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CRISPR-Cas9 knock-in of CDK12 G731E or G731R alleles

TH Ting Han

Last updated date: Sep 13, 2023 Views: 54 Forks: 0

original An abbreviated version of this protocol was published in eLife in Aug, 2020
Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation
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  1. Clone the sgRNA sequence targeting CDK12 (5’-GTGGACAAGTTTGACATTAT-3’) into the pSpCas9(BB)−2A-eGFP (PX458) vector (Addgene 48138).
  2. Order single-stranded oligodeoxynucleotides (G731E: 5’-ttatatacttggccataggttccttctccaataatTTCaataatgtcaaacttgtccacacagcgtttcccccagtc-3’; G731R: 5’-ttatatacttggccataggttccttctccaataatGCGaataatgtcaaacttgtccacacagcgtttcccccagtc-3’) from a commercial vendor.
  3.  Nucleofect 1 million A549 cells  (using 4D-Nucleofector, Lonza, Basel, Switzerland) with PX458-sgCDK12 (1 microgram) and single-stranded oligodeoxynucleotides (5 microliters of 100 micro-molar stock). 
  4. Afterwards, treat cells with 5 µM HQ461 for 2 weeks to select for cells with CDK12G731E/R knock-in. 
  5. Isolate clones that survive HQ461 treatment and use Sanger sequencing to confirm to G731E/R genomic conversion.
Copyright: © 2023 The Author(s); This is an open-access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Han, T(2023). CRISPR-Cas9 knock-in of CDK12 G731E or G731R alleles. Bio-protocol Preprint. bio-protocol.org/prep2418.
  2. Lv, L., Chen, P., Cao, L., Li, Y., Zeng, Z., Cui, Y., Wu, Q., Li, J., Wang, J., Dong, M., Qi, X. and Han, T.(2020). Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation. eLife. DOI: 10.7554/eLife.59994

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