Evaluation of the on-/off-target DNA cleavage induced by Cas9 variants

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dc.contributor.authorNiklas, Selfjord
dc.contributor.departmentChalmers tekniska högskola / Institutionen för biologi och biotekniksv
dc.contributor.examinerSiewers, Verena
dc.contributor.supervisorAkçakaya, Pinar
dc.date.accessioned2019-11-21T10:11:09Z
dc.date.available2019-11-21T10:11:09Z
dc.date.issued2019sv
dc.date.submitted2019
dc.description.abstractThe CRISPR-Cas9 system of gene editing has future potential for therapeutic applications in humans. Exploration of novel Cas9 variants is currently expanding and improving the capabilities of genome engineering. A major obstacle to the clinical translation of Cas9 gene therapy is unintended mutations at off-target sites. The specificity of guide RNAs and Cas9 variants needs to be carefully evaluated prior to their therapeutic use. This thesis work aimed to explore on/off-target activity of Cas9 variants. In the first part, we have evaluated on/off-target profiles of two previously described guide RNAs known to be specific and promiscuous in single cells by sequencing the off-target sites in Cas9 treated mouse embryos. We have found that highly efficient editing with a specific guide can be achieved in embryos with no detectable off-targets. In addition, we have shown that highly efficient editing by a promiscuous guide can induce off-targets in embryos, and off-target profiles of individual embryos can be decidedly distinct. In the second part, we have evaluated the on/off-target activity of a novel Cas9 variant, AzCas9. We have demonstrated that AzCas9 can efficiently cleave the intended target site in mouse cells, and are currently evaluating its specificity in the mouse genome with a promiscuous guide using ‘circularization for in vitro reporting of cleavage effects by sequencing’ (CIRCLE-seq).sv
dc.identifier.coursecodeBBTX60sv
dc.identifier.urihttps://hdl.handle.net/20.500.12380/300554
dc.language.isoengsv
dc.setspec.uppsokLifeEarthScience
dc.subjectCRISPRsv
dc.subjectCas9sv
dc.subjectgene editingsv
dc.subjectcell transfectionsv
dc.subjectoff-target detectionsv
dc.subjectCIRCLE-seqsv
dc.subjectnext-generation sequencingsv
dc.titleEvaluation of the on-/off-target DNA cleavage induced by Cas9 variantssv
dc.type.degreeExamensarbete för masterexamensv
dc.type.uppsokH
local.programmeBiotechnology (MPBIO), MSc
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Evaluation of the on-/off-target DNA cleavage induced by Cas9 variants
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