**3.3. CRISPR-Cas9 sgRNAs: "Superguides" for interfering with (onco)gene expression**

When a cancer cell is the target, a delivery strategy that can result in the expression of Cas9- and an oncogen-specific sgRNA in all infected cells is desirable. This is especially critical for *in vitro* gene therapy where the expansion processes from a selected edited cell are not available.- Similarly, it is also crucial for *in vivo* approaches in cancer therapies focused on disrupting a driver oncogene. If the efficiency of CRISPR/Cas9 reagents delivery to the cancer cell is acceptable, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the- oncogene. In this way, for the vast majority of knockout studies where the edited cells or- mice can be selected, the sgRNA targets different positions within the chosen exon, avoiding- boundaries. In most of these cases, the designs follow off-target criteria. However, for all those- cases where cellular selection is not an option and only one sgRNA can be used, the null effect- could be increased with a sgRNA targeting the exon boundary. Following this strategy, the- generation of null alleles would be increased by two ways: probability of producing a frameshift mutation and probability of breaking the canonical pre-mRNA splicing (**Figure 12**).-

It has long been known that mutations in splice-site consensus sequences can affect pre-mRNA- splicing patterns and can lead to generate null or deficient alleles [55]. In fact, pioneering genetic studies indicated that many of the thalassemia mutations in the β-globin gene affect splice- sites and give rise to aberrant splicing patterns [56, 57]. Recent studies have demonstrated that- a splicing mutation in the STAR gene is a loss-of-function mutation that produces an aberrant protein [58]. Besides, non-sense-mediated mRNA decay (NMD), a conserved biological- mechanism that degrades transcripts containing premature translation termination codons, could help secure the null effect when a DSB is induced in splice sites. In addition to transcripts- derived from nonsense alleles, the substrates of the NMD pathway also include pre-mRNAs- that enter the cytoplasm with introns intact [59]. Several mutations of splice donor sites that- cause loss of gene function have recently been identified. A novel mutation at a splice donor- site that was predicted to lead to skipping of exon 10 of the PLA2G6 gene was found in a homozygous state in infantile neuroaxonal dystrophy patients. This variant was correlated with very- strong loss of function, providing further evidence of its pathogenicity [60]. Mutations in the-

**Figure 12.** CRISPR/Cas9 design against sequences involved in intron processing.-

ectodysplasin A1 gene (EDA-A1) at the splice donor site have been described in patients with- hypohidrotic ectodermal dysplasia. The mutation resulted in the production of a truncated- EDA-A1 protein caused by the complete omission of exon 3. This novel functional skippingsplicing EDA mutation was considered to be the cause of the pathological phenotype [61]. Studies in a family with premature ovarian failure identified a variant that alters a splice donor- site. This variant resulted in a predicted loss of function of the MCM9 gene, which is involved- in homologous recombination and repair of double-stranded DNA breaks [62].

As we have mentioned before, not all indels targeting the exon coding sequences necessarily give rise to premature stop codons. However, if DSBs are induced near the boundaries of the target exon, then the canonical splicing pathway could also be altered. In that case, to the probability of producing frameshift, mutations should be added that of interfering with canonical pre-mRNA splicing (**Figure 12B**). Even if the CRISPR/Cas9-induced mutation did not produce a frameshift mutation, at least this strategy would offer the possibility of producing nonfunctional oncogenes by splice-pathway alteration. It has recently been shown that CRISPR/Cas9-mediated alterations at exon boundaries may also result in altered splicing of the respective pre-mRNA, most likely due to mutations of splice-regulatory sequences. Using the human FLOT-1 gene as an example, the authors demonstrated that such altered splicing products also give rise to aberrant protein products with loss of function [63].

An unpublished study has compared the efficiency of generatingnull alleles by CRISPR/Cas9 sgRNAs targeting exon boundaries. The authors compared the efficiency of producing null alleles inducing DSBs in a central position of the critical exon with DSBs close to the splice donor site on the exon. The study, which was carried out in a variety of genes, species and systems, revealed an increase in knockout efficiency using sgRNA guides targeting the splicedonor site of the chosen exon.
