The relationship between Nickase's biological mechanism of action and Buffer

The relationship between Nickase's biological mechanism of action and Buffer

文: 黃俊淵 / 聚和國際 R&D 實習生
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CRISPRs (Clustered regularly interspaced short palindromic repeats) are a small repeat of DNA fragments with equal-length spacer sequences, which are part of the bacterial immune system. When some bacteria are infected by the virus and survive, CRISPR can be used to remember the virus DNA that has been infected, recognize and destroy the virus before it integrates into bacterial DNA.

This feature is widely used in DNA and RNA editing technology (described by second paragraph), compared with the earlier gene editing technology (ZFNs, TALENs) using enzymes, CRISPR is extremely fast and has become a key focus of biotechnology in recent years.

CRISPR ‘s Characteristic:

CRISPR technology is named after different nickases, such as CRISPR/Cas9 or CRISPR/Cpf1, three different types of CRISPR/Cas systems have been discovered, of which Type II relies on Cas9 and guide RNA to form a complex to operate (Figure 1)[1].

(Figure 1) A CRISPR/Cas9 recognition complex consisting of guide RNA (consisting of crRNA and tracerRNA or sgRNA) and Cas9 [1].

 

First, the target DNA sequence is identified by the Protospacer sequence of the guide RNA (crRNA tracerRNA), After the target is matched, Cas9 snips the three amino acids (NGG) immediately after the Protospacer in the target DNA, which is called the double-stranded DNA of the Protospacer adjacent motif (PAM). Sequence, and then DNA repair is carried out by the original repair mechanism in the cell through the original repair in the cell (Figure 2).

(Figure 2) CRISPR operation process
 

Nickase types are used with their corresponding Buffers:

In order to control RNA hybridization, folding and enzymatic interaction, polyamide RNA is produced by RNA and NAI-N3 (azide-substituted acylating agent) to prevent hybridization with complementary nucleic acid. This step is called Masking. To restore RNA folding and activity, use Water-soluble phosphine induces Staudinger reduction of azide, which can spontaneously remove the acyl group [2]. This step is Unmasking. Staudinger reduction can adjust the activity of gRNA in the pH range of 6.9-8.9 [3], using Biological buffers need to meet a number of conditions such as appropriate solubility, permeability pKa, no complex formation, and no reaction with enzymes to be suitable buffers (not all conditions are not listed, see [4][5] ]). Different Nickase types and operating procedures will use different buffers. Taking CRISPR/Cas9 as an example, HEPES-KOH and HEPES are used as buffers for gRNA transcription purification and masking steps, while the subsequent unmasking and DNA cleavage use HEPES-KOH and HEPES as buffers. Cleavage was performed in Tris-HCl and 1× NEBufferTM3.1 buffer [3].
 

參考資料:

  1. Redman, M. King, A. King, D. et al.. What is CRISPR/Cas9? Arch Dis Child Educ Pract Ed. 2016 Aug; 101(4): 213–215. doi: 10.1136/archdischild-2016-310459.
  2. Kadina, A. Kietrys, A. Eric, T. RNA Cloaking by Reversible Acylation. Angew Chem Int Ed Engl. 2018 Mar; 57(12): 3059-3063. doi: 10.1002/anie.201708696.
  3. Wang, R. Wu, Y. Zhou, X. et al.. Conditional control of RNA-guided nucleic acid cleavage and gene editing. Nature Communications 2020; 11(91). doi: 10.1038/s41467-019-13765-3.
  4. Good, N. E. & Izawa, S. [3] Hydrogen ion buffers. Photosynthesis and Nitrogen Fixation Part B. Methods in Enzymology 1972; 24: 53–68. doi: 10.1016/0076-6879(72)24054-x.
  5. Scopes, R. Verlag, S. Protein purification: Principles and practice, 3rd edition. FEBS Letters 1994 Oct; 352(3): 400-401. doi: 10.1016/0014-5793(94)80041-3.
  6. Zhao, Z. Li, C. Tong, F. Deng, J. huang, G. Sang, Y. Review of applications of CRISPR-Cas9 gene-editing technology in cancer research. Biological Procedures Online 2021; 23(14). doi: 10.1186/s12575-021-00151-x
Release date:2023.02.04