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Table 1 Advantages and disadvantages of gene editing tools

From: Gene therapy for cystic fibrosis: new tools for precision medicine

  ZFN TALEN CRISPR/Cas9 Base Editing Prime editing
Mechanism Type IIs restriction enzyme, FokI endonuclease, fused to pair of ZFN DNA binding domains
Recognize 18-36 bp of DNA sequence
Target DNA sequence break by protein-DNA interaction
Type IIs restriction enzyme, FokI endonuclease, fused to pair of TALEN DNA binding domains
Recognize 30-40 bp of DNA sequence
Target DNA sequence break by protein-DNA interaction
Few Cas endonuclease options for broader specificity and flexibility (Cas9, Cas12)
PAM sequence require to design sgRNA
Target DNA sequence break by DNA-RNA interaction
Direct conversion of a DNA base to another without DSBs at a target locus
Permanent conversion of C-G to T-A base pairs by cytosin base editor (CBEs)
Enzymatically convert A-T base pairs into G-C base pairs by adenine base editors (ABEs)
Fusion complex composed of a catalytically impaired Cas9 protein and an engineered reverse transcriptase
Can recognize DNA of any sequence size
Efficiency Low Low High High High
Advantages Currently being used in clinical trials for HIV and Hunter’s syndrome
Low immunity and Small protein size
Target any DNA sequence
Less cytotoxic effects
Highly predictable target sequence
Easy to design and possible to target only 1 bp of target sequence
Potentially target multiple genes simultaneously
No random insertion and deletions because do not require DNA break
High A > G and C > T conversion
No random insertion and deletions because do not require DNA break
Can be used to generate different mutation types (insertions, deletions, and point mutations)
Limitations Difficult due to extensive cloning needed to link two zinc finger modules together and expensive to design Sensitive to DNA methylation
Require pair of TALEN with two independent DNA binding sites
Require PAM site near the target DNA sequence to design gRNA
Off-target effect observed
Cas9 protein too large for AAV-based delivery
Only accounts for 4 out of 12 possible base-to-base conversions
Too large for AAV-based delivery
Difficult to edit DNA sequence that several A or C residues are nearby
High targeting efficiency but may depend on cell type
Too large for AAV-based delivery
Detection of undesired off-target effects and on-target mutation