Month: April 2021

In the lab of Jonathan Weissman a non-viral electroporation-based method was developed to effectively create gene knockouts in primary monocyte-derived dendritic cell cultures. The approach relies on CRISPR/Cas9 ribonucleoprotein (RNP) complexes and a Nucleofector-4D (Lonza) for delivery. The authors reportedly achieved gene disruption efficiencies over 94% based on the targeting of more than 300 genes. In CRISPR screens, genetic mechanisms were identified by which dendritic cells mediate immune responses to foreign entities. This underlines the potential of this approach to systematically unravel cellular immune signaling pathways.

Jost, M., et al. (2021) CRISPR-based functional genomics in human dendritic cells. eLife 10:e65856. https://doi.org/10.7554/eLife.65856

Keywords: CRISPR screen, dendritic cells, electroporation, RNP, immune response

Questions? Email: crispr@amsterdamumc.nl

April 22. A novel nuclease activated by low nucleotide levels

On Earth day, a study was published detailing the discovery of a novel sequence specific nuclease (GajA) in the bacterial defense against phages. Interestingly, nuclease activity of GajA is controlled by an ATPase-like domain and activation occurs when the nucleotide pool becomes depleted following replication and transcription by invading phages.

For more information, see Cheng, R., et al. (2021) A nucleotide-sensing endonuclease from the Gabijabacterial defense system. Nucleic Acids Research https://doi.org/10.1093/nar/gkab277

Keywords: CRISPR, Nuclease, Nucleotide Pool

April 20. Pre-clinical advances: CRISPR base editors to fix mutations Sickle Cell disease

In another tweak on the traditional CRISPR tool, Beam Therapeutics has recently unveiled a new CRISPR base editing tool specifically designed to directly edit the causative sickle hemoglobin point mutation.

For more information, see: Chu, S.H., et al. (2021) Editing of the Sickle Cell disease mutation. The CRISPR J.4: https://doi.org/10.1089/crispr.2020.0144

Keywords: CRISPR, base editing, Sickle Cell disease

March 16. New Tools: Comparison of prime editing versus templated gene editing in vivo

In a study published in Genome Biology, the authors compared prime editing and CRISPR-mediated homology-directed repair to create inactivating base pair changes. They found that prime editing eliminated indels and off target effects compared to CRISPR-mediated homology-directed repair.

For more information, see: Gao, P., et al. (2021) Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression. Genome Biol. 22: https://doi.org/10.1186/s13059-021-02304-3

Keywords: CRISPR, prime editing, homology directed repair

Questions? Email: crispr@amsterdamumc.nl

Simoneschi, D. et al. CRL4^AMBRA1 is a master regulator of D-type cyclins. Nature https://doi.org/10.1038/s41586-021-03445-y

Hayman, T.J., et al. STING enhances cell death through regulation of reactive oxygen species and DNA damage. Nat. Comm. https://doi.org/10.1038/s41467-021-22572-8

Lee, D-h., et al. Genome wide CRISPR screening reveals a role for sialylation in the tumorigenesis and chemoresistance of acute myeloid leukemia cells. Cancer Letters https://doi.org/10.1016/j.canlet.2021.04.006

Jung, H.R., et al. CRISPR screens identify a novel combination treatment targeting BCL-XL and WNT signaling for KRAS/BRAF-mutated colorectal cancers. Oncogene https://doi.org/10.1038/s41388-021-01777-7 Xu, Z. et al. CHK2 inhibition provides a strategy to suppress hematological toxicity from PARP inhibitors. Mol. Cancer Res. DOI: 10.1158/1541-7786.MCR-20-0791

In an important advancement for precision oncology, researchers at the Center for Cancer Research in the US have shown how their tumor analysis pipeline, called SELECT, was 80% successful in predicting cancer patient responses to targeted therapy in over 30 clinical trials.

SELECT stands for SynthEtic LEthality and rescue-mediated precision onCology via the Transcriptome. Using machine learning, this tool analyzes both DNA and RNA from tumors to identify synthetic lethal interactions The inclusion of RNA analysis can identify vulnerabilities not readily evident by tumor profiling using standard DNA panels. The authors started by looking at a pools of synthetic lethal drug targets used for precision medicine based on published genome wide RNA interference and CRISPR screens in cancer cell lines or identified through the application of small molecule inhibitors. The authors plan to include DNA methylation information in the tool and further improve predictions in the next few years.

For more information, see: Lee, J.S., et al. (2021) Synthetic lethality-mediated precision oncology via the tumor transcriptome. Cell https://doi.org/10.1016/j.cell.2021.03.030

Keywords: CRISPR screen, synthetic lethality, machine learning

Questions? Email: crispr@amsterdamumc.nl

Chromothripsis is a mutational process resulting in extensive chromosome rearrangement of one or a few chromosomes that can cause human congenital disease and cancer. A new study published in Nature Genetics shows that CRISPR/Cas9 editing can generate structural defects of the nucleus, micronuclei and chromosome bridges, which initiate chromothripsis. In actively dividing cells, using CRISPR to initiate double-strand breaks increased the formation of chromosomal aberrations up to 20 times. This study flags a new concern with on-target CRISPR editing that will need to be monitored and further studied, despite a lack of evidence of malignant transformation from CRISPR studies thus far.

For more information, see: Leibowitz, M.L., et al. (2021) Chromothripsis as an on-target consequence of CRISPR/Cas9 genome editing. Nat. Genet. https://doi.org/10.1038/s41588-021-00838-7

Keywords: CRISPR, DNA damage, chromothripsis

Questions? Email: crispr@amsterdamumc.nl

In the lab of Jonathan Weissman, a CRISPR-mediated gene silencing tool based on methylation of genomic DNA was developed and published in the journal Cell. The authors show that gene silencing through DNA methylation can be achieved by transient expression of a modified nuclease dead Cas9. This gene silencing was maintained after many cell divisions or differentiation into neurons.

For more information, see: Nuñez, J.K., et al. (2021) Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell DOI:https://doi.org/10.1016/j.cell.2021.03.025

Keywords: CRISPR, epigenetics, DNA methylation, cell therapy, dCas9

Questions? Email: crispr@amsterdamumc.nl

From the lab of Stephen West, a genome-wide CRISPR screen to dissect cellular sensitivity and resistance to PARP-inhibitors (PARPi) was recently published in Science. Using the Brunello genome-wide sgRNA library and cells deficient in the endonuclease MUS81, the authors identified that sensitivity to PARPi can be increased by preventing nucleotide pool polishing through DNPH1, which eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate. In addition, it was reported that synthetic lethality mediated by PARPi and loss of BRCA is fully dependent on the function of the glycosylase SMUG1.

For more information, see: Fugger, K., et al. (2021) Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA-deficient cells to PARP inhibitors. Science 372: 156-165. DOI: 10.1126/science.abb4542

Keywords: CRISPR screen, PARP inhibitor, BRCA

Questions? Email: crispr@amsterdamumc.nl

A large consortium of CRISPR scientists initiated the Somatic Cell Genome Editing (SCGE) Program in order to speed up the development and implementation of gene editing therapies. The initiative to develop safer and more effective gene clinical gene editing methods is supported by the United States National Institutes of Health (NIH). The consortium will consider various aspects of clinical gene editing for optimization, such as the assessment of different enzymes, delivery systems, target cells, efficiencies and safety, and the use of preclinical animal models. Importantly, the research outcomes will be achieved by relying on standardized methods of data collection and coordinated by a central communication hub, greatly facilitating data comparison and integration among involved labs. The aim of the SCGE consortium is to deliver a validated toolkit that will facilitate gene editing in the clinic.

For more information, see: Saha, K., et al. (2021) The NIH somatic cell genome editing program. Nature 592: 195–204. https://doi.org/10.1038/s41586-021-03191-1

Keywords: Gene editing, Therapy, NIH, Consortium, Toolkit

Questions? Email: crispr@amsterdamumc.nl

April 9. Loss of heterozygosity after CRISPR gene editing in early-stage human embryos. Alanis-Lobato, G., et al. (2021) https://doi.org/10.1073/pnas.2004832117

March 24. A preprint from the lab of Jennifer Doudna: Accelerated SARS-Cov-2 point of care detection using CRISPR nucleases. https://doi.org/10.1101/2021.03.19.21253328

Keywords: CRISPR, LOH, human embryos, Diagnostics, SARS-Cov-2

Questions? Email: crispr@amsterdamumc.nl