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CRISPR/Cas9 gene editing in hematology
repair, the resulting frameshift or nonsense mutations can give rise to truncated proteins that have a gain- or loss-of- function.20 This is a considerably faster approach than con- ventional homologous recombination-based gene target- ing to create gene knock-outs and, given its simplicity, CRISPR/Cas9 is poised to become the method of choice
Table 2. Comparison of the most widely used Cas nucleases.
for knock-out studies in most cases. Moreover, CRISPR/Cas9 is superior to RNA interference approaches for deciphering gene function, since the latter produce hypomorphic phenotypes that do not always mirror the complete loss-of-function that often occurs with genetic mutations.21
Cas nuclease
Cas9
dCas9
Cas9 nickase
Cas12a (Cpf1)
Cas13a (C2c2)
Identified from*
Streptococcus pyogenes
Mutant form of Cas9
Mutant form of Cas9
Acidaminococcus sp. Lachnospiraceae bacterium
Leptotrichia wadei Leptotrichia buccalis Leptotrichia shahii
Targeted molecule
DNA
DNA
DNA
DNA
RNA
Key features
• DSB proximal to PAM (blunt ends)
• Widely used in genome editing
• More off-targets than Cas9 variants (eSp-Cas9,
Cas9-HF1, Hypa-Cas9)
• Lacks endonuclease activity
• Works by recruiting enhancers, silencers, chromatin
modifiers
• Useful for single base genome mutagenesis
• Single-strand break
• One inactived nuclease domain
• Higher accuracy in gene integration using two nickases • Lower off-targets than Cas9
• DSB distal to PAM (staggered ends) • Cleaves first the non-target strand • No requirement for tracrRNA
• Lacks a DNase domain
• No requirement for HDR machinery or a PAM • Acts in non-dividing cells
• Cleaves additional RNA (only in bacteria)
Most frequent application
Knock-out Knock-in
Regulation of gene expression (CRISPRi/ CRISPRa)
Knock-in
Knock-out Knock-in
Regulation of gene expression
Description of the alternative Cas nucleases employed in genome editing. *Most common organisms in which the Cas nuclease has been isolated from. CRISPRi: CRISPR inter- ference; CRISPRa: CRISPR activation; DSB: double-strand break; tracrRNA: trans‐activating crRNA; crRNA: CRISPR RNA; HDR: homology- directed repair; eSp-Cas9: enhanced specificity Cas9; Cas9-HF1: high fidelity Cas9, Hypa-Cas9: hyper-accurate Cas9.
Table 3. Summary of delivery approaches for CRISPR/Cas9 components.
Delivery vehicle
Physical approaches
Microinjection
Electroporation
Viral-based approaches
Lentivirus
Adenovirus
Adeno-associated virus
Non-viral approaches
Lipid nanoparticles
Advantages
• Delivered directly into cell of interest • High efficiency
• Standardized protocols available • High efficiency with plasmids
• Robust, stable expression
• Allows delivery in complex and primary cells • Efficiency variable with construct length
• No genome integration • Transient expression • Reduced off-targets
• High efficiency
• No genome integration
• Reduced immunogenicity and cytotoxicity • Reduced off-targets
• High efficiency
• Simple manipulation • Low cost
• Reduced off-targets
• Deliver intact ribonucleoproteins
Disadvantages
• Time-consuming
• Requires expertise
• Limited to in vitro and ex vivo cells • Cell cytotoxicity
• Some cells are not susceptible
• Immune response, but low
• Limited packaging capacity (18 kb)
• Random genome integration
• Off-targets from Cas9-constitutive expression • Expertise and safety issues
• High immune response
• Limited packaging capacity (35 kb) • Expertise and safety issues
• Immune response, but very low
• Limited packaging capacity (4.5 kb) • Costly
• Expertise and safety issues
• Dependent on cell type • Endosomal degradation
Cell-penetrating peptides
• Variable penetrating efficiency
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