CRISPR / Cas12b method

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The CRISPR / Cas12b method is a biochemical method for cutting DNA . It is a variant of the CRISPR / Cas method using the endonuclease Cas12b .

principle

Cas12b (old name C2c1 , a Cas of type V, class II), like other Cas proteins ( Cas9 , Cpf1 ), binds an RNA with a crRNA repeat sequence and a crRNA spacer sequence and cuts double-stranded DNA in the immediate vicinity to generate it a sticky end - double-strand break . Other necessary factors are the presence of a tracrRNA and a Protospacer Adjacent Motif (PAM) in the target DNA to be cut. The crRNA spacer sequence following the crRNA repeat sequence bound by Cas12b binds to the target DNA.

Types

Cas12b from Alicyclobacillus acidoterrestris , abbreviated AacCas12b, consists of 1129 amino acids and binds to a PAM with the sequence TTN in the target DNA. The cut of the target DNA through 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. At 37 ° C the enzyme activity is comparatively low. 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 ). A shortening of an sgRNA by five nucleotides at the 5 'end thirty-folds the enzyme activity. 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.

Individual evidence

  1. ↑ a b c 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. ↑ a b c d e 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 .
  4. ↑ 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 .