TABLE 1.

Different approaches of CRISPR/Cas9 delivery by physical and viral methods elucidating their advantages and limitations

Delivery vehicle and method	Most common cargo	Capacity	Advantages	Limitations	Reference
Microinjection by needle	DNA plasmid; mRNA (Cas9 + sgRNA); protein (RNP)	nmol/L levels of Cas9 and sgRNA	Guaranteed delivery into cells of interest	Time‐consuming, difficult and generally in vitro only	[71]
Electroporation and nucleofection by electric current	DNA plasmid; mRNA (Cas9 + sgRNA)	nmol/L levels of Cas9 and sgRNA	Delivery to cell population; well‐known technique	Generally in vitro only and some cells are not amenable	[72]
Hydrodynamic delivery by high‐pressure injection	DNA plasmid; protein (RNP)	nmol/L levels of Cas9 and sgRNA	Virus‐free; low cost; easy	Non‐specific and traumatic to tissues	[73]
AAV by non‐enveloped ssDNA	DNA plasmid	<5 kb nucleic acid	Minimal immunogenicity	Low capacity	[74]
Adenovirus by non‐enveloped dsDNA	DNA plasmid	8 kb nucleic acid	High‐efficiency delivery	Inflammatory response and difficult scaled production	[75]
Lentivirus by enveloped RNA	DNA plasmid	Almost 10 kb, up to 18 kb nucleic acid	Persistent gene transfer	Prone to gene rearrangement and transgene silencing	[76]

Abbreviations: Cas9, CRISPR associated protein 9; sgRNA, single guide RNA; RNP, ribonucleoprotein; AAV, adeno‐associated virus; kb, kilobase.