Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs

Abstract

Hemophilia B (HB) is an X-linked recessive bleeding disorder, caused by F9 gene deficiency. Gene therapy combined with the CRISPR/Cas9 technology offers a potential cure for hemophilia B. Now the Cas9 nickase (Cas9n) shows a great advantage in reducing off-target effect compared with wild-type Cas9. In this study, we found that in the multicopy ribosomal DNA (rDNA) locus, the homology directed recombination (HDR) efficiency induced by sgRNA-Cas9n was much higher than sgRNA-Cas9, meanwhile without off-target in six predicted sites. After co-transfection into mESCs with sgRNA-Cas9n and a non-viral rDNA targeting vector pMrnF9, harboring the homology donor template and the human F9 expression cassette, a recombination efficiency of 66.7% was achieved and all targeted clones were confirmed to be site-specific integration of F9 in the rDNA locus by PCR and southern blotting. Targeted mESCs retained the main pluripotent properties and were then differentiated into hepatic progenitor like cells (HPLCs) and mature hepatocytes, which were characterized by hepatic markers and functional assays. Importantly, the differentiated cells could transcribe exogenous F9 and secrete coagulation factor IX (FIX) proteins, suggesting active transcription and stable inheritance of transgenes in the rDNA locus. After intrasplenical transplantation in severe combined immune deficiency (SCID) mice, targeted HPLCs could survive and migrate from spleen to liver, resulting in secretion of exogenous FIX into blood. In summary, we demonstrate an efficient and site-specific gene targeting strategy in rDNA locus for stem cell-based gene therapy for hemophilia B.

An effective double gene knock‐in strategy using small‐molecule L755507 in the medaka fish (Oryzias latipes)

“…Thus, this is the first report demonstrating that L755507 promotes HDR events in vivo. In contrast, we did not observe a significant increase in the that, unlike DNA DSB-dependent strategies using wild-type Cas9, the nicking-based approach via Cas9n prevented the activation of NHEJ in medaka as well as other organisms or cells (Gao et al, 2017;Murakami et al, 2020;Paulsen et al, 2017;Y. Wang et al, 2018).…”

CRISPR/Cas9 nickase‐mediated efficient and seamless knock‐in of lethal genes in the medaka fish Oryzias latipes

“…The Cas9n D10A and Cas9n H840A are the variants of Cas9 containing a single amino acid substitution, which permit generation of site‐specific nicks on a gRNA complementary or non‐complementary DNA single strand, respectively. The Cas9n‐based approach has been reported to be available for the HDR‐mediated knock‐in in human or mouse cells and cattle, which contributed to circumvent unwanted NHEJ‐mediated gene disruptions (Gao et al., 2017; Paulsen et al., 2017; Wang et al., 2018). In medaka, several studies have reported the successful knock‐in mediated by wild‐type Cas9‐based approaches (Gutierrez‐Triana et al., 2018; Murakami et al., 2017; Watakabe et al., 2018); however, no studies have achieved DSBs‐free knock‐in with the Cas9n in medaka and other teleost fish.…”

“…The Cas9n-based approach has been reported to be available for the HDR-mediated knock-in in human or mouse cells and cattle, which contributed to circumvent unwanted NHEJ-mediated gene disruptions (Gao et al, 2017;Paulsen et al, 2017;Wang et al, 2018).…”

A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila

“…nD10A has been used to generate paired DNA nicks and efficiently disrupts genes in Drosophila and cell culture (Gopalappa et al, 2018; Port et al, 2014). Nicks induced by nD10A promoted higher HDR rates than nH840A, where HDR was almost undetectable (Bothmer et al, 2017; Hyodo et al, 2020; Mali et al, 2013; Wang et al, 2021, 2018). Furthermore, nD10A can boost specificity while reducing off-target effects since it requires two gRNAs that target complementary DNA strands to either disrupt a gene function or trigger HDR.…”