Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells

Yudovich, David; Bäckström, Alexandra; Schmiderer, Ludwig; Žemaitis, Kristijonas; Subramaniam, Agatheeswaran; Larsson, Jonas

doi:10.1038/s41598-020-79724-x

Cited by 19 publications

(21 citation statements)

References 32 publications

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“…We initially planned to carry out the CRISPR-Cas9 editing of miRNAs by transducing CD34+ HSPCs with lentiviral vectors to express Cas9 and the gRNAs, followed by ex vivo erythroid differentiation. However, as reported earlier [76], we found that the transduction efficiency of CD34+ HSPCs with different Cas9 lentiviral vectors was very low. Therefore, we decided to use HUDEP-2 erythroid progenitor cells [51], which have been extensively used for experiments to study erythropoiesis using several molecular tools, including CRISPR-Cas9-based gene editing [77][78][79].…”

Section: Crispr-cas9-mediated Gene Editing Of Mirnassupporting

confidence: 82%

Comprehensive Analysis of microRNAs in Human Adult Erythropoiesis

Nath

Rayabaram

Ijee

et al. 2021

Cells

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MicroRNAs (miRNAs) are small non-coding RNAs, which play an important role in various cellular and developmental processes. The study of miRNAs in erythropoiesis is crucial to uncover the cellular pathways that are modulated during the different stages of erythroid differentiation. Using erythroid cells derived from human CD34+ hematopoietic stem and progenitor cells (HSPCs)and small RNA sequencing, our study unravels the various miRNAs involved in critical cellular pathways in erythroid maturation. We analyzed the occupancy of erythroid transcription factors and chromatin accessibility in the promoter and enhancer regions of the differentially expressed miRNAs to integrate miRNAs in the transcriptional circuitry of erythropoiesis. Analysis of the targets of the differentially expressed miRNAs revealed novel pathways in erythroid differentiation. Finally, we described the application of Clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) based editing of miRNAs to study their function in human erythropoiesis.

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Section: Crispr-cas9-mediated Gene Editing Of Mirnassupporting

confidence: 82%

Comprehensive Analysis of microRNAs in Human Adult Erythropoiesis

Nath

Rayabaram

Ijee

et al. 2021

Cells

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“…Here, we set out to explore different strategies to simultaneously knockout two genes using our recently published split CRISPR/Cas9 delivery system in cord blood (CB)-derived CD34 + HSPCs 14 . In our previous work, using a chimeric sgRNA backbone, we identified highly efficient sgRNAs targeting the two cell surface markers CD45 and CD44.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously reported on a split CRISPR/Cas9 delivery system, combining stable lentiviral sgRNA delivery and transient delivery of Cas9 mRNA, for efficient and traceable gene editing in primary CD34 + HSPCs 14 . The use of lentiviral sgRNA vectors containing fluorescent markers allows extension of this system to traceable multiplexed perturbations 15 .…”

Section: Introductionmentioning

confidence: 99%

Combinatorial gene targeting in primary human hematopoietic stem and progenitor cells

Bäckström

Yudovich

Žemaitis

et al. 2022

Sci Rep

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The CRISPR/Cas9 system offers enormous versatility for functional genomics but many applications have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, to establish a paradigm for multiplexed gene editing in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs), we used co-delivery of lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The fluorescent markers allow for tracking of either single- or double-edited cells, and we could achieve robust double knockout of the cell surface molecules CD45 and CD44 with an efficiency of ~ 70%. As a functional proof of concept, we demonstrate that this system can be used to model gene dependencies for cell survival, by simultaneously targeting the cohesin genes STAG1 and STAG2. Moreover, we show combinatorial effects with potential synergy for HSPC expansion by targeting the Aryl Hydrocarbon Receptor (AHR) in conjunction with members of the CoREST complex. Taken together, our traceable multiplexed CRISPR/Cas9 system enables studies of genetic dependencies and cooperation in primary HSPCs, and has important implications for modelling polygenic diseases, as well as investigation of the underlying mechanisms of gene interactions.

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“…Scientists can easily search for a particular gene on specific websites and select from a range of available vectors. In particular, the use of the lentivirus as a means of delivering DNA to transfection-resistant cells is an effective method for producing stable Cas9 and sgRNA cell lines [ 18 ].…”

Section: Reagents In Crispr/casmentioning

confidence: 99%

Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants

Alghuthaymi

Ahmad

Khan

et al. 2021

IJMS

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Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

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Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells

Cited by 19 publications

References 32 publications

Comprehensive Analysis of microRNAs in Human Adult Erythropoiesis

Comprehensive Analysis of microRNAs in Human Adult Erythropoiesis

Combinatorial gene targeting in primary human hematopoietic stem and progenitor cells

Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants

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