Discovered as an immune system against viral infection in domain bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) system has quickly become a crucial tool in biological research. Long before it became the focus of debate because of its use to generate gene-edited babies (1), scientists recognized CRISPR system as an efficient, accurate and programmable nuclease system capable to induce double strand breaks (DSBs) in various organisms, therefore with a high potential as a versatile tool for scientific studies as well as a powerful tool for medicinal and agricultural improvement. Already, there are abundant data on the potential of CRISPR system for application in crop improvement (2) as an alternative to genetically modified organisms (GMO) (3). Moreover, clinical trials to cure cancer patients by using CRISPR edited T cells are ongoing in the United States (ClinicalTrials. gov registry number: NCT03399448) and China (registry number: NCT03545815).
From restriction enzyme technique (Fig. 1A), the ability to manipulate genomic DNA in living cells had a pivotal role in the history of biological research. In the 1980s, directed mutation technique via homologous recombination revolutionized the field by allowing directed mutation in mammalian cells (4). Homologous recombination is performed by introducing trans-acting DNA material, usually containing a selection marker, by flanking homologous sequence matching target genomic DNA (Fig. 1B). This technology led to pivotal discoveries at that time. As an example, directed gene deletion by homologous recombination of mice stem cells allowed subsequent generation of transgenic mouse bearing deletion in genes of interest (5). However, this technique efficiency was characteristically low (6). Thus, further researches focused on overcoming those limitations, for example by using negative selection markers such as thymidine kinase or diphtheria toxin fragment A. However, efficiency increase remained modest [reviewed in (7)]. In the 1990s, Rouet, et al. discovered that
CRISPR system is originally an RNA-mediated defense system in bacteria and archaea (21). While first discovered in
In 2007, Barrangou, et al. reported that CRISPR spacers provide prokaryotes resistance against corresponding phage by mechanisms involving neighbor
Parallel to these studies, another focus of this field was to elucidate the mechanism underlying crRNA maturation, as many organisms with a working CRISPR/Cas9 system lacked essential Cas proteins thought to be essential for crRNA maturation in previous studies. Trans-activating CRISPR RNA (tracrRNA) were first identified as the third most abundant class of transcripts in the
In 2011, as all the crucial components of the CRISPR/Cas9 systems were identified, Siksnys’ lab successfully reconstructed a working CRISPR/Cas9 system in
While the CRISPR/Cas9 system is quickly becoming the tool of choice to perform targeted mutation, there are still limitations to address. One of these was raised and assessed early from the groups that developed the CRISPR technology (44-47). Off-target, that is nuclease activity at sites other than the programmed sites, can occur in the CRISPR/Cas9 system [the widely used CRISPR system, Class 2 type II from the
Researchers investigated various paths toward improving the Cas9 on-target efficiency. A straightforward method was to optimize Cas9 and gRNA amount and proportion (45), or directly deliver the Cas9 protein and sgRNA as ribonucleoprotein into cells (54). Additionally, while studies of the Cas9 variants from other organisms than the
Recently, new techniques using dCas9 variants fused to proteins are emerging as promising tools in gene editing. For example, base editing is a technique that uses dCas9 fused to a nucleobase deaminase enzyme or a DNA glycosylase able to convert a single base pair in targeted site, enabling precise point mutation (64). Prime editing, or search-and-replace genome editing is a technique that uses a dCas9 fused to a reverse transcriptase domain and a modified gRNA to insert a designed sequence within target site, therefore enabling precise sequence insertion without donor DNA (65). While they do not induce DSBs, these editing techniques are also prone to the same off-target issue as CRISPR/Cas9 system. Indeed, it was shown that base editing occurs at off-target sites in a frequency ranging from 0.07% to 100% in 38-58% genes in human cell (66). Interestingly, it was shown that improvement in base editing on-target efficiency can be achieved by optimizing the base editing domain rather than the dCas9 domain (67).
Finally, optimization can be achieved by sgRNA configuration. The five base pairs at proximity of the PAM region of the sgRNA are known as ‘seed regions’ more stringent in guiding the Cas9 complex to its target (51). While distal sequences from the PAM region are necessary to the Cas9 activity (53), those ‘seed regions’ context are crucial in determining the binding specificity. For example, U-rich seeds sequence increases specificity (51, 68) while high GCs content decreases Cas9 activity (68, 69). G is more favorable and C is less favorable as the base directly after the PAM. In contrast, at the fifth base from the PAM site, C is more preferred. From the 9th to 10th distal sequence from the PAM, A is favorable. At the 18th base from PAM, C is less favorable (51, 68, 70, 71). Those criteria can be used to design a target site for the gene of interest with minimum putative off-targets.
In order to use CRISPR/Cas9 system as a therapeutic tool, common agreement is that the risk of off-target should be assayed in a case specific manner. The impact of off-target mutation in patients will differ greatly based on the genes and tissues affected, as factors such as differential expression between tissues and pathological effect of genes differ greatly (72). Thus, to enable practical application, another focus was the development of tools that facilitate analysis of the off-targets using whole-genome sequencing (WGS) with improvement in cost and efficiency. To this end, several methods were developed including BLESS/BLISS which label and detect breaks
Another putative side effect of the targeted CRISPR/Cas9 system is on-target mis-regulation. The underlying mechanism of gene mutation by CRISPR/Cas9 systems is that Cas9 induces DSB in the genome that triggers repair pathways via the non-homologous joint end repair (NHEJ) or the less frequent homology directed repair (HDR) that occurs if a homologous template is nearby (78). In eukaryote, DSBs occur relatively frequently because of reactive oxygen species, radiation, replication error or mechanical stress. Thus, proteins that are involved NHEJ are intrinsically active (79). Repair by NHEJ usually results in imperfect repairs with insertion or deletion mutations (InDels), leading to a frameshift mutation that consequently results to a premature termination codon (PTC) within coding region. PTC triggers cell inherent nonsense-mediated mRNA decay (NMD) mechanism leading to complete knock out of the targeted gene by mRNA degradation within seconds (80, 81). On-target CRISPR/Cas9 mediated gene silencing is usually achieved by this mechanism.
However, a recent investigation showed that ∼50% of the cell lines from previous studies did not result in targeted gene knock out, but rather caused the production of truncated functional proteins. To reduce on-target mis-regulation, the authors recommended selecting target sites avoiding the internal ribosomal re-entry site, as InDels in those sites may result in the production of pseudo-mRNA. Also, exon splicing enhancers site should be avoided as target site as their deletion may result in exon skipping, thus generating truncated proteins rather than knock out (82). While other studies applied this exon skipping capability to introduce alteration in the targeted genes (83-86), the consensus is that in addition to the off-target mutations, these on-target mis-regulations should be carefully evaluated before application (84, 86).
For now, CRISPR/Cas9 system is known as the most convenient method to program target sites for mutation among developed techniques. Thus, CRISPR/Cas9 system is quickly becoming a prominent tool for basic research as well as for clinical and agricultural purposes. In this review, we discussed a few of the many studies that led to its development. Its basic principle is that it induce a targeted DBS in the genome that can go through two inherent mechanisms, NHEJ that ligate the break without a homologous template and HDR that use a homologous template, therefore that is less error-prone but has lower efficiency compared to NHEJ. Thus, NHEJ remains the most commonly used pathway despite its putative on/off-target side effect. Recent improvements have been initiated to increase the specificity of the Cas9 targeted DSB as well as to develop techniques to detect off-target at large scale, crucial to evaluating its safety for clinical and agricultural applications. While off-target mutation can be detected using large-scale analyses, on-target mis-regulation can only be assessed after mutation in a case specific manner. This shows that the CRISPR/Cas9 possible side-effects should be carefully assayed before application, and there is room for improving this highly effective targeted mutation technique.
This study was supported by grants awarded to the JWO (NRF-2017H1D3A1A01052995 and NRF-2016R1D1A1B0393 5382) and the JS (NRF-S201806S00067) by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology. We apologize to colleagues whose work could not be cited because of space limitations.
The authors have no conflicting interests.