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6.5 Strategies to Deliver CRISPR-Cas9 Machinery
Delivery of CRISPR-Cas system in vivo is one of the key parameters needed to be
evaluated for successful application of CRISPR in mitigating antimicrobial resistance. CRISPR-Cas9 system can be delivered into the target cell using various
methods. Few coherent strategies to transport CRISPR-Cas9 system are using a
vector which can be delivered with the help of a virus or non-viral perspectives like
transformation by electroporation or microinjection, delivery by liposomes and
nanoparticles.
6.5.1 Vectors
A vector can be a plasmid with genes encoding for Cas9 protein and guide
RNA. Advantage of using only one vector coding for both nuclease and guide RNA
is to eliminate performing transformation of vectors containing these genes multiple
times. High stability of this vector makes it an efficient yet a simple approach. The
challenges or hurdles using plasmid are:
• Time consuming: Plasmid has to code for the Cas9 protein and guide RNA, followed by action of CRISPR (Liu et al. 2017)
• Increased off the mark and undesirable effects (Fu et al. 2013; Cradick et al. 2013)
• It becomes imperative to deliver the plasmid to nucleus which is a challenge
A solution to above problems can be the use of mRNA which translates into Cas9
nuclease and guide RNA. This will allow for the formation of CRISPR-Cas9 assembly in a comparatively shorter time with low off target effects. Also, as the RNA is
destined to cytoplasm, the need to target nucleus is mitigated. However, the low
stability of mRNA is a challenge (Liu et al. 2017; Fang et al. 2014).
6.5.2 RNA Protein Complex
Delivery of Cas9 nuclease and guide RNA complex using a delivery vehicle brought a new
dimension in delivery systems of CRISPR-Cas9. The ribonucleoprotein complex model is
a broadly used approach. The perks of using this complex are less time consuming, fast
acting, performance with very high precision, decreased side/undesirable reactions.
Moreover there is no compulsion of optimizing the codon. The complex can be delivered
using liposomes, polymer based nanoparticles, metallic nanoparticles, etc. (Liu et al. 2017).
A. P. Sarma et al.
6.5 Strategies to Deliver CRISPR-Cas9 Machinery
Delivery of CRISPR-Cas system in vivo is one of the key parameters needed to be
evaluated for successful application of CRISPR in mitigating antimicrobial resistance. CRISPR-Cas9 system can be delivered into the target cell using various
methods. Few coherent strategies to transport CRISPR-Cas9 system are using a
vector which can be delivered with the help of a virus or non-viral perspectives like
transformation by electroporation or microinjection, delivery by liposomes and
nanoparticles.
6.5.1 Vectors
A vector can be a plasmid with genes encoding for Cas9 protein and guide
RNA. Advantage of using only one vector coding for both nuclease and guide RNA
is to eliminate performing transformation of vectors containing these genes multiple
times. High stability of this vector makes it an efficient yet a simple approach. The
challenges or hurdles using plasmid are:
• Time consuming: Plasmid has to code for the Cas9 protein and guide RNA, followed by action of CRISPR (Liu et al. 2017)
• Increased off the mark and undesirable effects (Fu et al. 2013; Cradick et al. 2013)
• It becomes imperative to deliver the plasmid to nucleus which is a challenge
A solution to above problems can be the use of mRNA which translates into Cas9
nuclease and guide RNA. This will allow for the formation of CRISPR-Cas9 assembly in a comparatively shorter time with low off target effects. Also, as the RNA is
destined to cytoplasm, the need to target nucleus is mitigated. However, the low
stability of mRNA is a challenge (Liu et al. 2017; Fang et al. 2014).
6.5.2 RNA Protein Complex
Delivery of Cas9 nuclease and guide RNA complex using a delivery vehicle brought a new
dimension in delivery systems of CRISPR-Cas9. The ribonucleoprotein complex model is
a broadly used approach. The perks of using this complex are less time consuming, fast
acting, performance with very high precision, decreased side/undesirable reactions.
Moreover there is no compulsion of optimizing the codon. The complex can be delivered
using liposomes, polymer based nanoparticles, metallic nanoparticles, etc. (Liu et al. 2017).
A. P. Sarma et al.