Efficient Genome Editing of Mouse Hematopoietic Cells Using Crispr/Cas9 Technology

Sarrou, S., Gibson, B. and Keeshan, K. (2016) Efficient Genome Editing of Mouse Hematopoietic Cells Using Crispr/Cas9 Technology. 21st Congress Of The European Hematology Association, Copenhagen, Denmark, 9-12 June 2013. p. 295.

Abstract

Background: A number of techniques have been used to study hematopoietic malignancies including viral overexpression of human oncogenes, RNA interference (RNAi) and transgenic animals. Each of these have their caveats for cell specific targeting, particularly RNAi approaches that are characterised by incomplete genetic inactivation without changing the genetic code. Genome editing technologies, including clustered regularly-interspaced short palindromic repeats (CRISPR), have been used to stably modulate the genome of several model organisms. The genome engineering of hematopoietic cells is complicated mainly due to the low efficiency of the non-viral delivery methods. Aims: To generate efficient and stable genome edited murine hematopoietic models using CRISPR/Cas9 technology. Methods: Two different guideRNAs (gRNAs) targeting either Mcm6 (gRNA5 and 8) or Sirt1 (gRNA3 and 9) were designed and cloned into the pLCRISPR.EFS.GFP lentiviral vector. These genes were chosen as they are involved in hematopoietic progenitor and stem cell (HSPC) properties. GFP+ 32D Cl3 mouse hematopoietic cells were FACS sorted at day 5 post-transduction. Knockout efficiency was confirmed by both western blotting and qRT-PCR. Functional analysis was performed by the SURVEYOR assay and Sanger sequencing. Murine HSPCs were isolated from 5FU treated C57BL/6 mice (CD45.2+), transduced with the most efficient gRNA for either Sirt1 or Mcm6 and injected into B6.SJL mice. Engraftment was measured by FACS analysis of blood sampling at 4 weeks post-transplant. Results: CRISPR/Cas9 efficiency was first tested on the mouse 32D cell line in vitro. Decreased protein and mRNA levels of Sirt1 were detected in 32D GFP+ bulk population transduced with gRNA9 (32D+gRNA9) at day 5 which was not evident with gRNA3. SURVEYOR assay revealed that Cas9-mediated cleavage efficiency was higher in 32D+gRNA9 cells (16.3% indels) compared to 32D+gRNA3 cells (12.5% indels). 32D+gRNA9 cells were further expanded and FACS sorted for single clones. 5/20 clones showed complete absence of Sirt1 at the protein level (25% of Cas9 efficiency). Efficient clones were further used for genotype analysis by Sanger sequencing to confirm the type of indels. On the other hand, we saw complete absence of Mcm6 protein expression in 32D GFP+ bulk cells transduced with gRNA5 or gRNA8, and >2-4-fold mRNA reduction was observed when transduced with gRNA5 and 8, respectively. Our CRISPR/Cas9 system was further tested in vivo using primary cells. Murine CD45.2+ HSPCs were isolated, transduced with equal viral titre of empty vector, Mcm6-gRNA8 or Sirt1-gRNA9 and transplanted into CD45.1+ mice. Blood sampling revealed high levels of engraftment as determined by CD45.2% and CRISPR transduction efficiency determined by the GFP levels at 4 weeks posttransplant. Our results show that CD45.2+GFP+ cells engrafted at 4 weeks showing effective CRISPR/Cas9 mediated transduction and transplantation of Mcm6 and Sirt1 knockout primary HSPCs. Summary/Conclusions: In this study we successfully generated in vitro and in vivo CRISPR/Cas9 models for efficient genome editing on hematopoietic cells. Our findings demonstrate a complete gene knockout in vitro but with variable Cas9-mediated cleavage which was further confirmed by our clonal analysis. This suggests that Cas9 function might depend on both the targeted gene and/or the gRNA. Also, transduction of HSPCs was sufficient to ensure engraftment of GFP+ cells. Long-term serial analysis will be further conducted to ensure stable expression and test Cas9-mediated cleavage in vivo

• Item Type: Conference or Workshop Item
• Additional Information: Published in Haematoligica 101(S1):295
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Keeshan, Dr Karen
• Authors: Sarrou, S., Gibson, B., and Keeshan, K.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• ISSN: 0390-6078

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