**3.2. CRISPR/Cas9 delivery and gene therapy**

The CRISPR/Cas9 complex can be introduced into cultured cells and single-cell embryos in the form of DNA, RNA, or protein [36]. The DNA encoding Cas9 and gRNA can be delivered into the cell using plasmid and viral expression vectors. RNA or protein has been introduced through microinjection, liposome-mediated transfection, electroporation, and nucleofection.

**Figure 10.** Schematic representation of Six6os1 WT and edited allele. Two sgRNAs were used to produce a deletion between exon 2 and 3. As a consequence, a premature stop codon appears at the beginning of exon 3. The edited allele can easily be detected by PCR [34].

However, the delivery formats of mRNA and protein pose certain technical challenges *in vivo*  and viral-based *in vivo* genome editing remain a popular choice for achieving the stable or elevated expression of Cas9 and its sgRNA [37].

Given the great potential of viral vectors in gene and cell therapy, five major classes of viral vectors—retroviruses [38], lentiviruses [39, 40], adenoviruses [41, 42], AAVs [43, 44], and baculoviruses [45, 46]—have been used to deliver CRISPR components into mammalian cells for targeted genome editing. The advantages and disadvantages of using these viral vectors for *in vivo* delivery of the CRISPR transgenes have been extensively reviewed [43, 47–49]. In **Table 2**, we list the general characteristics and applications of various viral delivery vectors.-

 Currently, adenoviral vectors and γ-retroviruses are the most commonly used delivery system- in gene therapy (**Figure 11**; **Table 3**) [1]. For Cas9 delivery, adenovirus (ad)- and retro/lentivirus- (rt/lt)-based vectors have the advantage of packaging sizes of up to 30kb (ad) and 7kb (rt/lt),- allowing the accommodation of the SpCas9 gene (∼4.2kb), one or more sgRNAs, and the cisacting regulatory sequences required for efficient expression. Nevertheless, several disadvantages- such as low titers (rt/lt), insertional oncogenesis (rt/lt), generation of a replication-competent lentivirus (rt/lt), immunogenicity, and toxicity (ad) are risks that should be taken account in *in vivo*  gene therapy.

In contrast, the AAV system has major advantages for research and therapeutics, including- very low immune response and toxicity. AAVs remain in the cell as episome, avoiding insertional mutagenesis by random integration into the host genome. In fact, there are no human diseases related to them, and they can exist long term as concatemers in nondividing cells for- stable transgene expression [50]. Given this, AAV is thought to be one of the most suitable viral- vectors for gene therapeutic applications and gene transfer *in vivo*. However, two limitations- restrict its use: packing size and tropism. AAV has a packaging capacity of only ∼4.8kb. This- makes it impossible to express the ∼4.2-kb SpCas9 gene and the sgRNA from a single AAV- vector. One approach is to use two AAV vectors: one to express SpCas9 and the other to encode- one or more sgRNAs [44]. A second approach is to use a different smaller Cas9, for example,- the ∼3.2-kb Cas9 gene encoded by *Staphylococcus aureus* (SaCas9) [35, 51]. In this sense, single


**Table 2.** Indels induced by each sgRNA predicted by the TIDE algorithm.-

**Figure 11.** Delivery systems commonly used in gene therapy clinical trials.-


**Table 3.** Viral delivery systems most commonly used in gene therapy.-

AAV vectors are able to express SaCas9, and one sgRNA has been described that appears to- be potentially very useful for *in vivo* gene editing. A single AAV vector with U6-driven sgRNA- and a TBG-driven SaCas9 expression cassette was used to target the cholesterol regulatory- gene Pcsk9in mouse liver. In this study, the authors observed modification in >40% genes,- accompanied by significant reductions in serum Pcsk9 and total cholesterol levels [35].

Another problem with AAV vectors is their limited tissue tropism, although this has gradually expanded with the identification of additional AAV variants from different species and the derivation of AAV recombinants with enhanced tropism for specific tissues [52, 53]. AAV serotypes with a strong tropism for hepatocytes, neurons, and epithelial and endothelial cells have been described, but the search for AAV variants that can efficiently infect HSC or lymphoid cells has yet to identify any candidates [54].

All these advantages have led to increases in the number of studies using AAV vectors to deliver the CRISPR components in animals and in clinical trials for gene therapy.-
