Scientific Advances: Pancreatic cancer: tracking the gene changes underlying metastasis

Researchers from the University of Pennsylvania have developed a new CRISPR-based method to help trace the lineage and gene expression patterns of individual cancer cells in a mouse model of metastatic pancreatic cancer. Combining CRISPR lineage tracing with single-cell RNA sequencing, they were able to detailed the changes in gene expression that accompany metastasis. Their findings reveal that genetic changes that drive metastasis include altered gene expression patterns in addition to genetic mutations. In particular, expression of S100 gene family propagated across clonally distinct metastatic subpopulations and enhanced the ability to metastasize.

For more information, see: Simeonov, P. et al. (2021) Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states, Cancer Cell.

Keywords: lineage tracing; phylogenetics; CRISPR; evolving barcodes; barcoding; metastasis;

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Scientific Advances: Lowering cholesterol, the CRISPR way

CRISPR base editors delivered via a single infusion of lipid nanoparticles to the liver efficiently reduced expression of the PCSK9 gene in monkeys. Researches observed a reduction of low-density lipoprotein cholesterol of approximately 60% that was stable after 8 months. Atherosclerotic cardiovascular disease is the leading cause of death worldwide.

Musunuru, K., Chadwick, A.C., Mizoguchi, T. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429–434 (2021).

Keywords: CRISPR, Base Editors, Cholesterol

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At a glance

May 13. Prime editing efficiencies in primary human cells and Zebrafish
For more information, see Petri, K., et al. (2021) CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol.

Keywords: CRISPR, prime editing, efficiencies

April 27. Clinical Trial: CRISPR disruption of GM-CSF in CAR-T therapy
For more information, see: Yi, Y., et al. (2021) CRISPR-edited CART with GM-CSF knockout and auto secretion of IL6 and IL1 blockers in patients with hematologic malignancy. Cell Discov. 7.

Keywords: CRISPR, CAR-T, GM-CSF, cytokine storm

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April 25 – May | Genome wide CRISPR Screens

  • Liscovitch-Brauer, N., et al. (2021) Profiling the genetic determinants of chromatin accessibility with scalable single-cell CRISPR screens. Nat. Biotechnol.
  • In this study the authors combine CRISPR technology to inactivate 105 chromatin-related genes with transposon activity to document chromatin accessibility in 30,000 single cells.
  • Zou, X., et al. (2021) A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage. Nat. Cancer
  • In human induced pluripotent stem cells, 43 genes involved in the DNA Damage Response were inactivated to reveal potential mutational signatures related to malignant transformation by whole-genome sequencing of 173 sub clones.
  • Nie, M., et al. (2021) Genome-wide CRISPR screens reveal synthetic lethal interaction between CREBBP and EP300 in diffuse large B-cell lymphoma. Cell. Death. Dis. 12, 419.
  • Keatinge, M., et al. (2021) CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury. PLOS Genetics 17: e1009515.
  • Researchers from the University of Edinburgh developed a CRISPR screening method to find inflammation-associated genes that were crucial for spinal regeneration in zebrafish.

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Scientific Advances: Potential novel gene therapy opportunities revealed by CRISPR CopyCatchers

Gene conversion events represent the exchange of genomic information from one chromosome to its homolog. Based on the effectiveness of CRISPR gene-drive technologies, the lab of Ethan Bier recently published a study in which they visualize gene conversion (GC) events between homologous chromosomes. Exchange of DNA via homology directed repair is thought to be a relatively rare event. However, after CRISPR-induced double strand break formation the authors report GC frequencies between 0-50% in various fruitfly tissues using the CopyCatcher fluorescent reporter construct at two different loci. In human 293 HEK cells CRISPR-induced breaks resulted in GC events in 4-8% of the cells as indicated by the CopyCatcher reporter. Notably, high c-MYC expression correlated with reduced CG activity. The authors propose the CRISPR-induced somatic gene conversion events may proof a valid therapeutic route to establish functional alleles in patients with two distinct mutant alleles. Indeed, natural gene correction events through GC have been reported in literature previously.

For more information, see: Li, Z., et al. (2021) CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion, Nat. Comm.

Keywords: CRISPR, gene conversion, fluorescent reporter

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Scientific advances: Retrons exceed CRISPR/Cas to screen barcoded gene variant libraries in bacteria

The lab of George Church at Harvard University has created a new gene editing tool called ‘Retron Library Recombineering’ (RLR) that generates up to millions of mutations simultaneously in bacteria. Described as a simpler, more flexible gene editing tool that can be used for highly multiplexed experiments, the system is based on another aspect of the bacterial immune system involving retrons, segments of DNA that are reverse transcribed to produce single-stranded DNA that detects infecting phages.

Max G. Schubert, M.G., et al. (2021) High-throughput functional variant screens via in vivo production of single-stranded DNA. PNAS DOI: 10.1073/pnas.2018181118

Keywords: retron; synthetic biology, bacteria

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Scientific advances / Screens / New Tools: Gene editing in difficult to manipulate dendritic cells

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.

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

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