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

Satoshi Watanabe,Kazuchika Miyoshi,Takayuki Sakurai,Masahiro Sato,Shingo Nakamura

doi:10.3390/ijms19041075

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.

Highlights

Gene targeting technology, which results in knockout (KO), is laborious and time-consuming
The cells judged as −/− exhibited no staining, while those judged as +/− or +/+ were positive for staining with the lectin. These results suggest the usefulness of IB4SAP for the enrichment of bi-allelic KO clones after transfection with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9)-related DNA + EndoGalC expression vector
Cells surviving after IB4SAP treatment must be those exhibiting strong expression of EndoGalC, its expression was transient

Summary

Introduction

Gene targeting technology, which results in knockout (KO), is laborious and time-consuming. It comprises several steps that include the construction of a target vector for successful gene targeting, transfection of embryonic stem (ES) cells with the constructed vector, selection of transfectants with appropriate drugs, molecular biological screening of successfully targeted clones, blastocyst injection with the targeted ES clone to generate chimeric mice, acquiring heterozygous mice by mating with normal mice, and generating homozygous KO mice by mating between the resulting heterozygous mice. It typically takes 2 years or more to obtain homozygous mice from the transfection of the target vector. Even in this case, it takes more than 2 years to obtain homozygous animals [1]

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