Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

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, including zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs). However, potential off-target activities are crucial shortcomings in the CRISPR system. Therefore, highly sensitive assays are needed to comprehensively analyze the off-target effects of CRISPR. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype-phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.

Mechanism of Off-Target Effects in Crispr/Cas9 System

Cas9 binds to unintended genomic sites for cleavage, termed as off-target effects. The target efficiency of CRISPR/Cas9 determined through 20 nucleotide sequences of gRNA and PAM sites adjacent to target loci. More than three mismatches between target sequences and 20 nucleotides of gRNA can result in off-target effects. It has demonstrated that 4 mismatches in PAM-distal end induce off-target effects. 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. Off-target effects cause a severe type of problem in the organism at the genomic level, large deletions and genomic rearrangements, which rarely could occur as a consequence of dsDNA breaks. Off-target effects could lead to lethal genetic mutations that cause loss of gene function, ultimately cancer cells in animals and undesirable phenotype (disease sensitivity) in plants.

Fig. 1 Effects of off-target mutation on animal and plant phenotype. (Naeem, M., et al, 2020)

Methods of Off-Target Detection

Detecting off-target sites in a highly sensitive and comprehensive manner remains a key challenge in the field of gene editing. Typically, in vivo/in vitro biochemical assays and algorithm-based computational methods are used to detect and quantify off-target effects to improve gene editing efficiency. For example, AID-Seq, PEM-Seq based detection techniques, etc. Choosing an appropriate off-target detection tool, such as biased and unbiased with predictive on-target and off-target sites, is more critical than the CRISPR delivery system. Most preferred off-target detecting biased and unbiased methods are Elevation and GUIDE-seq, respectively.

Method	Advantage	Disadvantage
T7E1 assay	Simple	Poor sensitivity, not cost-effective
Deep sequencing	Precise	Biased, misses potential off-target sites elsewhere in the genome
In silico prediction	Predicts some off-target mutation sites	Fails to predict bona-fide off-target sites
ChIP-seq	Unbiased detection of Cas9 binding sites genome-wide	Most off-target DNA-binding sites recognized by dCas9 are not cleaved at all by Cas9 in cells
GUIDE-seq	Unbiased, sensitive (0.1%), qualitative translocations, identifies breakpoint hotspots	False negatives present, limited by chromatin accessibility.
HTGTS	Identifies translocations	False negatives present, limited by chromatin accessibility.
IDLV	Programmable, sensitive (1%)	Many bona-fide off-target sites cannot be captured
Digenome-seq	Sensitive (0.1% or lower), unbiased and cost-effective	Not widely used
FISH	Quick	Less precise

Strategies for Minimizing Off-Target Effects

Various strategies have been reported to reduce RGEN off-target effects:

Guide RNA (gRNA) design and modification: Different strategies, such as GC contents, gRNA length, truncated gRNA and chemical modification have been developed to reduce off-target effects.
Improved Cas variants: Streptococcus pyogenes Cas9 (SpCas9), which is the more broadly utilized Cas9, generates genome-wide off-target mutations, which led scientists to develop variant SpCas9 s and other Cas9 orthologous that can solve the issue to some extent.

Fig. 2 Characteristics of Cas9 variants and their specificity. (Naeem, M., et al, 2020)

CRISPR delivery methods: The delivery methods used to introduce the sgRNA/Cas9 editing machinery has a role in the ultimate accuracy of the system which include improved viral CRISPR delivery methods (adeno-associated virus (AAV), adeno viruses (AdV), etc.) and improved non-viral CRISPR delivery methods (liposome, Lipid nanoparticles, vesicles, polyethyleneimine (PEI) magnetic nanoparticles (MNPs), etc.).
Base editors: The base editing technique comprises dCas9, deaminase (catalytical base modification enzyme) and sgRNA. Compared to HDR, these base editing tools have shown significant gene editing efficiency.
Prime editing: Recently, 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.
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. AcrIIA4 decreases off-target modifications by four times without reducing on-target effects by adjusting the time when AcrIIA4 or Cas9 added to assay.

References:

Naeem, M.; et al. Latest developed strategies to minimize the off-target effects in CRISPR-Cas-mediated genome editing. Cells. 2020, 9(7): 1608.
Zhang, X. H.; et al. Off-target effects in CRISPR/Cas9-mediated genome engineering. Molecular Therapy-Nucleic Acids. 2015, 4.

* For research use only. Not intended for any clinical use.

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Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

Mechanism of Off-Target Effects in Crispr/Cas9 System

Methods of Off-Target Detection

Strategies for Minimizing Off-Target Effects

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