Cas9 nickase represents a specialized version of the Cas9 protein used in gene editing, engineered for enhanced precision. Unlike its wild-type counterpart, this modified enzyme cuts only one of the two DNA strands, creating a “nick” rather than a complete double-strand break. This specific modification is a significant advancement for developing gene-editing tools that offer improved safety and accuracy in altering genetic material.
From DNA Scissors to a DNA Scalpel
Standard Cas9, often referred to as “DNA scissors,” creates a double-strand break (DSB) by cutting both strands of the DNA helix. This cutting action is performed by two distinct molecular “blades” or catalytic domains within the Cas9 protein: the HNH domain and the RuvC domain. Each of these domains is responsible for cleaving one of the DNA strands. The HNH domain cuts the DNA strand complementary to the guide RNA, while the RuvC domain cleaves the non-targeting strand.
A Cas9 nickase is engineered by introducing a specific mutation into one of these two catalytic domains, rendering it inactive. For example, a D10A mutation inactivates the RuvC domain, causing the enzyme to nick only the targeting strand, while an H840A mutation inactivates the HNH domain, resulting in a nick on only the non-targeting strand. Double-strand breaks are repaired by non-homologous end joining (NHEJ), an error-prone process that can lead to insertions or deletions. In contrast, single-strand nicks are repaired with high fidelity using the undamaged complementary strand as a precise template.
The Paired Nickase Strategy
A single nick in the DNA is repaired by the cell, making it insufficient to introduce a desired genetic change. To overcome this, scientists use the “paired nickase” or “double nicking” approach. This strategy uses two separate guide RNAs, each directing a Cas9 nickase enzyme to a specific location on opposite DNA strands, in close proximity. The individual nicks produced by each enzyme are offset, collectively creating a staggered double-strand break.
This staggered break promotes the cell to activate the Homology-Directed Repair (HDR) pathway. HDR is a high-fidelity repair mechanism that uses a homologous DNA template to precisely repair the break, allowing for the integration of new genetic information. Unlike the error-prone NHEJ pathway, HDR can introduce specific, desired genetic modifications with high accuracy. The precise placement of these two nicks is important for efficient genome editing.
Reducing Off-Target Effects
The paired nickase strategy reduces unintended cuts, known as “off-target effects,” in the genome. With standard Cas9, even a single off-target double-strand break can result in harmful mutations or chromosomal rearrangements. This risk arises because the wild-type enzyme only needs to bind and cut at one unintended site to cause damage.
The paired nickase system significantly lowers this risk because for an off-target double-strand break to occur, two separate nickase enzymes would have to bind and cut incorrectly in close proximity to each other. This requirement for two independent, simultaneous off-target events makes the system statistically far less likely to cause unwanted mutations than a single off-target event by standard Cas9. It is similar to needing two different keys to open a single lock simultaneously.
A Building Block for Advanced Gene Editors
The Cas9 nickase serves as a fundamental component for developing next-generation gene-editing tools. One such innovation is Base Editors, which fuse a Cas9 nickase with an enzyme that chemically modifies a single DNA base. The nickase guides the system to the target DNA and unwinds the strands, allowing the modifying enzyme to alter a specific nucleotide without creating a double-strand break.
Another advanced tool built upon the Cas9 nickase is Prime Editors. These systems utilize a Cas9 nickase to create a single nick on one DNA strand. This nick then provides a primer for an attached reverse transcriptase enzyme to directly “write” new genetic information into the target site using an RNA template. The nickase’s ability to open the DNA helix without fully breaking both strands has enabled the creation of these sophisticated editing methods.