Summary in reference to: In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9 Lukasz Swiech, PhD Yinqing Li Matthias Heidenreich, PhD John Trombetta Abhishek Banerjee Mriganka Sur, PhD Naomi Habib, PhD Feng Zhang, PhD Nature Biotechnology, 19 October 2014, Epub ahead of print. http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3055.html Since being applied as a genome editing tool in 20121, CRISPR (Clustered regularly interspaced short palindromic repeats) systems have been widely applied in multiple eukaryotic systems, including C.elegans, yeast, drosophila, zebrafish and mice. In March of 20142, CRISPR-Cas was used to successfully correct a Fah point mutation in hepatocytes in the hereditary tyrosinemia (HT1) mouse model via hydrodynamic injection, which is less likely to be used for clinical trials. The question of how to efficiently and safely deliver CRISPR systems remains an obstacle for therapeutic application. Swiech et. al. recently used adeno-associated viral (AAV) vectors to deliver CRISPR-Cas9 into mice brains to successfully target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) into the adult mice brains in vivo3. Due to packaging limits, SpCas9 (AAV-SpCas9) and sgRNA expression cassettes (AAV-SpGuide) were packaged in two separate viral vectors. The efficacy of dual vector systems have been tested, and a co-transduction efficiency of ~75% was achieved in primary mouse cortical neurons in vitro, and 80% co-transduction efficiency in hippocampal granule cells of the adult mouse brain was detected 4 weeks after stereotactic AAV injection. The authors observed an important ~70% deduction of MeCP2-positive cells in the dentate gyrus (DG) of adult mice injected with AAV-SpCas9 and Mecp2-targeting sgRNA. Western blotting showed that total MeCP2 protein levels in the DG were decreased by 60%. Also, contextual fear-conditioning paradigm (CFC) behavioral tests revealed that mice injected with dual AAV CRISPR-Cas9 targeting MeCP2 in the DG have impaired contextual learning similar to MeCP2 mutant mice. Swiech et. al. also tested the efficacy of multiplex genome editing in mice brains. AAV-SpCas9 and AAV-SpGuides targeting Dnmt3a, Dnmt1 and Dnmt3b were stereotactically injected into the DG of adult mice. Eight weeks later, modification rates of ~75% for Dnmt1, ~75% for Dnmt3a, and ~50% for Dnmt3b were detected in AAV transduced cells via next generation sequencing(NGS) based indel analysis. Importantly, off- target rate is only detected in about 0–1.6% of the top predicted off-target loci by indel analysis. Reference: 1Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., Charpentier, E. (2012). ‘A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity’. Science 337 (6096): 816–821. 2Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014 Jun 5,157(6):1262-78. 3Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, Grompe M, Koteliansky V, Sharp PA, Jacks T, Anderson DG. Nat Biotechnol. 2014 Jun,32(6):551-3. 4Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur M, Zhang F. Nat Biotechnol. 2014 Oct 19. doi: 10.1038/nbt.3055. [Epub ahead of print] American Society of Gene & Cell Therapy 555 East Wells Street, Suite 1100 | Milwaukee, Wisconsin 53202-3823 USA Phone: 414.278.1341 | Phone: 414.276.3349 | Email: email@example.com You have received this message because you have had previous contact with the American Society of Gene & Cell Therapy. If you do not wish to be included in our mailing list, please forward this message to firstname.lastname@example.org with the word ‘Remove’ in the subject line. Source.