The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones

Abstract

Recent advances in genome editing systems such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) have facilitated genomic modification in mammalian cells. However, most systems employ transient treatment with selective drugs such as puromycin to obtain the desired genome-edited cells, which often allows some untransfected cells to survive and decreases the efficiency of generating genome-edited cells. Here, we developed a novel targeted toxin-based drug-free selection system for the enrichment of genome-edited cells. Cells were transfected with three expression vectors, each of which carries a guide RNA (gRNA), humanized Cas9 (hCas9) gene, or Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene. Once EndoGalC is expressed in a cell, it digests the cell-surface α-Gal epitope, which is specifically recognized by BS-I-B4 lectin (IB4). Three days after transfection, these cells were treated with cytotoxin saporin-conjugated IB4 (IB4SAP) for 30 min at 37 °C prior to cultivation in a normal medium. Untransfected cells and those weakly expressing EndoGalC will die due to the internalization of saporin. Cells transiently expressing EndoGalC strongly survive, and some of these surviving clones are expected to be genome-edited bi-allelic knockout (KO) clones due to their strong co-expression of gRNA and hCas9. When porcine α-1,3-galactosyltransferase gene, which can synthesize the α-Gal epitope, was attempted to be knocked out, 16.7% and 36.7% of the surviving clones were bi-allelic and mono-allelic knockout (KO) cells, respectively, which was in contrast to the isolation of clones in the absence of IB4SAP treatment. Namely, 0% and 13.3% of the resulting clones were bi-allelic and mono-allelic KO cells, respectively. A similar tendency was seen when other target genes such as DiGeorge syndrome critical region gene 2 and transforming growth factor-β receptor type 1 gene were targeted to be knocked out. Our results indicate that a combination of the CRISPR/Cas9 system and targeted toxin technology using IB4SAP allows efficient enrichment of genome-edited clones, particularly bi-allelic KO clones.

Application of CRISPR-Cas9 gene editing for congenital heart disease

“…embryonic stem cells using CRISPR-Cas9 to validate and confirm the role of this protein in these processes. [106][107][108]…”

“…CRISPR-Cas9 knockout of Tbx1 in murine stem cells (E14-Tg2a) enabled chromatin remodeling studies and transcriptome analyses to understand the pathogenesis of this syndrome [ 105 ]. Similarly, the putative adhesion receptor protein DGCR2, which has shown muscular defects as well as the risk for developing schizophrenia in DiGeorge syndrome, was deleted in mouse TT2 embryonic stem cells using CRISPR-Cas9 to validate and confirm the role of this protein in these processes [ 106 - 108 ].…”

Development of a Novel Pipette Tip-Aided Cell Cloning Method for The Effective Isolation of Genome-Edited Porcine Cell

“…Testis-derived porcine fibroblasts [27] were cultured in Dulbecco's modified Eagle's medium or Ham's F-12 medium (#048-29785; Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (#A5955; Sigma-Aldrich Co. Ltd., St. Louis, MO, USA) [hereafter referred to as the PF medium] at 37 °C in 5% CO2 atmosphere.…”

“…This query could be resolved by referring to a few previous reports. Watanabe et al [27] employed a paper method [3] for isolating colonies and demonstrated the successful destruction of the DiGeorge syndrome critical region gene 2 (Dgcr2) locus in mouse ESCs after co-transfection with the vectors carrying the Cas9 gene and the gRNA (targeted to Dgcr2), introduction of the gene encoding endo--galactosidase (capable of cleaving the -Gal epitope), and subsequent selection using IB4SAP. Furthermore, the transforming growth factor-β receptor type 1 (TGFβRI) gene was successfully destroyed in porcine adipocyte precursor cells (PAPCs) using similar technology.…”

Transplacental Gene Delivery (TPGD) as a Noninvasive Tool for Fetal Gene Manipulation in Mice

“…Among currently available genome editing tools, CRISPR/Cas9 is widely used to manipulate GOI in a variety of cells and organisms [74,75,76,77,78,79]. It requires the use of synthetic gRNAs that bind to specific chromosomal DNA sites with Cas9 endonuclease [80,81].…”