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Karolinska Genome Engineering

At the Karolinska Genome Engineering Facility (KGE) we are specialists in CRISPR/Cas technology, and it is our mission to facilitate and accelerate your research by providing flexible and affordable gene-editing services in cells. Your cell type, your edit – from strategy design to delivery of a cell pool with quantified allele frequency.

Workflow KGE

KGE is a branch of the High Throughput Genome Engineering (HTGE), a national SciLifeLab facility.

Application examples

  • Reporter cell lines (fluorescent proteins, luciferase, tags, etc.)
  • Disease relevant cellular model systems such as knock-outs, knock-ins, creation/reversion of pathogenic mutations
  • Cellular model systems to facilitate genetic screening and drug screening
  • Validation of hits from genetic screens or drug screens
  • Isogenic cell lines; isogenic disease models
  • Anything you can think of. Feel free to contact us with any additional applications you may be interested in.

Services

Strategy design

In discussion with the client, we determine the optimal guide sequences and recombination template (dsDNA, ssDNA, ssODN). We then custom design the optimal genome engineering strategy for each project, with an emphasis on easy downstream screening for correctly edited cell clones. Examples of strategies are:

  • CRISPR-cut/non-homologous end joining (NHEJ) to create knock-outs
  • CRISPR-cut/homology dependent repair (HDR) for knock-ins, small mutations, conditional alleles, etc
  • CRISPR-inhibition (CRISPR-i) for stable or inducible knock-downs
  • CRISPR-activation (CRISPR-a) for stable or inducible upregulation

Provision of reagents

KGE aims to provide the best reagents at the lowest possible cost. Our own R&D aims to replace commercial products with in-house reagents to increase editing efficiency while reducing the risk of off-target mutations. In particular, we are currently working on setting up in-house synthesis of long, single-stranded DNA templates based on rolling-circle amplification (Ducani C. and Högberg B., 2017).

We provide the following reagents:

Cas enzymes, both as recombinant proteins and lentiviral expression vectors

  • WT SpCas9
  • SpCas9-D10A nickase
  • SpCas9-H840A nickase
  • HiFi SpCas9 R691A
  • Cpf1/Cas12a

Guide RNAs, either as RNA (sgRNA or split crRNA/tracrRNA); or cloned into lentiviral vectors

HDR templates as ssODN, dsDNA or long ssDNA

Optimization of delivery method and delivery into target cells

With input from the client, we determine the best way of delivery of the editing components into cells. We provide liposomal transfection reagents, lentiviral delivery systems (full support for all lentiviral work in collaboration with the VirusTech core facility), or nucleofection.

Quantifications by droplet digital PCR

We use BioRad’s QX200 droplet digital PCR system to determine gene copy number prior to editing, and to quantify the frequency of the edited allele in a population of cells after editing.

Workflow of a typical project

 

A typical project consists of three phases. First, the client contacts KGE for the initial project discussion. The project prioritization is established and the client provides mycoplasma-tested parental cells.

In the second phase, KGE will design and synthesize guide RNAs and donor templates. Cell culture will be established, baseline copy number assessment by ddPCR will be performed and delivery method of the plasmids optimized. During editing & validation the editing components will be delivered into the target cells, followed by quantification of edited allele frequency (ddPCR). Edited cells will be returned to the client. The pool will be tested for mycoplasma and delivery of edited cells will be done as a pool or as single cells sorted in 96 well plates.

In the final post-edit phase, the client will expand the single cell clones and screen for the correct edit.

Creation of genetically modified mouse strains – collaboration with the Karolinska Center for Transgene Technologies

KGE has established a collaboration with the Karolinska Center for Transgene Technologies (KCTT). KGE’s CRISPR expertise combined with KCTT’s mouse expertise provides a seamless service pipeline from CRISPR strategy design to transgenic animals. KGE provides strategy and reagents, and first tests both in mouse cell lines. If successful, KCTT takes over and injects mouse zygotes with the designed CRISPR/Cas components and repair templates for one-step creation of genetically modified mice.

Accessibility and project prioritization

KGE is open to all researchers from academia, healthcare or industry. While internal projects within Karolinska Institutet and Karolinska University Hospital are prioritized, KGE is happy to accept external projects pending available resources. Project requests can be submitted to KGE at any time by email. KGE’s project prioritization committee ranks projects based on factors such as:

  • Internal versus external project
  • Currently available resources
  • Expected difficulty and chance of success
  • Downstream application of the resulting cellular models

Cost model and pricing

KGE is a not-for-profit research facility. Charges are based on Karolinska Institutets’s full cost model for commissioned research with subsidies applied. Industry and private clients will be charged at full cost. For further details, please contact KGE.

Organisation

Together with our SciLifeLab branch (High Throughput Genome Engineering, HTGE), KGE is part of the Department of Medical Biochemistry and Biophysics (MBB). HTGE is a national facility financed by the SciLifeLab, performing genome-wide or targeted loss- and gain-of-function CRISPR screens in cells. HTGE supports researchers from all over Sweden and beyond. KGE is funded by Karolinska Institutet and is a local core facility prioritising internal users.

People

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Bernhard Schmierer PhD, Scientific Director and Head of KGE and HTGE

Bernhard started working with the CRISPR technology in 2013, shortly after publication of the seminal papers describing CRISPR/Cas9 application in eukaryotic cells. Since then, his work has focused on both CRISPR precision genome engineering and the applications of CRISPR in genetic screening. In 2017, he became Scientific Director and Head of the national SciLifeLab facility High Throughput Genome Engineering (HTGE), which provides genetic screens using CRISPR to the Swedish research community. An unmet need for a precision genome engineering service prompted him to found KGE as a financially separate, yet scientifically closely linked facility.

 

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Georgia Tsikala PhD, Staff Scientist at KGE

Georgia joined the KGE facility in February 2019 after completing a PhD and postdoctoral research in respiratory system development. She has a strong background in molecular genetics, as well as in cell and developmental biology. She enjoys strategy design, optimisation and development of new technologies.

 

 

 

 

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Allegra Lord PhD, Staff Scientist at HTGE

Allegra joined the HTGE facility in August 2018, after completing a PhD and a postdoctoral fellowship focusing on the genetic basis for hematopoietic disease. Allegra brings expertise in cell culture, murine models, and flow cytometry, as well as a passion for technology development.

 

 

 

 

 

 

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Jenna Persson PhD, Staff Scientist at HTGE

Jenna has been working with CRISPR-based genetic screening since 2016, when she introduced CRISPR screening as a SciLifeLab facility service. Prior to joining HTGE Jenna worked with chromatin biology and DNA topology.

 

 

 

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Soniya Dhanjal PhD, Staff Scientist at HTGE

Soniya joined HTGE in March 2019. Previously she has been working as a Lab Manager and Post Doctoral Fellow in the field of DNA repair and cancer, thus bringing in organizational skills and experience as a molecular biologist.

 

 

 

Contact details and visiting address

Please contact us with your request at kge@mbb.ki.se.

Karolinska Genome Engineering (KGE)

Department of Medical Biochemistry and Biophysics

Biomedicum 9B, B0951

Solnavägen 9

171 65 Solna