Improving Homology-Directed Repair in Genome Editing Experiments by Influencing the Cell Cycle

Genome editing is currently widely used in biomedical research; however, the use of this method in the clinic is still limited because of its low efficiency and possible side effects. Moreover, the correction of mutations that cause diseases in humans seems to be extremely important and promising. Numerous attempts to improve the efficiency of homology-directed repair-mediated correction of mutations in mammalian cells have focused on influencing the cell cycle. Homology-directed repair is known to occur only in the late S and G2 phases of the cell cycle, so researchers are looking for safe ways to enrich the cell culture with cells in these phases of the cell cycle. This review surveys the main approaches to influencing the cell cycle in genome editing experiments (predominantly using Cas9), for example, the use of cell cycle synchronizers, mitogens, substances that affect cyclin-dependent kinases, hypothermia, inhibition of p53, etc. Despite the fact that all these approaches have a reversible effect on the cell cycle, it is necessary to use them with caution, since cells during the arrest of the cell cycle can accumulate mutations, which can potentially lead to their malignant transformation

Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7

This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency

Exome, transcriptome and miRNA analysis don’t reveal any molecular markers of TKI efficacy in primary CML patients

Abstract Background Approximately 5–20% of chronic myeloid leukemia (CML) patients demonstrate primary resistance or intolerance to imatinib. None of the existing predictive scores gives a good prognosis of TKI efficacy. Gene polymorphisms, expression and microRNAs are known to be involved in the pathogenesis of TKI resistance in CML. The aim of our study is to find new molecular markers of TKI therapy efficacy in CML patients. Methods Newly diagnosed patients with Ph+ CML in chronic phase were included in this study. Optimal and non-optimal responses to TKI were estimated according to ELN 2013 recommendation. We performed genotyping of selected polymorphisms in 62 blood samples of CML patients, expression profiling of 33 RNA samples extracted from blood and miRNA profiling of 800 miRNA in 12 blood samples of CML patients. Results The frequencies of genotypes at the studied loci did not differ between groups of patients with an optimal and non-optimal response to TKI therapy. Analysis of the expression of 34,681 genes revealed 26 differently expressed genes (p < 0.05) in groups of patients with different TKI responses, but differences were very small and were not confirmed by qPCR. Finally, we did not find difference in miRNA expression between the groups. Conclusions Using modern high-throughput methods such as whole-exome sequencing, transcriptome and miRNA analysis, we could not find reliable molecular markers for early prediction of TKI efficiency in Ph+ CML patients

Copy number variation analysis in cytochromes and glutathione S-transferases may predict efficacy of tyrosine kinase inhibitors in chronic myeloid leukemia

<div><p>Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of <i>BCR/ABL</i> fusion gene in leukemic cells, which promotes uncontrolled cell proliferation. Up to 20% of CML patients show primary resistance or non-optimal response to tyrosine kinase inhibitor (TKI) therapy. We investigated the association between copy number variation (CNV) in glutathione S-transferases (GST) and cytochromes (CYP) and the response rate to TKI. We enrolled 47 patients with CML: 31 with an optimal response and 16 with failure at 6 months in accordance with European LeukemiaNet 2013 recommendations. CNV detection was performed using SALSA MLPA P128-C1 Cytochrome P450 probe mix. Patients with optimal response and with failure of TKI therapy showed different frequencies of wild type and mutated CYPs and GST (p<0.0013). Validation in the group of 15 patients proved high prognostic value (p = 0.02): positive and negative predictive value 83% and 78%; sensitivity and specificity 71% and 88%. Wild type genotypes of CYP and GST associate with a worse response to TKI treatment in CML patients. This test can be recommended for further clinical trials.</p></div

Diagnostic value of CNV in <i>CYP1A2</i>, <i>CYP2A6</i>, <i>CYP2C19</i>, <i>CYP2C9</i>, <i>CYP2D6</i>, <i>CYP2E1</i>, <i>CYP3A4</i>, <i>CYP3A5</i>, <i>GSTM1</i>, <i>GSTP1 and GSTT1</i> for prediction of optimal response and failure of TKI therapy in CML patients (<i>P</i> = 0.0001).

<p>Diagnostic value of CNV in <i>CYP1A2</i>, <i>CYP2A6</i>, <i>CYP2C19</i>, <i>CYP2C9</i>, <i>CYP2D6</i>, <i>CYP2E1</i>, <i>CYP3A4</i>, <i>CYP3A5</i>, <i>GSTM1</i>, <i>GSTP1 and GSTT1</i> for prediction of optimal response and failure of TKI therapy in CML patients (<i>P</i> = 0.0001).</p

Difference in CNV status of CYP and GST genes (<i>P</i>< 0.0013) in optimal response, TKI failure and control groups of CML patients.

<p>Difference in CNV status of CYP and GST genes (<i>P</i>< 0.0013) in optimal response, TKI failure and control groups of CML patients.</p

Outcomes of TKIs treatment in validation group of CML patients.

<p>CML = chronic myeloid leukemia; CNV = copy number variation; TKI = tyrosine kinase inhibitor; CyR = cytogenetic response; CCyR = complete cytogenetic response.</p

Analyzed genes.

<p>Analyzed genes.</p

P.F508del editing in cells from cystic fibrosis patients.

Development of genome editing methods created new opportunities for the development of etiology-based therapies of hereditary diseases. Here, we demonstrate that CRISPR/Cas9 can correct p.F508del mutation in the CFTR gene in the CFTE29o- cells and induced pluripotent stem cells (iPSCs) derived from patients with cystic fibrosis (CF). We used several combinations of Cas9, sgRNA and ssODN and measured editing efficiency in the endogenous CFTR gene and in the co-transfected plasmid containing the CFTR locus with the p.F508del mutation. The non-homologous end joining (NHEJ) frequency in the CFTR gene in the CFTE29o- cells varied from 1.25% to 2.54% of alleles. The best homology-directed repair (HDR) frequency in the endogenous CFTR locus was 1.42% of alleles. In iPSCs, the NHEJ frequency in the CFTR gene varied from 5.5% to 12.13% of alleles. The best HDR efficacy was 2.38% of alleles. Our results show that p.F508del mutation editing using CRISPR/Cas9 in CF patient-derived iPSCs is a relatively rare event and subsequent cell selection and cultivation should be carried out