Merck

Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery.

Bio-protocol (2017-04-05)
Giel Vandemoortele, Delphine De Sutter, Sven Eyckerman
RESUMEN

The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol, we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a C-terminal tag sequence in a human cell line.

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Millipore
Benzonase® Nuclease, ≥250 units/μL, ≥90% (SDS-PAGE), recombinant, expressed in E. coli, buffered aqueous glycerol solution
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PerfectHyb Plus Hybridization Buffer, for Northern and Southern blotting, solution
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Sodium citrate tribasic dihydrate, for molecular biology, ≥99%
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Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
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Magnesium chloride, anhydrous, ≥98%
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SuRE/Cut Buffer H, solution, pkg of 5 × 1 mL
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Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
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Bovine Serum Albumin, cold ethanol fraction, pH 5.2, ≥96%
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Ethylenediaminetetraacetic acid disodium salt dihydrate, suitable for electrophoresis, for molecular biology, 99.0-101.0% (titration)
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HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
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Sodium dodecyl sulfate, ACS reagent, ≥99.0%
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Puromycin dihydrochloride from Streptomyces alboniger, powder, BioReagent, suitable for cell culture
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Sodium hydroxide, ACS reagent, ≥97.0%, pellets