Recent Advancements in Reducing the Off-Target Effect of CRISPR-Cas9 Genome Editing

Figures & data

Figure 1 Overview of CRISPR-Cas9 mediated genome editing. The synthetic sgRNA directs Cas9 endonuclease to the targeted genomic DNA then the Cas9 protein introduces a double-strand break (DSB) downstream to the PAM sequence (NGG) by its two distinct nuclease domains. Finally, either Non-homology End Joining (NHEJ) or Homology Directed Repair (HDR) host-mediated mechanisms could repair the DSB. In the absence of a repair template, an error-prone NHEJ pathway is activated which may cause indels (red lines) and frequently result in disruption of gene function. In the presence of a donor template containing a sequence of interest, the error-free HDR pathway is activated and provides precise gene modification.

Figure 2 Potential outcomes in the off-target of CRISPR-Cas9. Off-target editing occurs when Cas9 with a particular sgRNA binds and edits at a place other than its target sequence. This may have unanticipated serious consequences, such as mutation, oncogene activation, immune response, and activation/inactivation of undesired genes.

Table 1 Summary of Recent Strategies to Reduce the Off-Target Effect of CRISPR-Cas9

Strategy	Brief Description	Reference (s)
Optimizing sgRNA length	Extending the length of sgRNA, which is responsible for recognizing the target DNA sequence for Cas9 cleavage, enhanced specificity and reduced off-target DNA cleavage in the CRISPR-Cas9 gene editing system.	[44]
Improved Cas variants	In compared to the commonly used wild type SpCas9, using either natural or engineered CRISPR-Cas nucleases results in higher precision.	[45]
Application of prime editors	Using an RNA-guided DNA-nicking domain (Cas9 nickase) linked with a reverse transcriptase complexed with a prime editing guide RNA (pegRNA), prime editing allows for targeted insertions of a few bases, deletions, and base-to-base conversions in human cells.	[46]
Utilization of Anti-CRISPR proteins	A chemically induced anti-CRISPR protein (AcrIIA4) is engineered to prevent Cas9 DNA binding, significantly improving the specificity and biosafety of the CRISPR-Cas9 system. In mammalian cells, an anti-CRISPR protein employed to interfere with Cas9 DNA binding activity drastically reduced off-target activity.	[47]
Utilization of SpCas9	It was shown that enhanced specificity SpCas9 (eSpCas9) variants lessen off-target effects while maintaining strong on-target cleavage.	[48]

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