Evaluating and enhancing target specificity of gene-editing nucleases and deaminases

Some detection methods for off-target prediction are applicable for other family of Cas nucleases, such as Cas12a (Cpf1), which also create DSBs on off-target sites.

“Some of these detection methods for off-targets prediction is applicable for other family of Cas nucleases, such as Cas12a (Cpf1), which also create DSBs on off-target sites (Kim et al., 2019).”

The accuracy of SITE-seq in finding off-target sites is low, with only 10% positive hits that could be validated by targeted sequencing.

“Nevertheless, the accuracy of SITE-seq in finding off-target sites is still low, with only 10% positive hits that could be validated by targeted sequencing (Kim et al., 2019).”

SITE-seq reduces the background noise of Digenome-seq and requires much less sequencing coverage (~0.62-2.46 million reads for human genome).

“SITE-seq reduces the background noise of Digenome-seq and requires much less sequencing coverage (∼0.62–2.46 million reads for human genome).”

CIRCLE-seq demands only ~4-5 million reads for a successful analysis for human genome.

“CIRCLE-seq demands only ∼4–5 million reads for a successful analysis for human genome (Kim et al., 2019).”

IDLV displays a propensity to integrate into the vicinity of DSBs.

“One such labeling tool is called the integrase-defective lentiviral vectors (IDLV), which displays the propensity to integrate into the vicinity of DSBs (Wang et al., 2015; Kim et al., 2019).”

Digenome-seq requires high sequencing coverage (~400-500 million reads for human genome), so its sequencing cost can be relatively high.

“Due to the high background of non-specific DSBs in the purified DNA samples, Digenome-seq requires high sequencing coverage (∼400–500 million reads for human genome) thus the sequencing cost can be relatively high (Kim et al., 2019).”

BLESS demonstrates a false positive rate lower than 1%.

“BLESS demonstrates a false positive rate lower than 1% (Crosetto et al., 2013; Yan et al., 2017; Kim et al., 2019), validating the accuracy of this method.”

Extra guanine nucleotides at the 5' end of sgRNAs may hinder the interaction between the Cas9/sgRNA complex and the DNA at off-target sites.

“may hinder the interaction between the Cas9/sgRNA complex and the DNA at the off-target sites”