Cas12b

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Alicyclobacillus acidoterrestris CRISPR-associated endonuclease Cas12b
other names

AacC2c1, AacCas12b

Mass / length primary structure 1,129 amino acids , 130,611 Da
Identifier
External IDs
Enzyme classification
EC, category 3.1.-.-

Cas12b (from CRISPR-associated , synonymous C2c1 ) is an enzyme from the group of endonucleases and a ribonucleoprotein . It occurs in Alicyclobacillus acidoterrestris and Bacillus hisashii , among others . It is used in the CRISPR / Cas method , more precisely: in the CRISPR / Cas12b method .

properties

Cas12b is a Class II Type VB Cas protein and is found in bacteria as part of the adaptive antiviral immune response with CRISPR . It binds RNA with a crRNA repeat sequence, which in turn binds a tracrRNA . The crRNA repeat sequence is followed by a crRNA spacer sequence that binds to viral DNA by base pairing . The endonuclease function then creates a double-strand break in the viral DNA . When using Cas12b as part of the CRISPR / Cas method, the crRNA spacer sequence is changed in order to bind to a target DNA. A Protospacer Adjacent Motif (PAM) with the sequence TTN ( thymidine-thymidine -any nucleotide ) must be present in the target DNA . The cut of the target DNA creates a sticky end with a 5 'overhang of 6-8 nucleotides 14-17 nucleotides downstream from the PAM on the non-target strand or 23-24 nucleotides downstream from the PAM on the target strand. Cas12b cuts DNA at a temperature between 37 and 60 ° C, the temperature optimum is 48 ° C. Compared to Cas9 or Cpf1 (synonymous Cas12a), Cas12b generates fewer unspecific cuts.

Cas12b from Bacillus hisashii , abbreviated to BhCas12b, with 1108 amino acids is smaller than SpCas9 (1368 amino acids) and therefore also has a smaller gene, which makes it easier to use in viral vectors (especially AAV vectors ). However, the generated wild-type , only of BhCas12b at 37 ° C single-strand breaks . Therefore, a mutant of BhCas12b called BhCas12b v4 was developed (K846R / S893R / E837G), which produces double-strand breaks and less unspecific cuts even at the body temperature of mammals.

literature

  • I. Jain, L. Minakhin, V. Mekler, V. Sitnik, N. Rubanova, K. Severinov, E. Semenova: Defining the seed sequence of the Cas12b CRISPR-Cas effector complex. In: RNA biology. [electronic publication before printing] August 2018, doi : 10.1080 / 15476286.2018.1495492 , PMID 30022698 .

Individual evidence

  1. ↑ a b c d cas12b - CRISPR-associated endonuclease Cas12b - Alicyclobacillus acidoterrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) - cas12b gene. In: uniprot.org. October 16, 2013, accessed January 24, 2019 .
  2. ↑ S. Shmakov, OO Abudayyeh, KS Makarova, YI Wolf, JS Gootenberg, E. Semenova, L. Minakhin, J. Joung, S. Konermann, K. Severinov, F. Zhang, EV Koonin: Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas systems. In: Molecular cell. Volume 60, number 3, November 2015, pp. 385-397, doi : 10.1016 / j.molcel.2015.10.008 , PMID 26593719 , PMC 4660269 (free full text).
  3. ↑ L. Liu, P. Chen, M. Wang, X. Li, J. Wang, M. Yin, Y. Wang: C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism. In: Molecular cell. Volume 65, Number 2, January 2017, pp. 310-322, doi : 10.1016 / j.molcel.2016.11.040 , PMID 27989439 .
  4. ↑ a b c Jonathan Strecker, Sara Jones, Balwina Koopal, Jonathan Schmid-Burgk, Bernd Zetsche, Linyi Gao, Kira S. Makarova, Eugene V. Koonin & Feng Zhang: Engineering of CRISPR-Cas12b for human genome editing. In: Nature Communications . Volume 10, number 1, January 2019, p. 212, doi : 10.1038 / s41467-018-08224-4 , PMID 30670702 .