How to reduce off-target effects and increase CRISPR editing efficiency?

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 emerged as a substantial tool due to its simplicity in use, less cost and extraordinary efficiency than the conventional gene-editing tools.However, potential off-target activities are crucial shortcomings in the CRISPR system. Researchers have proposed two types of off-target effects, the first types of off-target effects likely to occur due to the sequence homology of the target loci and the next types of off-target sites occur in the genome other than the target site.

Reduce off-target effects and increase CRISPR :

1)Guide RNA (gRNA) Modification and Engineering

a)GC Content of sgRNA: The GC contents between 40%–60% in the gRNA sequence increase the on-target activity  because higher GC content stabilizes DNA: RNA duplex and destabilize the off-target binding.

b)Length and Mismatches: Many studies revealed that unwanted mutations could be affected by the length of gRNA, such as its length up to 17 nucleotides revealed a higher genome-editing efficiency. In contrast, its length (18–20 bp) revealed low genome editing efficiency

c)Truncated gRNA

Off-target events reported decreased by 500-fold, without affecting on target accuracy, by shortening the gRNA length over the first 20 bp to 17/18 bp . On the contrary, unintended minimum changes were observed in mammalian cells when 17 bp sgRNA was used.

d) Chemical Modification of gRNA

Incorporation of 2ʹ-O-methyl-3ʹ- phosphonoacetate in the gRNA ribose-phosphate backbone leads to site-specific modification causing 40–120-fold reductions in off-target cleavage while preserving on-target performance 

2)Improved Cas Variants

a)SpCas9 and SaCas9 Variants: genome editing with SaCas9 may have reduced the chances of off-target mutations 

b)Improved Cas9 Orthologous with Broader PAM Capability and Specificity:Recombinant CRISPR-Cpf1 Ribonucleoprotein (CRISPR-Cpf1-RNP) decreased off-target activity in mouse cells

3)Improved CRISPR Delivery Methods

a) Improved Viral CRISPR Delivery Methods:Adeno viruses (AdV) naturally show meager potential to integrate into the target cell genome, a characteristic that is advantageous for limiting off-target effects.

b)Improved Non-Viral CRISPR Delivery Methods:if the Cas9 protein and gRNAs delivered as an RNP complex, the editing time window can be shortened, which reduces the off-target effects.Electroporation showed low numbers of off-target mutations in plant protoplasts when the sgRNA and Cas9 delivered as RNP complexes

4)Anti-CRISPR Proteins

Anti CRISPR (Acr) proteins are natural CRISPR/Cas system inhibitors which are encoded by various mobile genetic elements (MGEs), that inhibit the CRISPR-Cas immune function at various stages.

5) Prime Editing

a new gene-editing method (Prime editing) developed by combining reverse transcriptase programmed with prime editing gRNA (pegRNA) and Cas-nickase nuclease that can edit or “search and replace” bases in mammalian cells without a double-strand break and DNA donor template with less collateral damage.