Comparison of gene disruption induced by cytosine base editing-mediated iSTOP with CRISPR/Cas9-mediated frameshift.

ABSTRACT:

Objectives

Recently developed CRISPR-dependent cytosine base editor (CBE), converting four codons (CAA, CAG, CGA and TGG) into stop codons without DNA double-strand breaks (DSB), serves as an efficient gene disruption strategy besides uncontrollable CRISPR-mediated frameshift. However, the detailed difference of gene knockout between the two systems has not been clarified.

Materials and methods

Here, we selected some sgRNAs with different position background, then HEK293T cells were transfected with CBE/Cas9 plasmids together with sgRNAs. GFP-positive cells were harvested by fluorescence-activated cell sorting (FACS) 48 hours after transfection. Genomic DNA was collected for deep sequencing to analyse editing efficiency and genotype. RNA and protein were extracted to analyse

SUBMITTER: Dang L 

PROVIDER: S-EPMC7260061 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature