**2. CRISPR-Cas9 technology, DSBs, and gene interruption**

The CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and is currently the best tool for generating cell lines and animal models of human diseases. The main advantages of this technology are its simplicity, versatility, and efficiency compared with other gene-modifying technologies. CRISPR/Cas9 technology is usually used to introduce targeted DSBs in any biological system [16], and the only requirement for Cas9-mediated DNA recognition and cleavage is the presence of a short protospacer adjacent motif (PAM) immediately 3′ to the targeted DNA sequence [17] (**Figure 3**).-

Following the creation of a DSB within the coding sequence of a gene, mechanisms of DNA- repair can induce insertions and deletions (indels), resulting in frameshift or nonsense mutations [18]. Basically, the repair of DSBs involves four possible mechanisms (**Figure 4**). The first- mechanism is standard nonhomologous end-joining (C-NHEJ). In this mechanism, the DSB is- repaired by blunt-end ligation independently of sequence homology and requires DNA ligase- IV action (**Figure 1A**). C-NHEJ can occur throughout the cell cycle but is dominant in G0/G1-

**Figure 3.** CRISPR/Cas9 ribonucleoprotein complex. Cas9 nuclease is driven to the target DNA sequence by an sgRNA molecule, composedby crRNA (blue) and trackRNA (green). The target sequence must be followed immediately by a protospacer adjacent motif sequence (PAM). After hybridization of 20nt of the cRNA with the target sequence, the nuclease performs a double-stranded break 3nt upstream of the PAM sequence.-

**Figure 4.** Approaches to repair DNA double-strand breaks. When DNA resection is blocked, C-NHEJ (classic nonhomologous end joining) is well established, whereas if the resection does occur, DNA damage is repaired by HR (homologous recombination), SSA (single-strand annealing) or alt-EJ (alternative end joining) [18].

and G2 and is associated with 1–4 bp deletions [19], which could produce frameshift mutations. Alternatively, the DSB end can be resected, leaving 3′ single-stranded DNA (ssDNA) overhangs.- The resected DSB can be repaired by three possible mechanisms: homologous recombination- (HR), single-strand annealing (SSA), and alternative end joining (alt-EJ). HR predominates inthe mid-S and mid-G2 cell cycle phases, where the amount of DNA replication is highest and- when the sister template is available [20]. HR uses a template for repair and so requires strand- invasion mediated by the recombinase RAD51 (**Figure 4**) [21]. It may be possible to exploit this property to edit mutations, delivering the appropriate template joined to the CRISPR/Cas9- system inside the target cell. The resected DSB can also be repaired by mutagenic repair pathways, namely SSA or alt-EJ.-SSA mediates end joining between interspersed nucleotide repeats- in the genome and involves reannealing of Replication Protein A (RPA)-covered ssDNA by the- RAD52 protein. SSA is typically associated with large deletions (**Figure 4**) [21]. The alt-EJ mechanism is not well understood but has an apparent predilection for joining DSBs on different- chromosomes, thereby generating chromosomal translocations and mutagenic rearrangements (**Figure 4**) [22]. Early evidence for alt-EJ came from studies, showing that yeast and mammalian- cells deficient in C-NHEJ were still able to repair DSBs via end joining [23].

As a consequence of its efficiency at inducing DSB, CRISPR/Cas9 technology has gained a reputation as the "gold standard" for creating null alleles in both *in vivo* and *in vitro*. These null alleles can arise from frameshift mutations, premature stop codons, and/or non-sensemediated decay on the target gene, resulting in loss of function. Currently, CRISPR/Cas9 is extensively used to engineer gene knockouts, but due to the variable size of the NHEJ-induced indel, generating a full KO in one step is not always achieved. In fact, full KO generation requires off-frame mutations in both alleles, and this is a matter of probability because several mutations could preserve the reading frame (e.g., +3 or −3 mutations). This undesirable effect

**Figure 5.** Experimental design of the first CRISPR/Cas9-edited cell injection in humans. Immune precursor cells were isolated from blood and *in vitro* CRISPR/Cas9 edited to eliminate PD-1 gene. Modified cells were then reinfused into the patient [25].

may be irrelevant in assays in which the knockout cell can be selected, or the null allele of the animal model can be segregated [24]. The first clinical trial using CRISPR for gene suppression and cancer therapy enrolled its first patient at Sichuan University's West China Hospital in Chengdu in 2016 [25]. In this study, the safety of PD-1 knockout CRISPR-engineered T cells *ex vivo* was evaluated when treating metastatic non–small cell lung cancer that had progressed after employing all standard treatments. Patients enrolled in the gene-editing trial provided peripheral blood lymphocytes, and PD-1 knockout of T-cells by CRISPR/Cas9 was performed *ex vivo*. In this trial, the edited lymphocytes were selected, expanded, and subsequently infused back into the patients (**Figure 5**).-

Nevertheless, there are several situations, either *in vivo* or *in vitro*, where cell selection and expansion are not an option, and obtaining a high knockout/gene inactivation efficiency is crucial [26, 27]. Hematological cancer therapies based on specific oncogenic silencing within primitive pluripotent stem cells could be the best example of these situations. In this pathological cell context, the highly efficient interruption of the oncogenic open reading frame (ORF) could be an effective therapeutic option. It would even be more important for those tumors directed by a single oncogenic event, as is the case for several leukemias or sarcomas, which are directed by specific fusion oncoproteins [28, 29].
