Table 3.

Technical limitations in CRISPR-Cas9 application and their effects.

Pitfall	Reason (s)	Effects	Reference
Off-target effects	(1) Improper concentration ratio between Cas9 and sgRNA may cause off-target cleavage. (2) PAM sites may lead to undesired cleavage of DNA regions.	Unexpected mutations	Sternberg et al., 2014
Cas9 codons	Insufficient Cas9 codon optimization	Inefficient translation of Cas9 proteins	Feng et al., 2013, 2014; Schiml et al., 2014
Vectors	Mostly CRISPR/Cas9 systems use exogenous promoters for Cas9 and sgRNA expression. Vectors with optimal promoters should be selected.	Improper vectors can stop system proceedings.	Shan et al., 2014
Gene homologs	Gene family members may complicate target sequences to be edited.	False editing of target sequence.	Song et al., 2016
Epigenetic factors	DNA methylation or histone modification occurs not in regions with complex DNA compositions, such as those with repetitive sequences.	limit protein binding or RNA pairing	Song et al., 2016