Part:BBa_K3286040

F. novicida dCpf1 (dCas12a) protein

Endonuclease dead version of the Francisella novicida cpf1 gene [1]

The Fn-dCpf1 protein can highly efficient bind target DNA, but due to mutations in the DNA cleavage domains unable to cleave target DNA. The Fn-dCpf1 protein can be used as a transcriptional regulator for gene expression by blockage of RNA polymerase initiation (targeting a promoter region) or RNA polymerase elongation (targeting the coding sequence). The DNA cleavage domains of the F. novicida Cpf1 protein were inactivated by the introduction of two mutations in the RuvC I and the RuvC II domains (D917A, E1006A) [2].

1. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., … Zhang, F. (2015). Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell, 163(3), 759–771. https://doi.org/10.1016/j.cell.2015.09.038
2. Leenay, R. T., Maksimchuk, K. R., Slotkowski, R. A., Agrawal, R. N., Gomaa, A. A., Briner, A. E., … Beisel, C. L. (2016). Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. Molecular Cell, 62(1), 137–147. https://doi.org/10.1016/j.molcel.2016.02.031

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 16
  Illegal PstI site found at 280
  Illegal PstI site found at 1543
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 16
  Illegal NheI site found at 1549
  Illegal PstI site found at 280
  Illegal PstI site found at 1543
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 16
  Illegal BglII site found at 1502
  Illegal BglII site found at 1536
  Illegal BglII site found at 1622
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 16
  Illegal PstI site found at 280
  Illegal PstI site found at 1543
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 16
  Illegal PstI site found at 280
  Illegal PstI site found at 1543
  Illegal NgoMIV site found at 3231
  
• 1000
  COMPATIBLE WITH RFC[1000]

Contribution from iGEM 2021 RDFZ-China

Group: iGEM 2021 RDFZ-China
Author: Jiangshan Gong

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dCpf1 is an efficient tool for multiplex gene regulation. In the research, connecting 24nt guide sequences targeting three independent segments within the coding region of the sf-GFP gene by the 36nt DR sequences and co-expressed under a constitutive promoter (A), the researchers found that crRNAs targeting any one of the three segments resulted in varied but significant gene repression(10–100 fold). Repression was further augmented by doubly or triplycombined crRNAs, presumably through a stronger blockage of transcription elongation (C). Strikingly, the triply combined crRNAs completely abolished GFP expression (>300-fold reduction). The fold reduction by multiplex targeting, relative to individual targeting, was between additive and multiplicative. These results suggested that co-transcribed crRNAs targeting multiple DNA segments can be utilized by dCpf1 to combinatorially augment gene repression.

Reference: Chensi Miao, Huiwei Zhao, Long Qian, Chunbo Lou, Systematically investigating the key features of the DNase deactivated Cpf1 for tunable transcription regulation in prokaryotic cells, Synthetic and Systems Biotechnology, Volume 4, Issue 1, 2019, Pages 1-9, ISSN 2405-805X, https://doi.org/10.1016/j.synbio.2018.11.002.