Table 2.

Characteristics of genome-editing technologies using programmable nucleases

Gene editing	Principle of use	Advantages or application	Limitation
CRISPR-Cas9 guided gene editing: (1)NHEJ; and (2)HDR	Cas9 enzyme (an endonuclease) cleaves ds- DNA at a specific site as determined by the specific sequence of the guide RNA. Genome editing is done when the cell tries to repair the dsB (either via NHEJ or HDR)	Has the potential to edit genes in almost any cell type in vivo; Has potential in every field, notably infections[54], genetic disease[55], cancer[56] etc.; CRISPR-Cas9 can also be used for large scale loss-of-function gene screen: Catalytically inactive Cas9 (dCas9) can be directed by guide RNA, bind to specific genes to reversibly suppress or activate gene transcription (by fusion of transcription activators or suppressors with dCas9)[57]; Epigenetic modulators (e.g., DNA methylase) can also be fused with dCas9 to achieve controlled epigenetic modulations. Cas-9 NHEJ is simpler and efficient; Cas-9 HDR is more precise but lower efficiency than NHEJ. The mutant version of the Cas9 called Cas9 nickase can be used to minimize the risk of off-targets	The off-target activity of RNA-guided endonuclease-induced mutations[58]. Off-target mutations with a frequency below 0.5% cannot be detected by current off-target detection techniques[59]
Augmented CRISPR-Cas12a system	Cas12a cuts target ds- DNA. However, unlike Cas9, Cas12a subsequently becomes activated and causes indiscriminate cleavage of ssDNA causing collateral damage. SARS-CoV-2 RNA DETECTR Assay: samples from upper airway swabs are processed using simultaneous reverse transcription and isothermal amplification with loop-mediated amplification (RT-LAMP). Subsequently the Cas12 enzyme is added	CRISPR-Cas12a system can be used to create new drug or cell delivery systems and bio-sensing (e.g., to detect methicillin-resistant Staphylococcus aureus, Ebola virus[60]. Emergency Use Authorization (EUA) Only for qualitative detection of nucleic acid from the SARS-CoV-2 in upper respiratory specimens[61,62]	Limited research data and application. The technology is still in its infancy
CRISPR-Cas 13	CRISPR-Cas 13 system can be used via SHERLOCK technique for ultra-sensitive detection of RNA or DNA from the clinical samples	SherlockTM CRISPR SARS-CoV-2 kit: Emergency Use Authorization (EUA) qualitative for detection of nucleic acid fromSARS-CoV-2 in upper respiratory specimens[63,64]
Prime editors	It uses a catalytically impaired Cas9 which is fused to an engineered reverse transcriptase and prime editing guide RNA. The guide RNA specifies the target site and encodes the desired sequence	Prime editing is associated with fewer off-target edits when compared with conventional CRISPR-Cas system[65]. Anzalone et al[66] applied prime editing in human cells to correct the primary genetic causes of sickle cell disease and Tay-Sachs disease. It does not require double-strand breaks or donor DNA templates	Research literature on application of prime editing is limited. Unlike conventional CRISPR-Cas system prime editing may not be able to provide large DNA insertions or deletions[65]
Zinc finger nucleases	Zinc finger nuclease (dimer of zinc finger hybrid bound to restriction endonuclease) is a programmable nuclease that cleaves specific sites in DNA. They recognize the target sequence through protein-DNA interaction	Potential for plant genome editing for crop improvement[67]	Necessity to engineer novel proteins for each target site: Expensive; Difficult to reproduce
TALENS	TAL proteins have TAL effector DNA-binding domain fused to a DNA cleavage domain. TALENs create dsBs that require repair by NHEJ or HDR	The DNA-binding specificity of TALEs is easier to engineer than zinc-fingerProteins[68]	Necessity to engineer novel proteins for each target site. TALENs are large and pose packaging challenge in viral delivery systems[69]

HDR: Homology-directed repair; NHEJ: Nonhomologous end-joining; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2; TALENs: Transcription activator-like effector nucleases; dsBs: Double stranded breaks; ssDNA: Single stranded DNA; TAL: Transcription activator-like; SHERLOCK: Specific High Sensitivity Enzymatic Reporter UnLOCKing.