User:Michael.vermeulen0/sandbox

Off-target effects of genome editing are nonspecific and unintended genetic modifications that can arise through the use of engineered nucleases technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA (“target”), which they cleave (or "cut"), creating a double-stranded chromosomal break (DSB) that summons the cells DNA repair mechanisms (non-homologous end joining (NHEJ) and homologous recombination (HR)) and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects are comprised of unintended point mutations, deletions , insertions , inversions and translocations.

Designer nuclease systems such as CRISPR-cas9 are becoming increasingly popular research tools as a result of their simplicity, scalability and affordability. With this being said, off-target genetic modifications are frequent in these systems and can alter the function of otherwise intact genes. Multiple studies using early CRISPR-cas9 agents found that greater than 50% of RNA-guided endonuclease-induced mutations were not occurring on-target. The Cas9 guide RNA (gRNA) recognizes a 22 bp target DNA sequence, which it binds and cleaves to ‘edit’ the DNA sequence. However, target sequence binding can tolerate mismatches up to several base pairs, meaning there are often thousands of possible binding sites which present several experimental and safety concerns. In the research sphere off-target effects can confound variables in biological studies leading to non-reproducible results. In the clinical sphere the major concerns surround the disruption of vital coding regions leading to genotoxic effects such as cancer. Accordingly, the improvement of the specificity of genome editing tools and the detection of off-target effects are rapidly progressing research areas. Such research incorporates designer nuclease development and discovery, computational prediction programs and databases  , and high-throughput sequencing  to reduce and anticipate mutational occurrence. Many designer nuclease tools are still in their relative infancy and as their molecular properties and in vivo behaviors become better understood they will become increasingly precise and predictable.

Methods to detect off-target mutations and increase specificity
The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease is effective however it induces unwanted off-target mutations at high frequencies. Several engineering and screening methods have been described in an effort to reduce genome-wide off-target mutations including nuclease mutation, protospacer adjacent motif (PAM) sequence modification, guide RNA (gRNA) truncation and novel nuclease discovery. For example, in 2013, Fu et al. reported that by truncating the gRNA from <20 bp in length to 17 or 18 bp the target specificity of the nuclease could increase up to 5,000 fold and mismatch occurrences above 3 bases rarely, if ever occur.

Cas9 nickases
The spCas9 nuclease can also be mutated in a variety of ways to improve specificity and control. Nuclease domains can be mutated independently of each other into what are known as Cas9 nickases. These nucleases have one active and inactive nuclease domain which result in a complex that performs single strand cleavage. Cas9 nickases can be employed in tandem (known as paired nickases), which perform two single strand cuts on alternate strands. Using this strategy both Cas9 nickases must co-localize, bind and cleave their target, which drastically reduces the probability of off-target indels. Also, DSBs have long overhangs instead of blunt ends which provide improved control of targeted insertions.

Fok1-dCas9 and dimerization nucleases
As monomeric nucleases often involve high levels of off-target effects, dimerization is an attractive strategy. In a dimer system, both nucleases must bind to their individual targets or ‘half-sites’ and then interact and dimerize to initiate cleavage which greatly decreases the probability of off-target effects. In 2014, Tsai et al. described a novel method that incorporated the reliability of dimerization-dependent FokI nuclease domains, used in ZFNs and TALENs, with the simplicity of CRISPR-cas9. The FokI nuclease was originally found in Flavobacterium okeanokoites, and will only cleave DNA given dimerization activation. Basically, the researchers fused this nuclease to a CRISPR complex with an inactive Cas9 nuclease (Fok1-dCas9). The gRNA directs the CRISPR complex to the target site however the cut is made by dimerized Fok1. It is estimated that the Fok1-dCas9 strategy reduces detectable off-target effects by 10,000 fold, which makes it effective for applications requiring highly precise and specific genome editing.

Nuclease mutation
In addition to gRNA target, Cas9 requires binding to a specific 2-6 nucleotide sequence known as the protospacer adjacent motif (PAM). In commonly used SpCas9 systems the PAM motif is 5’ NGG 3’, where N represents any of the four DNA nucleotides. The requirement of the PAM sequence can cause specificity limitations as some regions will not have an available target sequence to make a desired genetic modification. In 2016, Kleinstiver et al. were able to edit the PAM sequence to non-canonical NAG and NGA motifs which not only improved the specificity but also reduced off-target effects. The researchers noticed a D1135E mutant that appeared to alter PAM specificities. Further analysis found that the D1135E reduced off-target effects and increased the specificity of SpCas9. In another 2016 paper, Kleinstiver et al. reported an additional variant, SpCas9-HF1, which also results in favorable improvements to Cas9 specificity. The researchers tested several combinations of substitutions known to form non-specific DNA contacts (N497A, R661A, Q695A, and Q926A). They found that a quadruple substitution of these residues (later named SpCas9-HF1), had extremely low levels of off-target effects as detected by GUIDE-seq experiments. Variants such as SpCas9-HF1 and D1135E,  and others like it can be combined, tested and readily added to existing SpCas9 vectors to reduce the rates of off-target mutations. Additionally, many of the engineering strategies listed above can be combined to create increasingly robust and reliable RNA-guided nuclease editing tools.

CRISPRi and CRISPRa
Research on Cas9 has allowed for the development of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). These systems can precisely alter gene transcription at the DNA level without inflicting irreversible genetic alterations. Furthermore, by directly acting on DNA they are generally more specific and predictable compared to RNAi. Although CRISPRi/a cannot be used for many genome editing in all experiments, they can act as a effective alternatives in some cases. CRISPRi and CRISPRa use a deactivated Cas9 (dCas9) enzyme that cannot cut DNA, but can deliver transcriptional activators and repressors to modulate desired gene expression with high precision. Currently, Off-target effects of CRISPRi are minimal, and show a reduced response and sensitivity to single-base mismatches. Importantly, when non-specific effects do inevitably occur they are reversible, time-dependent, and less damaging than DNA editing, making them effective alternatives that can limit the off-target burden when possible. In 2017, Cox et al. reported that CRISPR-cas13b, using a type IV CRISPR-Cas system (as opposed to the commonly used type II) could target and edit specific RNA sequences. Although minimal research has been done in this system, such an RNA editing platform has the ability to specifically edit mRNA and therefore protein translation without altering genetic code. The represents a promising technology that if successful would reduce also reduce the burden of irreversible off-target mutations.

Gene Therapy
In order for gene editing technologies to make the leap towards safe and widespread use in the clinic, the rate of off-target modification needs to be rendered obsolete. The safety of gene therapy treatment is of utmost concern especially during clinical trials as if any off-target modifications are detected the further development of a candidate product can be blocked. Perhaps the most well-known example of modern gene therapy is CAR-T therapy, used for the treatment of B-cell lymphoma. To limit the rate of off-target cleavage the therapy uses a highly specific and finely tuned TALEN which has proven to have little to no background off-target interaction. The immunotherapy is an ex vivo procedure, which means that the patient's immune cells (in this case T-cells) are extracted and edited using designer nucleases. While TALEN system development is expensive and time consuming, research and engineering modifications have drastically limited their rate of off-target interaction. However, patients receiving the treatment are still monitored frequently, and will be for the next 15 years so that off-target effects, and immunogenic responses can be analyzed and brought into consideration as new gene therapies are brought to clinical trial.

CCR5 ZFN-modified autologous helper T cell trials
In 2014, Tebas et al. enrolled 12 patients with acquired immune deficiency syndrome (AIDS) into a phase I/II clinical trial to test the safety and effectiveness of administering ZFN-modified autologous helper T cells. Through targeted deletions, the custom ZFN disables the C-C chemokine receptor 5 (CCR5) gene, which encodes a co-receptor that is used by the HIV virus to enter the cell. As a result of the high degree of sequence homology between C-C chemokine receptors, this ZFN also cleaves CCR2, leading to off-target ∼15kb deletions and genomic rearrangements. The impacts of these CCR2 modifications are still not known, and to date there have been no reported side effects however, CCR2 is known to have many critical roles in neural, and metabolic systems.

Controversy
The increased use of genome editing and its eventual translation towards clinical use has evoked controversy surrounding the true off-target burden of the technologies.

Schaefer et al. 2017
On May 30th, 2017 a two-page correspondence article was published in Nature Methods that reported an unusually high number of off-target SNVs and indels after sequencing mice that were previously involved in an in vivo gene repair experiment. The previous experiment, completed by the same group, successfully restored the vision of blind mouse strain (rd1) by correcting the Y347X mutation in the Pde6b gene using a CRISPR-cas9 system. After completing the experiment two genetically corrected mice were whole genome sequenced and compared to a control and known mouse strain genomes. Greater than 1,600 SNV s, and 128 indels were discovered, many of them overlapping between the two edited mice suggesting that the off-target effects were not random in origin. Additionally, algorithms attempting to predicting the location of off-target mutations failed to correctly predict an overwhelming majority. Many experts disagreed with the paper and criticized through journal articles and social media, suggesting that unusual CRISPR treatments were used in the initial paper and the sample size was very low (n=2). Nature Methods has issued two editorial notes on the paper. In the past, several other papers have analyzed the frequency of off-target mutations in response to CRISPR editing and although significant rates off-target rates were found, they are much smaller than what Schaefer et al. found. Nonetheless, off-target rates are consistently higher in vivo when compared to cell culture experiments, and are thought to be particularly common in humans.