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A Rapid, Simple, High‐throughput Compatible Approach to Generating CRISPR/Cas9 Knock‐out Cell Lines
Author(s) -
Wu Meiye,
Okino Steven,
Uy Gerald,
Woo Deanna,
Shulewitz Mark,
Wang Yan
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.649.1
Subject(s) - crispr , cas9 , computational biology , genome engineering , context (archaeology) , genome editing , biology , genomic dna , genome , workflow , digital polymerase chain reaction , functional genomics , genomics , genetics , dna , computer science , polymerase chain reaction , gene , database , paleontology
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated endonuclease Cas9 have emerged as a revolutionary genome engineering tool. Using CRISPR/Cas9 to induce mutations in protein encoding genomic DNA regions is a powerful method for studying protein function in the native cellular context. However, the existing workflows for generating CRISPR/Cas9 knock‐out cell lines are cumbersome and labor intensive, comprising of arduous rounds of cloning, limiting dilutions, and surveyor assays performed on dozens if not hundreds of clones. In order for large scale CRISPR/Cas9 studies to become practical and accurate for the genomics community, a streamlined process must be developed and validated at both genomic and protein levels. Here, we report a novel workflow that leverages the power of droplet digital PCR (ddPCR) and high‐resolution melt analysis (HRM) to produce DNA sequence and western blot validated CRISPR/Cas9 knock‐out cell lines in under 5 weeks. Support or Funding Information Bio‐Rad, Inc Internal R&D support. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .