Nickase: the Next Tool in Gene Editing

Introduction

CRISPR-Cas9 revolutionized the field of genetic engineering with its ability to cleave DNA and has been lauded for its potential to treat genetic disorders [1]. However, the CRISPR system has significant drawbacks, as the double-strand breaks it induces have a relatively high rate of mutations and off-target activity [2]. Thus, multiple research teams have tried to develop gene editing systems that can be used alongside CRISPR or as an alternative. One such alternative is NICER, a technique utilizing an endonuclease called nickase [3]. An endonuclease is an enzyme that can cleave DNA, and nickase specifically causes single strand cuts [3]. Nickase has much lower levels of mutagenicity and a high level of specificity that rivals CRISPR-Cas9. Burgeoning developments in its usage have led to new strategies for treating genetic disorders [2]. While all of these systems are in research infancy, further developing them may lead to real progress in treating genetic disorders. This review will survey the various developments made in the nickase pipeline, specifically how they make up for CRISPR’s drawbacks, and its potential for treating genetic disorders with more accuracy and efficiency than current therapeutic tools.

The Problem of CRISPR-Cas9

In broad terms, the CRISPR system originated in bacteria to defend the integrity of their genetic material from bacteriophages, which are viruses that infect bacteria with foreign DNA [4]. “CRISPR” itself stands for “clustered regularly interspaced palindromic repeats,” which refers to how it can target certain repeated nucleotide sequences in DNA for cutting [4]. Essentially, guide RNA shows the endonuclease Cas9 where to cut, inducing a double-strand break in the DNA [4]. Once DNA has been cleaved, it may be repaired by two main pathways. Saito et al. describes how the first repair pathway is triggered by double-strand breaks, called NHEJ [1]. This stands for “non-homologous end joining” and is notoriously mutation-prone [1]. The second, IHR, stands for “inter-homolog repair” and has a much lower rate of mutagenic mistakes [1]. IHR may be further broken down into variants, one of which is interhomolog homologous recombination, known as IH-HR. It is of particular interest because a homologous DNA strand may be used as a template strand such that any unwanted mutations on the target DNA can be referenced against the “healthy” template and repaired [1]. 

CRISPR has a significant number of drawbacks despite its widespread use in clinical trials and research settings. NHEJ is the primary cause of most of these drawbacks, as it is the primary repair system triggered by double-strand breaks–it causes genomic instability, insertion-deletion errors, and structural instability [3]. Roy et al. further notes that some alleles simply cannot be repaired with NHEJ due to irreparable reading frame mutations, which makes CRISPR an unviable tool for some genetic disorders and mutagenic events [4]. CRISPR itself is significantly limited by a need for PAM sites [5]. PAM sites are particular sequences of DNA that act as markers for Cas9, telling the endonuclease where to cut. These PAM sites are scattered throughout DNA and when attempting to do high-specificity gene editing, a lack of viable, nearby PAM sites can prove difficult; additionally, these locations may vary from person to person, which makes standardizing treatment difficult [5]. Thus, Shin et al. notes that because there is such variation in the human genotype, gene editing tools must be more selective, efficient, and less mistake-prone than current tools [5]. 

Enter: NICER and Nickase

At its core, NICER is a nickase-based system that induces single-strand breaks rather than double-strand breaks, in contrast to CRISPR-Cas9 [3]. Tomita et al.’s study showed that utilizing single-strand breaks actually induces IHR-HR and rarely, if ever, causes NHEJ repair to occur, causing a significantly lower number of mutagenic events [3]. Tomita et al. utilized the Genome Analysis Toolkit, which shows changes in DNA sequences, to check the number of mutations caused by CRISPR and NICER [3]. CRISPR-Cas9 caused 2.53⋅10^-2 off-target insertion-deletion mutations of around 10 bp. However, none of the three nickases tested by the team demonstrated mutations when assayed [3]. In essence, CRISPR-Cas9 caused small mutagenic events at a significant rate, and these mutagenic events were not even in the region of interest. Rather, they occurred in other regions of the chromosome. If a patient were to undergo CRISPR gene therapy, there is a statistically significant chance that other parts of their genome would be disadvantageously affected by the treatment. 

The nickase system caused statistically none. This lack of mutagenic tendencies was explained by a lack of deaminases and reverse transcriptases needed for single-strand repair, both of which are enzymes affect non-target DNA sequences [3]. Roy et al. supports this conclusion, stating that through their own study, alleles tend to remain intact when nickase is used, while CRISPR-Cas9 often leads to higher levels of mutation of the DNA [4]. According to the data, NICER was able to repair long deletions, where one strand was missing a significant number of base pairs, as well as just excising DNA [3]. Thus, NICER has a broad, promising scope when it comes to editing DNA. 

As for when NICER does make mistakes and cuts off-target, three separate studies agreed that single-strand breaks, referred to as “nicks,” are easily repaired without NHEJ [2, 3, 4]. Any mistakes made by CRISPR are necessarily double-strand breaks that have a high chance of causing mutagenic events, making NICER a more appealing option. In terms of sheer efficiency, nickase-based systems can still outperform CRISPR, as supported by Roy et al. who stated that their testing of the nickase D10A resulted in 46% corrected mutations versus Cas9’s 22% [4]. They determined this by conducting genetic cleavage experiments on Drosophila somatic cells, finding that D10A, the same endonuclease used in the NICER team’s study, was vastly more precise at targeting DNA sequences than CRISPR-Cas9 [2, 3].

Nickase Applications

Shin et al. found that there was a number of out of frame mutations caused by the CRISPR silencing technique, but Wang et al.’s study found that one can use high specificity dual nickases, a technique using nickases to cut on either strand [2, 3, 5]. The dual-nickase study has been used to test nickase in vivo. Shamsara et al. studied hemophilia in mice and demonstrated that nickase genomic cleavage can be utilized instead of Cas9 to knock out a gene [6]. After extracting DNA from three lab mice and sequencing it, they designed guide RNA that could point to a coagulation factor, or a gene promoting blood coagulation [6]. After mating the mice, researchers injected recombinant DNA vectors, which included nickase and guide RNA, into fertilized zygotes [6]. Then they analyzed the DNA of the offspring to find that the target gene had in fact been knocked out [6]. There was also a significant decrease in blood coagulation, demonstrating that the paired nickase strategy had worked to induce hemophilia, with only seven mice out of the 67 offspring still exhibiting normal levels of coagulation [6]. Notably, hemophilia in mice is a recessive genetic disorder, which means both copies of the allele are mutagenic. While NICER focuses specifically on multiple nicks on a single strand of DNA, paired nickases can induce double-strand breaks that tend to be less mutagenic and more specific than CRISPR’s double-strand breaks according to Tomita et al. 2023 et al., which Wang et a.l’s study agrees with [2, 3]. This is because nickase-induced double-strand breaks are one-ended, so they can be repaired with alternatives to NHEJ, such as fork reversal or remodeling [3]. Thus, nickase-based systems may be used to excise the problematic components of a mutagenic allele to silence it. 

Tomita et al. add to these advancements, as they demonstrated developments in using nickase to treat heterozygous disorders, which is when only one copy of the allele is mutagenic [3]. This suggests that nickase can be used as a gene-editing tool just as widely as CRISPR can.

In fact, an in vitro study using CRISPR-Cas9 nickase to treat a human genetic order was conducted by Leal et al. They state that the significant risks of CRISPR’s off-target effects remain a major concern for using gene therapy to treat Morquio Syndrome [7]. Morquio Syndrome, a lysosomal storage disorder that hinders development, has historically been treated with enzyme replacement therapy, but using gene therapy may be even more effective [7]. To this end, Leal et al. focused on using nickase because of how much more effective it is in cutting the target sequence, but also how it has much lower off-target effects than CRISPR [7]. Using the double-nicking technique mentioned in Wang et al.’s study, gene therapy was promisingly completed on a kidney cell line with no off-target effects–this consisted of using nickase to recombine DNA until the cell had a healthy phenotype [2, 7]. Thus, it is not just in lab mice and in bacteria that nickase has been used for successful gene therapy and gene editing. It has been successful in human cell lines and treating human diseases in preclinical testing.

Further Developments

Moreover, developments in the nickase pipeline have been improving the accuracy and efficiency of such enzymes. Wang et al. looked into the evolution of nickases from Cas9 variants, a process that has already been studied and standardized; indeed, it involves the excising of two domains from a Cas9 endonuclease to make it a nickase instead [2]. Out of the several nickase evolutions studied, the one engineered from Sniper Cas9–a more selective, accurate variation of the wildtype Cas9 protein–performed the best [2]. The protein called eSpCas9(1.1)^D10A was very specific and efficient in its editing [2]. This suggests that the NICER system could be further improved upon by using more accurate nickases, ones that are currently in the development pipeline. 

Hu et al. has demonstrated that nickase can also make up for how CRISPR is constrained to PAM sites. Because PAM sites direct genetic cleavage, CRISPR-Cas9’s cutting ability is limited to sites with PAM sequences nearby [8]. When a Cas9 nickase is used in conjunction with Cas12, it does not have this problem, as it does not rely on PAM sites [8]. Hu et al. developed a possible diagnostic test for MRSA, a drug-resistant and often fatal bacterial infection whose current diagnostics are prohibitively slow and inaccurate [9]. Their strategy involved conjugating a Cas9 nickase with Cas12 to nick and extract the DNA of the MRSA bacteria, which can then be amplified and identified much more quickly and accurately than current diagnostic tests [9]. The current CRISPR-Cas9 system used can only cut DNA at sites next to PAM sequences, which can be limiting, as an allele of interest may not have PAM sites in useful areas. However, this demonstrates that nickase can be used to overcome this limitation, which can be useful for many genetic therapies down the line. 

CRISPR is also problematic because it cannot be used to treat mutations in mitochondrial DNA, which is not located in the nucleus of the cell like genomic DNA [10]. Using gene therapy has been problematic in editing these mutations because of the off-target effects and difficulties in guiding the system into mitochondria, and because double-strand breaks, which CRISPR causes, can disastrously degrade mitochondrial DNA [10]. The study found that single-strand breaks caused by nickase, however, can generate specific base pair substitutions in mitochondrial DNA [10]. Thus, nickase is a valuable tool in treating mitochondrial genetic disorders on top of disorders caused by mutations in genomic DNA. It is fundamentally the single-strand breaks caused by nickase that makes it such a promising candidate, as espoused by the Osaka University study on NICER in particular, and Wang et al.’s study [2, 3, 10]. 

Even More Alternatives?

There are multiple alternative systems in development that are attempting to make up for CRISPR’s deficiencies that are not as effective as NICER. For example, Fanzors are endonuclease proteins that are native to eukaryotic hosts, while CRISPR-Cas9 is a prokaryotic system originally; this alleviates some concerns about the incorporation of exogenous DNA, which does not come from the target organism, but the system itself significantly less efficient than CRISPR [1]. There have also been attempts to use Argonautes, a specific type of protein found in bacteria, to cleave DNA [8]. However, research for these is in infancy, and the only real improvement Argonautes have over CRISPR is the lack of dependency on PAM sites, which, as demonstrated by the Hu study, can already be accounted for by using nickase in conjunction with Cas12 [8, 9].

Conclusion

As such, nickase has proved itself as a more attractive alternative to the typical CRISPR-Cas9 gene editing system, notably because of its lower rate of off-target mutations, its induction of single-strand breaks, and its high efficiency of editing the target sequence. More research should be done utilizing nickase and systems like NICER in clinical settings, especially in treating heterozygous genetic disorders. Furthermore, as shown by Hu et al.’s study, nickase can also be used as a diagnostic tool for infectious diseases [9]. Nickase has great potential to become a helpful tool for gene therapy and diagnostic tests with its enhanced accuracy and more broadly applicable abilities.

About the Author: Isabella Acosta

Isabella is a Biochemistry and Molecular Biology major with an intended East Asian Studies minor. They chose their major because of their interest in biotechnology and gene editing, which further led them to writing this literature review on NICER. They hope readers will learn more about the alternatives to the CRISPR-cas9 gene editing system and come away with a better understanding of the limitations of such systems. Isabella’s professional goals include attending grad school and working in industry. Currently, they are an undergraduate member of the Britt Lab studying metalloenzymes with Dr. Melissa Bollmeyer. 

Author's Note

I wrote this literature review as part of UWP 102B, taught by Professor Amy Goodman-Bide, during Fall Quarter 2023. When I first learned about genetics and gene editing, I was in middle school and CRISPR had just exploded onto the scene. Everyone spoke with such hope for CRISPR-Cas9 revolutionizing the field of medicine, and while it certainly has done so in recent years, I wanted to write a literature review on what other tools were emerging in CRISPR’s wake. Once I heard about NICER, a slightly different gene editing system, I decided to explore how it had evolved. Thus, in this literature review, I analyze the developments in nickase-based gene editing as alternatives or supporting tools for CRISPR, as NICER is an extremely novel technique resulting from years of research into utilizing nickase.

References

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2. Wang Q, Liu J, Janssen JM, Le Bouteiller M, Frock L et al. 2021. Precise and broad scope genome editing based on high-specificity Cas9 nickases. Nucleic Acids Research. 49(2): 1173-1198. https://doi.org/10.1093/nar/gkaa1236
3. Tomita A, Sasanuma H, Owa T, Nakazawa Y et al. 2023. Inducing multiple nicks promotes interhomolog homologous recombination to correct heterozygous mutations in somatic cells. Nature Communications. 14: 5607. https://doi.org/10.1038/s41467-023-41048-5. 
4. Roy S, Juste SS, Sneider M, Auradkar A et al. 2022. Cas9/Nickase-induced allelic conversion by homologous chromosome-templated repair in Drosophila somatic cells. Science Advances. 8(26): eabo0721. https://doi.org/10.1126/sciadv.abo0721. 
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6. Shamsara M, Jamshidizad A, Rahim-Tayfeh A, Davari M, Rajabi Zangi A, Masoumi F, Zomorodipour A. Generation of mouse model of hemophilia a by introducing novel mutations, using crispr/nickase gene targeting system. Cell J. 2023. 25(9): 655-659. https://doi.org/10.22074/CELLJ.2023.1999800.1278.
7. Leal AF, Alméciga-Díaz CJ. Efficient CRISPR/Cas9 nickase-mediated genome editing In an in vitro model of mucopolysaccharidosis IVA. Gene Therapy. 2023. 30:107–114. https://doi.org/10.1038/s41434-022-00344-3.
8. Lee KZ, Mechikoff MA, Kikla A, Liu A et al. 2021. NgAgo processes guided DNA nicking activity. Nucleic Acids Research. 49(17): 9926-9937. https://doi.org/10.1093/nar/gkab757.
9. Hu Y, Qiao Y, Li XQ, Xiang Z et al. Development of an inducible Cas9 nickase and PAM-free Cas12a platform for bacterial diagnostics. Talanta. 2023. 265. https://doi.org/10.1016/j.talanta.2023.124931. 
10. Yi Z, Zhang X, Tang W, Yu Y et al. Strand-selective base editing of human mitochondrial DNA using mitoBEs. Nature Biotechnology. 2023. 42:498–509. https://doi.org/10.1038/s41587-023-01791-y.