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Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples.

ABSTRACT: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated nucleases (Cas) are among the most promising technologies for the treatment of hemoglobinopathies including Sickle Cell Disease (SCD). We are only beginning to identify the molecular variables that influence the specificity and the efficiency of CRISPR- directed gene editing, including the position of the cleavage site and the inherent variability among patient samples selected for CRISPR-directed gene editing. Here, we target the beta globin gene in human CD34+ cells to assess the impact of these two variables and find that both contribute to the global diversity of genetic outcomes. Our study demonstrates a unique genetic profile of indels that is generated based on where along the beta globin gene attempts are made to correct the SCD single base mutation. Interestingly, even within the same patient sample, the location of where along the beta globin gene the DNA is cut, HDR activity varies widely. Our data establish a framework upon which realistic protocols inform strategies for gene editing for SCD overcoming the practical hurdles that often impede clinical success.

SUBMITTER: Modarai SR

PROVIDER: S-EPMC7902267 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples.

Modarai Shirin R SR Kanda Sambee S Bloh Kevin K Opdenaker Lynn M LM Kmiec Eric B EB

Gene therapy 20200901 1-2

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated nucleases (Cas) are among the most promising technologies for the treatment of hemoglobinopathies including Sickle Cell Disease (SCD). We are only beginning to identify the molecular variables that influence the specificity and the efficiency of CRISPR- directed gene editing, including the position of the cleavage site and the inherent variability among patient samples selected for CRISPR-di ...[more]

PMID: 32873924

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Project description:BackgroundHepatic knockdown of the proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) gene or the angiopoietin-like 3 ( ANGPTL3 ) gene has been demonstrated to reduce blood low-density lipoprotein cholesterol (LDL-C) levels, and hepatic knockdown of the angiotensinogen ( AGT ) gene has been demonstrated to reduce blood pressure. Genome editing can productively target each of these three genes in hepatocytes in the liver, offering the possibility of durable "one-and-done" therapies for hypercholesterolemia and hypertension. However, concerns around making permanent gene sequence changes via DNA strand breaks might hinder acceptance of these therapies. Epigenome editing offers an alternative approach to gene inactivation, via silencing of gene expression by methylation of the promoter region, but the long-term durability of epigenome editing remains to be established.MethodsWe assessed the ability of epigenome editing to durably reduce the expression of the human PCSK9, ANGPTL3 , and AGT genes in HuH-7 hepatoma cells. Using the CRISPRoff epigenome editor, we identified guide RNAs that produced efficient gene knockdown immediately after transfection. We assessed the durability of gene expression and methylation changes through serial cell passages.ResultsCells treated with CRISPRoff and PCSK9 guide RNAs were maintained for up to 124 cell doublings and demonstrated durable knockdown of gene expression and increased CpG dinucleotide methylation in the promoter, exon 1, and intron 1 regions. In contrast, cells treated with CRISPRoff and ANGPTL3 guide RNAs experienced only transient knockdown of gene expression. Cells treated with CRISPRoff and AGT guide RNAs also experienced transient knockdown of gene expression; although initially there was increased CpG methylation throughout the early part of the gene, this methylation was geographically heterogeneous-transient in the promoter, and stable in intron 1.ConclusionsThis work demonstrates precise and durable gene regulation via methylation, supporting a new therapeutic approach for protection against cardiovascular disease via knockdown of genes such as PCSK9 . However, the durability of knockdown with methylation changes is not generalizable across target genes, likely limiting the therapeutic potential of epigenome editing compared to other modalities.

Project description:Background: Hepatic knockdown of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene or the angiopoietin-like 3 (ANGPTL3) gene has been demonstrated to reduce blood low-density lipoprotein cholesterol (LDL-C) levels, and hepatic knockdown of the angiotensinogen (AGT) gene has been demonstrated to reduce blood pressure. Genome editing can productively target each of these three genes in hepatocytes in the liver, offering the possibility of durable “one-and-done” therapies for hypercholesterolemia and hypertension. However, concerns around making permanent gene sequence changes via DNA strand breaks might hinder acceptance of these therapies. Epigenome editing offers an alternative approach to gene inactivation, via silencing of gene expression by methylation of the promoter region, but the long-term durability of epigenome editing remains to be established. Objectives and Results: We assessed the ability of epigenome editing to durably reduce the expression of the human PCSK9, ANGPTL3, and AGT genes in HuH-7 hepatoma cells. Cells treated with the CRISPRoff epigenome editor and PCSK9 guide RNAs were maintained for up to 124 cell doublings and demonstrated durable knockdown of gene expression and increased CpG dinucleotide methylation in the promoter, exon 1, and intron 1 regions. In contrast, cells treated with CRISPRoff and ANGPTL3 guide RNAs experienced only transient knockdown of gene expression. Cells treated with CRISPRoff and AGT guide RNAs also experienced transient knockdown of gene expression; although initially there was increased CpG methylation throughout the early part of the gene, this methylation was geographically heterogeneous—transient in the promoter, and stable in intron 1.Conclusions: This work demonstrates precise and durable gene regulation via methylation, supporting a new therapeutic approach for protection against cardiovascular disease via knockdown of genes such as PCSK9. However, the durability of knockdown with methylation changes is not generalizable across target genes, likely limiting the therapeutic potential of epigenome editing compared to other modalities.

Dataset Information

Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples.

Publications

Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples.

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