Targeting Speci fi city of the CRISPR Cas 9 System
Year: 2017

CRISPR/Cas9 system has accelerated research across many fields since its demonstration for genome editing. CRISPR also offers vast therapeutic potential, but an important hurdle of this technology is the off target mutations it can induce. In this viewpoint, we will discuss recent strategies for improving CRISPR specificity, emphasizing how a complete mechanistic understanding of CRISPR/ Cas9 can benefit such efforts. We also propose that agreeing upon a consensus protocol with the highest specificity could benefit researchers working on CRISPR based therapies. In addition to improving CRISPR/Cas9 specificity, accurate detection of off target events is also crucial, and we will discuss various unbiased off target detection methods in terms of their advantages and disadvantages. We suggest that using a combination of cell based and cell free methods can prove more useful. In addition, we point out that improving predictive algorithms for off target sites would require pooling of the available off target analysis data and standardization of the protocols used for obtaining the data. Moreover, we highlight the risk of insertional mutagenesis for gene correction applications requiring the use of donor DNA. We conclude by discussing future prospects for the field, as well as steps that can be taken to overcome the aforementioned challenges. D of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) as a genome editing tool was one of the most significant breakthroughs in biology. CRISPR system consists of a Cas9 single guide RNA (sgRNA) complex, which recognizes a 20 bp target sequence with a downstream protospacer adjacent motif (PAM) and induces a site specific double strand break (DSB) Although gene editing through nuclease induced DSBs was feasible using zinc finger nucleases (ZFNs) or transcription activator like effector nucleases (TALENs) the cost effectiveness and ease of using CRISPR/ Cas9 made it a very attractive tool. CRISPR is applicable to a wide variety of organisms, which can be useful for various purposes, from crop engineering to human gene therapy. However, despite all the promises CRISPR has to offer, there are still debates over the extent of its off target activity. Another debate was recently started after a provocative publication by Schaefer et al. which claims that CRISPR/Cas9 can create more than a thousand unintended mutations. Even though multiple scientists suggested that the reported abnormally high off target activity was due to the flaws in experimental design and data analysis, it is still true that off target mutations are an important caveat of CRISPR/Cas9 that needs to be addressed. Especially for therapeutic applications of CRISPR, even a low frequency of unintended mutations might have deleterious effects and improving CRISPR specificity is essential. In this article, we highlight recent developments in the field regarding improvements in the specificity of CRISPR/Cas9 and off target detection methods. We also suggest strategies and considerations for further optimizations of CRISPR/Cas9. In addition, we emphasize that donor DNAs used for gene correction purposes can be another source of unintended mutations. RECENT STRATEGIES FOR IMPROVING SPECIFICITY OF THE CRISPR/CAS9 SYSTEM There have been many efforts for increasing CRISPR's specificity including rational Cas9 engineering, Cas9 directed evolution, discovery and use of more specific Cas9 variants, creating obligate heterodimers by using Cas9 nickase or dCas9 FokI fusion proteins, and modification of the sgRNA by truncation or changing the scaffold sequence  Another approach is limiting the time of CRISPR/ Cas9 activity by transfecting synthetic or in vitro transcribed sgRNA together with Cas9 protein or mRNA, instead of their expression from a plasmid. Inducible Cas9 variants can also be used for controlling the exposure time of the cells to the Cas9 activity. Another promising recent finding was antiCRISPR proteins, which could decrease off target effects without significantly affecting on target efficiency. It is hard to claim complete lack of off target effects for any of these methods and the goal should be continuous refinement of the CRISPR specificity. For more efficient rational engineering, Received: July 31, 2017 Published: September 15, 2017  DOI: 10.1021/acssynbio.7b00270 ACS Synth. Biol. 2017, 6, 1609 1613 a complete understanding of the CRISPR/Cas9 system would be helpful. While a great deal of information has been gained from recent studies of the structural, biochemical and mechanistic features of CRISPR/Cas9, questions still remain about the conformational changes associated with Cas9 transitioning from the apo state to the holo form. Specifically, details regarding the structural transitions of the catalytic HNH domain remain unclear. Furthermore, while higher fidelity variants of Cas9 have been engineered, their mechanism of ontarget/off target discrimination was incompletely understood, limiting the potential for further advances. Hence, more efforts for the full characterization of the CRISPR/Cas9 system are necessary and can help designing better strategies to decrease off target effects without compromising on target activity. COMPARING THE METHODS TO IMPROVE SPECIFICITY REQUIRES STANDARDIZATION In order to determine the best available method for improving specificity, the next step should be a side by side comparison of the above mentioned strategies. A direct comparison of the data across laboratories could be more practical; however, a true comparison would necessitate standardization in the genome editing practices. Factors that might affect the reported specificity results, such as Cas9 amount, delivery method, the controls used, and the cell line and locus tested, should be standardized and any deviations from the standard protocols should be reported for an objective and strict comparison of various tools to improve CRISPR specificity. A careful analysis of the strategies to improve CRISPR specificity can also be helpful to define best practices for preclinical studies using CRISPR. It is also possible that the effect of these methods on specificity might be additive, which should be examined. As more strategies to improve the specificity are developed, they can be incorporated into the agreed upon protocols for further refinement. Researchers working on the CRISPR based therapeutic strategies should use the best practices available to report specificity data since their results have a direct impact on the clinical translation of the CRISPR technology. Using suboptimal conditions would subsequently lead to lower specificity, which would underestimate the current advances in the field and be misleading to the community.   In one example, directed evolution is utilized to obtain Cas9 variants recognizing longer and more unique alternative PAM sequences. In another example, Cas9 is engineered by introducing rational mutations based on the Cas9 crystal structure and its known mechanism of action. SpCas9 HF1 and eSpCas9 are two such variants obtained by rational protein engineering (corresponding mutations are shown in orange and teal, respectively, on the Cas9 protein map) Cas9 FokI fusion proteins and Cas9 nickase are other strategies that can improve specificity since they require target recognition by two sgRNAs simultaneously. Inducible Cas9 variants can be used for increasing specificity by limiting Cas9 activity. Some Cas9 orthologs (SaCas9, StCas9 and NmCas9) and CRISPR proteins were also discovered that could have high target specificity. (B) Modifications to the sgRNA could be another approach to increase specificity. Truncated spacers with 17 18 nt sequence can be more specific since they may have less tolerance to mismatches. Although not explored well, modifications to functional sequences within the scaffold can also improve specificity. ACS Synthetic Biology Viewpoint DOI: 10.1021/acssynbio.7b00270 ACS Synth. Biol. 2017, 6, 1609 1613 1610 ACCURACY AND SENSITIVITY OF THE METHODS FOR DETERMINING OFF TARGET MUTATIONS Development of accurate and comprehensive off target detection methods has been another focus in the CRISPR/ Cas9 field. The earliest examples were based on evaluation of top predicted off target sites, which is not comprehensive. Recently various methods to identify genome wide off target events in an unbiased manner were developed  In the integrase defective lentiviral vector (IDLV) capture method, DSB sites were labeled by integration of the doublestranded IDLV DNA and these sites of integration were determined by high throughput sequencing. However, this method suffers from low integration efficiency of IDLVs and potential false positives due to random integration. GUIDE Seq (genome wide unbiased identification of DSBs enabled by sequencing) was a similar but a more sensitive approach that was based on capturing a small double stranded oligodeoxynucleotide (dsODN) barcode instead of IDLV. Although GUIDE Seq and IDLVs can also capture CRISPR independent DSBs in the genome, use of proper controls, such as IDLV transduction or dsODN transfection only, can help identify such background DSBs. A more significant issue with GUIDESeq is that it is technically difficult to adapt it for in vivo experiments since it requires codelivery of dsODN. HTGTS (high throughput, genome wide translocation sequencing) is a method that could be compatible with in vivo studies since it relies on detecting translocations induced by CRISPR/Cas9 and does not require additional components. However, translocations as a result of gene editing are very rare events which could be difficult to detect. BLESS (breaks l