Designing Homologous Repair Templates

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Modifications of Cas9-target sites can be achieved by supplying a homologous repair template in addition to sgRNA and Cas9. Homologous repair templates are plasmids containing 2-3 kb of homology surrounding the target sequence that are then altered to have the desired mutations or “knock-ins” such as selectable markers or fluorescent tags. For initial amplification of the region of homology, The homologous region can be isolated by PCR amplification from genomic DNA with a high-fidelity polymerase and then cloned into your plasmid backbone. NEB suggests the use Q5 High Fidelity DNA Polymerase products for this amplification followed by the use of the PCR Cloning Kit (NEB #E1202) to clone this product into a plasmid. Further modifications can then be introduced using NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621) or by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (NEB #E0554).

Homology Directed Repair Template (HDR) Template Preparation

GE_HDR_0215

Homology Directed Repair Templates can be prepared by PCR amplification of a region of genomic DNA centered around the location of the desired modification. PCR products are then inserted and joined into a vector backbone. Knock-ins, selectable markers, tags, and other changes can then be introduced through techniques such as Gibson Assembly® or site-directed mutagenesis.