By Daniel Lo
For the use of CRISPR-Cas, the size and specificity of the sgRNAs make it a highly precise tool for binding to target DNA sequences. The 20 nucleotide sgRNA is an easily modifiable component within the system, and multiple genetic targets can be targeted in the same experiment through the addition of the Cas9 endonuclease and any number of sequence-specific sgRNAs (Fire A, 1998). For these reasons, the CRISPR-Cas system is a highly robust process, and the use of CRISPRi and CRISPRa libraries in achieving loss and gain of function screens respectively have been achieved on the genome scale with ease.
One study that highlights the efficacy of using CRISPR-Cas systems in discovering cancer drug targets was conducted by Shi and his colleagues in 2015 (Shi et al, 2015). Multiple previous studies involving CRISPR-Cas induced mutations tended to target only the 5’ exons of genes, which led to the generation of in-frame gene variants with identical functions as the original gene (Fedorov et al, 2020; Wolf et al, 2016; Sanson et al, 2016; Shalem et al, 2014). This renders the CRISPR-Cas mutation ineffective, as the original phenotype is maintained. Therefore, to bypass this limitation, Shi et al. conducted their experiments by instead specifically targeting the exon regions of genes that coded for functional protein domains.
Through this method of selective targeting, the authors achieved more null mutations than past studies which provided better insight into the function and significance of the target gene. The group carried out the study with a diploid Cas9-expressing acute myeloid leukemia cell line of mouse origin (RN2), and generated sgRNAs that targeted 192 different exonal regions coding for different functional domains within genes that were responsible for chromatin regulation and modification. After transfecting the mouse cells with different sgRNAs, the authors selected for cells with a stunted growth phenotype, meaning that the target region of the sgRNA had an important or potentially essential function in cell growth and division. The group identified 25 exon domains in proteins within the RN2 cell line that were previously overlooked as leukemia dependencies. These include the Dot11, Ehmt1 and Brd4 proteins, and cells that were treated with the sgRNAs targeting these proteins demonstrated a slower growth. The team confirmed the link between these genes and growth phenotype with deep sequencing analyses, showing that the targeted gene region experienced a mutation that led to a loss of its function. With these results, the group suggested that using CRISPR-Cas9 screens in a functional domain-focused fashion can allow for the elucidation of essential domains in genes that can be subjected to pharmacological inhibition and can be applied to the study of different cancer systems and their protein dependencies. They also argued that based on their findings, the severity of negative selection phenotypes (as shown by the stunted growth of the cells) after different sgRNA treatments is an indication of the importance of the function of the targeted domain.
Their study was successful in demonstrating that the specificity of the sgRNAs matter when it comes to creating a CRISPR-Cas9 cleavage of a gene. This is also a crucial finding for optimizing the targeting of genes that encode for bigger proteins with multiple domains, since simply employing random sgRNAs to target the exons of such gene sequences may not provide researchers with the desired effect of knocking down the function of the protein product. For example, the indel mutations that result from the double strand break can actually compensate for the original breakage and lead to a change in base sequence that does not affect the reading frame of the gene sections encoding for important protein domains. The team also further explains that their technique in targeting genes for CRISPR-Cas cleavage can be effective in identifying distinct active sites in enzymatic proteins.
Although this study by Shi and colleagues seem to be a strong indication towards the importance of the exact location that is targeted by sgRNA, CRISPR-Cas is still considered a technique that is yet to be fully optimised. Previous studies have noted that the sgRNAs in CRISPR-Cas systems can bind to incorrect target areas and tolerate up to five mismatched base pairings (Hollen et al , 2002). Therefore, it may be possible that the cells that were treated with certain sgRNAs designed by their team experienced off target effects, where other regions of the genome that were not sequenced by them afterwards experienced mutations that led to altered phenotypes that could also contribute to their observations of a positive or negative selection.
In addition, a study from 2014 has described varying efficiencies of CRISPR-Cas genome editing depending on the method in which the Cas9 protein and sgRNA are delivered to cells (Jackson et al, 2006). For example, they have shown that directly delivering the purified components into cells (namely the Cas9 protein and sgRNAs) resulted in the least number of undesired off target effects. On the other hand, introducing plasmid sequences encoding for the CRISPR-Cas components is comparatively less desirable as they are easily degraded in cells after delivery. It is unknown whether the method used by Shi et al., which was to allow for stable Cas9 expression and subsequent lentiviral delivery of sgRNA was the most efficient method of introducing CRISPR-Cas to the cell system. This further adds on to the possibility of unforeseen and unrealised off target effects that may have contributed to their observations of positive/negative selection.
Moreover, mounting evidence is suggesting that the frequency and likelihood of off target effects that are brought forth by CRISPR-Cas gene editing is correlated to different cell types (Ui-Tei et al, 2020). For example, cell lines with intact DNA repair proteins and pathways, such as healthy human pluripotent stem cells, were shown to have less off target genetic changes and mutations through whole genome sequencing after CRISPR-Cas treatment, whereas transformed cell types such as cancer cells display a higher rate of off target effects. This means that studies involving CRISPR-Cas gene editing of different cell types may yield different results and effects due to the nature of their DNA repair mechanisms and functionality. Since Shi’s team used murine leukemia cells with an unknown profile and functioning of DNA repair proteins and pathways, it is again possible for off target effects to have occurred in the background without being detected.
Despite the limitations of the study and the inherent shortcomings of the CRISPR-Cas system, Shi et al have still soundly demonstrated that where the sgRNA targets the gene exon is also important, since not all the exonal regions targeted can be altered or silenced successfully. Thus,
this study can potentially be hailed as a stepping-stone in refining the use of CRISPR-Cas as a method of gene screening for cancer drug discovery.
References:
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