Genome Editing CRISPR Cas9

Designing Homologous Repair Templates

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).

HDR Template

Product table

PRODUCT Prod. # SIZE PRICE
Q5® High-Fidelity DNA Polymerase M0491S 100 units 99 € In den Shop wechseln Produktinformationen
M0491L 500 units 435 € In den Shop wechseln Produktinformationen
Q5® Hot Start High-Fidelity DNA Polymerase M0493S 100 units 115 € In den Shop wechseln Produktinformationen
M0493L 500 units 521 € In den Shop wechseln Produktinformationen
NEB PCR Cloning Kit E1202S 20 rxns 299 € In den Shop wechseln Produktinformationen
NEB PCR Cloning Kit (Without Competent Cells) E1203S 20 rxns 152 € In den Shop wechseln Produktinformationen
Q5® Site-Directed Mutagenesis Kit E0554S 10 rxns 199 € In den Shop wechseln Produktinformationen
Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) E0552S 10 rxns 136 € In den Shop wechseln Produktinformationen
NEBuilder® HiFi DNA Assembly Master Mix E2621S 10 rxns 162 € In den Shop wechseln Produktinformationen
E2621L 50 rxns 648 € In den Shop wechseln Produktinformationen
E2621X 250 rxns 2.589 € In den Shop wechseln Produktinformationen
NEBuilder® HiFi DNA Assembly Cloning Kit E5520S 10 rxns 192 € In den Shop wechseln Produktinformationen

As of: 01.01.2021

Further information can be found in our Technical Resources section or at neb.com. Information on trademarks can be found here.