Part:BBa_K2872019

Cas12a protein

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1198
  Illegal PstI site found at 1696
  Illegal PstI site found at 3403
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1198
  Illegal PstI site found at 1696
  Illegal PstI site found at 3403
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 938
  Illegal BglII site found at 1271
  Illegal BglII site found at 1841
  Illegal BglII site found at 2108
  Illegal BglII site found at 2729
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1198
  Illegal PstI site found at 1696
  Illegal PstI site found at 3403
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1198
  Illegal PstI site found at 1696
  Illegal PstI site found at 3403
  Illegal NgoMIV site found at 1000
  Illegal NgoMIV site found at 1999
  Illegal NgoMIV site found at 2701
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1329
  Illegal SapI.rc site found at 1389
  

Part Information

This part was synthesized using Cas12a DNA coding sequence from pY016 (pcDNA3.1-hLbCpf1) plasmid (Addgene plasmid # 69988) [1]

Biology and Usage

Cas12a is a programmable DNA endonuclease guided by a single guide RNA (gRNA) and can be used in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules [2]

• Targeting requires a gRNA complementary to the target site as well as a 5' TTTN protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence.
• Cleavage occurs ~18 bases 3' of the PAM and leaves 5' overhanging ends.
• Cas12 a relies on a short (40-44 base) guide RNA to initiate DNA degradation.
• Cas12a is active from 16 to 48°C and maintains activity at lower temperatures than the Acidaminococcus orthologs, permitting editing in ectothermic organisms such as zebra fish and xenopus.
• High concentration liquid format can be used for microinjection, electroporation and lipofection [3]

There are 2 classes of effectors in the microbial adaptive immune system; class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Cas12a (Cpf1) is a putative class 2 CRISPR effector which mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. We chose Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) -along with Acidaminococcus- because it was found to have efficient genome-editing activity in human cells. [4]

Source

Lachnospiraceae bacterium ND2006

References

[1] Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038. 10.1016/j.cell.2015.09.038 PubMed 26422227
[2] Chen, J. S., Ma, E., Harrington, L. B., Costa, M. D., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018, April 27). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Retrieved from http://science.sciencemag.org/content/360/6387/436
[3] Biolabs, N. E. (n.d.). EnGen® Lba Cas12a (Cpf1). Retrieved from https://international.neb.com/products/m0653-engen-lba-cas12a-cpf1#Product Information_Notes
[4] Lassner, M. (2017). Faculty of 1000 evaluation for Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. F1000 - Post-publication Peer Review of the Biomedical Literature. doi:10.3410/f.725812264.793537694