Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

Details

• Alternative Title:
  Nature
• Personal Author:
  Konermann, Silvana ; Brigham, Mark D. ; Trevino, Alexandro E. ; Joung, Julia ; Abudayyeh, Omar O. ; Barcena, Clea ; Hsu, Patrick D. ; Habib, Naomi ; Gootenberg, Jonathan S. ; Nishimasu, Hiroshi ; Nureki, Osamu ; Zhang, Feng
• Description:
  Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.
• Subjects:
  Cell Line, Tumor Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-Associated Proteins CRISPR-Cas Systems DNA, Complementary Drug Resistance, Neoplasm Gene Expression Regulation, Neoplastic Gene Library Genetic Engineering Genetic Loci Genetic Testing Genome, Human Humans Indoles Melanoma Proto-Oncogene Proteins B-raf Reproducibility Of Results RNA, Untranslated Sulfonamides Transcriptional Activation Up-Regulation

  More +
• Source:
  Nature. 517(7536):583-588.
• Document Type:
  Journal Article
• Funding:
  DP1 MH100706/MH/NIMH NIH HHS/United States ; DP1-MH100706/DP/NCCDPHP CDC HHS/United States ; R01 NS062849/NS/NINDS NIH HHS/United States ; R01 NS073124/NS/NINDS NIH HHS/United States ; R01-NS07312401/NS/NINDS NIH HHS/United States
• Volume:
  517
• Issue:
  7536
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:00240015f8e83fdc18d2356f7aaa2060e230d14be0f83ff74a6b8bda785ee102
• Download URL:
  https://stacks.cdc.gov/view/cdc/30833/cdc_30833_DS1.pdf
• File Type:
  [PDF - 6.41 MB ]