Nickase and CRISPR: Revolutionary Gene Editing Tools

What is CRISPR?

CRISPR(clustered regularly interspaced short palindromic repeat) is a kind of genome editing (genome editing) technology. After the target gene is deleted, the cut gene fragments are connected by the repair mechanism of bacterial genetic material. He can edit in cells and has extensive research in biotechnology and clinical medicine.

CRISPR is divided into three types, the most simple and efficient one is microbial CRISPR type II - CRISPR-cas9 of the adaptive immune system. It has been successfully designed for genome editing applications in various animals, plants and bacteria [1].

 

CRISPR-cas9 Gene Editing: A New Technology

CRISPR-cas9 is formed by the Cas9 endonuclease protein from Streptococcus pyogenes and a small-guide RNA (sgRNA) to form a complex, and the complex will recognize complementary DNA. The main recognition of DNA sequence is through the sequence of the Protospacer on the 5' end of the gRNA, and each protospacer has a short protospacer-adjacent motif (PAM) on both sides. The cas9 complex recognizes the PAM and induces the target DNA double bond breakage (Double-strand Break, DSB) to facilitate gene editing. Double bond breaks can be via non-homologous end joining. (Non-homologous End Joining, NHEJ) or homology directed repair (Homology Directed Repair, HDR). NHEJ is prone to random deletions or insertions, has a high error rate, and has great editing limitations; HDR uses homologous template recombination, which can replace sequences, although accurate but only occurs during cell division with low efficiency^[1].
 

(Figure 1) Gene editing mechanism of CRISPR-Cas9^[4].
 

Even though sgRNAs can often be designed to be highly specific for the desired target site, there is much evidence that cas9 has a high probability (off-target effects), implying that gene editing alters off-target genes, this phenomenon might confound experimental results and limit the utility of the CRISPR-Cas9 system. There are many studies investigating various methods to reduce this effect, cas9 nickase is one of them^[2][3].

 

The biological mechanism of action of Nickase

CRISPR has various Nickases according to different technologies. Take cas9 as an example. In order to reduce the off-target effects of wild-type cas9, Cas9 inactivates one of the original two catalytic sites, Cas9 inactivates one of the original two catalytic sites, and the mutation converts it into a nickase (Cas9 nickase) that can cut single-stranded DNA. Cas9 Nickase can be divided into RuvCD10A and HNHN863A, the mechanism is the same as that of the general endonuclease, the difference is that the wild-type cleavage is at one site on each of the RuvC and HNH double chains, but D10A will inactivate one chain of RuvC, only in the sgRNA cleavage on the complementary strand; while N863A was found to inactivate HNH and cleavage on the non-target strand. Because Cas9 Nickase only cuts one DNA strand, resulting in a Single-strand Break (SSB) that can be repaired. When the single-strand is cut, it will be repaired by the HDR pathway, using the complete complementary DNA strand as a template, off-target effects are minimized without causing indels^[1].

(Figure 2.a) The wild-type cas9 endonuclease is cleaved at two sites^[3].


(Figure 3) D10A inactivates RuvC and just makes a hole in the reciprocal strand of sgRNA;
while N863A was found to inactivate HNH and make a hole in the non-target strand^[1].
 

References
[1] Alexandro E Trevino, Feng Zhang . (2014). Genome editing using Cas9 nickases. Methods Enzymology. Vol. 546:161-74. doi: 10.1016/B978-0-12-801185-0.00008-8.

[2] Chiang, TW., le Sage, C., Larrieu, D. et al. (2016). CRISPR-Cas9^D10A nickase-based genotypic and phenotypic screening to enhance genome editing. Scientific Reports .Sci Rep 6,24356. doi.org/10.1038/srep24356.

[3] Komor AC, Badran AH, Liu DR. (2017).CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell. 168(1-2):20-36. doi: 10.1016/j.cell.2016.10.044.

[4] Cribbs, Adam & SMW, Perera. 2017. Science and Bioethics of CRISPR-Cas9 Gene Editing: An Analysis Towards Separating Facts and Fiction. The Yale journal of biology and medicine.90:625-634. PMID: 29259526; PMCID: PMC5733851.