractory cancer , 2020, Science.

[17]  G. Cullot,et al.  CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations , 2019, Nature Communications.

[18]  N. Ferrara,et al.  VEGF in Signaling and Disease: Beyond Discovery and Development , 2019, Cell.

[19]  J. Taipale,et al.  CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response , 2018, Nature Medicine.

[20]  Gregory McAllister,et al.  p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells , 2018, Nature Medicine.

[21]  Haitao Wang,et al.  FOXO Signaling Pathways as Therapeutic Targets in Cancer , 2017, International journal of biological sciences.

[22]  David M. Sabatini,et al.  mTOR Signaling in Growth, Metabolism, and Disease , 2017, Cell.

[23]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

[24]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[25]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[26]  Jennifer Doudna,et al.  RNA-programmed genome editing in human cells , 2013, eLife.

[27]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[28]  Philippe Horvath,et al.  The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli , 2011, Nucleic acids research.

[29]  K. Makino,et al.  Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product , 1987, Journal of bacteriology.