Tag-seq: a convenient and scalable method for genome-wide specificity assessment of CRISPR/Cas nucleases

Abstract

Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPR-associated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases. However, current methods for off-target discovery are tedious and costly, restricting their widespread applications. Here we present an easy alternative method for CRISPR off-target detection by tracing the integrated oligonucleotide Tag using next-generation-sequencing (CRISPR-Tag-seq, or Tag-seq). Tag-seq enables rapid and convenient profiling of nuclease-induced DSBs by incorporating the optimized double-stranded oligodeoxynucleotide sequence (termed Tag), adapters, and PCR primers. Moreover, we employ a one-step procedure for library preparation in Tag-seq, which can be applied in the routine workflow of a molecular biology laboratory. We further show that Tag-seq successfully determines the cleavage specificity of SpCas9 variants and Cas12a/Cpf1 in a large-scale manner, and discover the integration sites of exogenous genes introduced by the Sleeping Beauty transposon. Our results demonstrate that Tag-seq is an efficient and scalable approach to genome-wide identification of Cas-nuclease-induced off-targets.

Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity

“…To globally evaluate the editing specificity of As Cas12a-KK and As Cas12a-KA, we performed Tag-seq experiments [ 65 ] to assess seventeen different sgRNAs targeting different sites in the endogenous human EMX1 , DNMT1 , RUNX1 , PD1 , CTLA4 , CD47 , SIRPa , CCR5 , and CXCR4 genes (Fig. 1 c and Additional file 2 : Figure S1e), as these sites had been well-studied or were of clinical relevance.…”

“…Melanoma cell line A375 is a homozygous genotype with BRAF-V600E [ 74 ] (Additional file 2 : Figure S5a). By using a mut-sgRNA, Tag-PCR assay [ 65 ] roughly displayed that the As Cas12a nucleases (WT, RKA, and HF) retained high editing selectivity at this site, as they did not cut wild-type but mutated sequence, whereas Cas9 recognized and cut both wild-type and mutated alleles (Additional file 2 : Figure S5b, c). Next, to more accurately assess the editing selectivity of the Cas-nucleases ( Sp Cas9-WT, As Cas12a-WT, and all the As Cas12a mutants in this study), Tag-seq experiments were performed by using the Cas9- and Cas12a-sgRNA (both contained WT- and mut-sgRNAs) in both BRAF +/+ HEK293T and BRAF V600E/V600E A375 cells (Fig.…”

“…Tag-seq experiments were performed and analyzed as previously described [ 65 ]. Briefly, HEK293T cells were transfected by PEI with 20 nM Tag, 1000 ng of Cas nuclease, and 1000 ng single sgRNA or a pool sgRNAs (30–50 ng/sgRNA) per well in a six-well plate.…”

“…Tag-PCR was used to roughly determine the editing events of the CRISPR-Cas systems, which reflected the insertion efficiency of the Tag at the editing sites [ 65 ]. Briefly, cell transfection was the same as the Tag-seq method.…”

Target-matched increased fidelity SpCas9 variants can edit any target without genome-wide off-target effects

“…Finally, the most important result of this study is that by employing target-matched IFNs we are able to ensure maximal specificity editing for practically any target sequence, that is accessible to WT SpCas9, without any genome-wide off-target effects. Several clever and effective genome-wide off-target detecting methods have been developed in vitro and in cellulo 11,20,[46][47][48][49][50][51][52][53][54][55][56][57][58] . While in vitro methods tend to report more off-target sites, they are prone to identifying a high number hits with uncertain relevance and require extensive validations.…”