Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System

In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.This study was funded by the Spanish ISCIII Health Research Fund and the European Regional Development Fund (FEDER) through research grants PI15/02015, PI18/00337 (F.M.) and PI18/00330 (K.B.) The CECEyU and CSyF of the Junta de Andalucía EDER/European Cohesion Fund (FSE) for Andalusia provided the following research grants: 2016000073391-TRA, 2016000073332-TRA, PI-57069, CARTPI-0001-201 and PAIDI-Bio326 (F.M.) and PI-0014-2016 (K.B). K.B. held a Nicolas Monardes regional Ministry of Health contract (0006/2018). S.S.H is funded thanks to Fundación Poco Frecuente donations. A.A.-G. and N.M.-P. are funded by Spanish Ministry of Science and Innovation (SMSI) through fellowships FPU17/04327 and FPU17/02268 respectively. I.R.H is funded by the ISCIII through a PFIS fellowship (FI19/00163). M.C.-G. is funded by (SMSI) through fellowship GJ (PEJ-2018-001760-A).Ye

Analysis of p.Tyr307Asn variant in the LRP10 gene in Parkinson’s disease in southern Spain

Lipoprotein receptor-related protein 10 (LRP10) has been proposed as a novel causative gene for autosomal dominant Parkinson’s disease (PD), and the c.919T>A (p.Tyr307Asn) variant has been identified as possibly involved in the development of familial PD and PD with dementia. We screened for the p.Tyr307Asn variant in a southern Spain population of 679 PD patients, of who 129 were familial cases, and 1217 unrelated healthy controls. A total of 3 carriers of the LRP10 p.Tyr307Asn variant were identified: 1 PD patient and 2 healthy controls. Together with the absence of a family history of PD, this finding might suggest a low penetrance variant as well as a limited role for p.Tyr307Asn in PD in our cohort. Nevertheless, a family history of Alzheimer’s disease in the LRP10 p.Tyr307Asn carriers provides evidence for a possible association with dementia.This work was supported by the Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (ISCIII-FEDER) [PI14/01823, PI16/01575, PI18/01898, PI19/01576], the Consejería de Economía, Innovación, Ciencia y Empleo de la Junta de Andalucía [CVI-02526, CTS-7685], the by the “Juan Rodés” program [B-0007-2019] and Daniel Macías-García by the “Río Ho Consejería de Salud y Bienestar Social de la Junta de Andalucía” [PI-0471-2013, PE-0210-2018, PI-0459-2018, PE-0186-2019], and the Fundación Alicia Koplowitz. Pilar Gómez-Garre was supported by the “Miguel Servet” program [MSII14/00018] (from ISCIII-FEDER) and “Nicolás Monardes” program [C-0048-2017] (from Andalusian Regional Ministry of Health). Silvia Jesús was supported by the “Juan Rodés” program [B-0007-2019] and Daniel Macías-García by the “Río Hortega” program [CM18/00142] (both from ISCIII-FEDER). Maria Teresa Periñán was supported by the Spanish Ministry of Education [FPU16/05061]