1. INTRODUCTION
Figure 2: Kcnv1 Knock Down In Cortical
Neuron SliceCulture
Figure 1: Screening Of Different CRISPR
sgRNA On KCNV1 cDNAOn HEKCells
Figure 3: Comparison Of CRISPR Efficiency
In Different Systems
FUTURE DIRECTIONS
REFERENCES
1. Layer V Neurons in Mouse Cortex Projecting to Different Targets Have Distinct Physiological Properties. Alexis M. Hattox, Sacha B.
Nelson. Journal of Neurophysiology Published 1 December 2007 Vol. 98 no. 6, 3330-3340 DOI: 10.1152/jn.00397.2007
2. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Heckl D,
Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, Ebert BL. Nat Biotechnol. 2014 Jun 22.
doi: 10.1038/nbt.2951. 10.1038/nbt.2951
3. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Maddalo D, Manchado E, Concepcion CP,
Bonetti C, Vidigal JA, Han YC, Ogrodowski P, Crippa A, Rekhtman N, de Stanchina E, Lowe SW, Ventura A. Nature. 2014 Dec
18;516(7531):423-7. doi: 10.1038/nature13902. Epub 2014 Oct 22. 10.1038/nature13902
1Department of Biology, Brandeis University
Maitreya Das1, Vera Valakh1 and Sacha Nelson1
Construction and Optimization of CRISPR mediated KCNV1 Knock Out Models to
Study its Phenotypiceffects on Post-mitotic Pyramidal Neurons
ACKNOWLEDGEMENTS
• Lucia Capano for Slice Culture and Molecular Cloning
• Erin Clark for FACS
sgRNA 8 showed the highest efficiency for causing a
mutation
sgRNA 8 showed relative highest efficiency, but overall
mutation efficiency was very low
• CRISPR efficiency was reduced in post
mitotic neurons
• Level of Cas9 expression may have
affected efficiency
• Sorted cells from micro-dissected Layer V
and VI showed greater mutation efficiency
may reflect inaccessibility of gene in cells
not expressing it, or dilution from cells not
expressing Cas9
• Use of multiple guides to achieve more efficient knock out
• Changing level of Cas9 expression for genomic KCNV1 (in slice culture)
• Comparing CRISPR efficiency on KCNV1 with CRISPR efficiency on a generally
expressed gene
• Two sub-types of pyramidal neurons in layer V of the
neocortex differ in their morphology, projections and
firing properties
• Adapting neurons decrease their firing rate during
constant current injection; non-adapting neurons do not.
This property is believed to result from potassium currents
that build up during repetitive firing.
• KCNV1 is an auxiliary potassium channel subunit
expressed in adapting, but not non-adapting sub-types
Non$Adapting Adapting
Hattox and Nelson 2007
• To determine whether or not KCNV1 contributes to
adaptation we aim to use CRISPR-mediated knockout in
post mitotic neurons in slice cultures from mouse cortex.