transfection, isolation, and screening of the cenp-e knockout hela cells

CRISPR/Cas9 시스템을 사용한 Centromere-associated protein-e 유전자 편집

Engineered genome editing mediates the targeted modifications of genes in a variety of cells and organisms. In eukaryotes, site-specific mutagenesis can be introduced by the applications of sequence-specific nucleases that stimulate homologous recombination of target DNA1. In recent years, several genome editing technologies, including zinc finger nucleases (ZFNs)2,3, transcription activator-like effector nucleases (TALENs)4,5, and homing meganucleases6,7, have been engineered to cleave genomes at specific sites, but these approaches require complex protein engineering and redundant experimental procedures. Studies have shown that the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an efficient gene editing technology, which specifically mediates RNA-guided, site-specific DNA cleavage in a wide variety of cells and species8,9,10,11. The CRISPR/Cas9 gene knockout technology has revolutionized the fields of basic biology, biotechnology, and medicine12.
Bacteria and most archaea have evolved an RNA-based adaptive immune system that uses CRISPR and Cas proteins to identify and destroy viruses and plasmids13. Streptococcus pyogenes Cas9 (SpCas9) endonuclease contains the RuvC-like Holliday junction resolvase (RuvC) and His-Asn-His (HNH) domain, which can efficiently mediate sequence-specific, double-stranded breaks (DSBs) by providing a synthetic single-guide RNA (sgRNA) containing CRISPR RNAs (crRNA) and trans-activating crRNA (tracrRNA)14,15,16. DSBs can be repaired through the indel-forming non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway, which introduces multiple mutations, including insertions, deletions, or scar-less single nucleotide substitutions, in mammalian cells1,8. Both the error-prone NHEJ and the high-fidelity HDR pathway can be used to mediate gene knockout through insertions or deletions, which can cause frameshift mutations and premature stop codons10.
Kinesin-7 CENP-E is required for kinetochore-microtubule attachment and chromosome alignment during cell division17,18,19. Antibody microinjection20,21, siRNA depletion22,23, chemical inhibition24,25,26, and genetic deletion27,28,29 of CENP-E leads to chromosome misalignment, the activation of spindle assembly checkpoint and mitotic defects, which results in aneuploidy and chromosomal instability19,30. In mice, CENP-E deletion results in abnormal development and embryonic lethality at the very early stages of development27,29,31. Genetic deletion of CENP-E usually leads to chromosome misalignment and cell death26,27,29, which is an obstacle in studying the functions and mechanisms of the CENP-E proteins.
A recent study has established a conditional CENP-E knockout cell line using an Auxin-inducible CRISPR/Cas9 gene-editing method32, which enables rapid degradation of CENP-E proteins in a relatively short time33. However, to date, stable CENP-E knockout cell lines have not been established, which is an unresolved technical challenge in CENP-E biology. Considering genetic robustness34, genetic compensation responses35,36,37, and complex intracellular environments, as the direct consequences of complete deletion of CENP-E may be complex and unpredictable, it is important to establish CENP-E knockout cell lines for the investigation of mechanisms of chromosome alignment, spindle assembly checkpoint, and downstream signaling pathways.
The discovery and applications of CENP-E inhibitors are important for cancer treatment. To date, seven types of CENP-E inhibitors have been found and synthesized, including GSK923295 and its derivatives24,25, PF-277138,39, imidazo[1,2-a]pyridine scaffold derivatives40,41, compound-A42,43, syntelin44,45, UA6278446, and benzo[d]pyrrolo[2,1-b]thiazole derivatives47. Among these inhibitors, GSK923295 is an allosteric and efficient CENP-E inhibitor that binds to the motor domain of CENP-E and inhibits CENP-E microtubule-stimulated ATPase activity with a Ki of 3.2 &#177; 0.2 nM24,25. However, compared with the inhibitory effects of GSK923295 on cultured cancer cells, the therapeutic effects of GSK923295 in clinical cancer patients are not ideal48,49, which also raised concerns about the specificity of GSK923295 for CENP-E. Moreover, the specificity and side effects of other CENP-E inhibitors on the CENP-E proteins are key issues in cancer research.
In this study, we have completely knocked out the CENP-E gene in HeLa cells using the CRISPR/Cas9 system. Three optimized phenotype-based screening strategies have been established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, to improve the screening efficiency and success rate of CENP-E gene editing. Furthermore, CENP-E knockout cell lines can be used to test the specificity of candidate compounds for CENP-E.

저자 스포트라이트: CENP-E Knockout HeLa 세포 확립 – Kinesin-7 CENP-E 생물학 및 그 억제제를 연구하기 위한 새로운 접근 방식

CRISPR/Cas9 시스템을 사용한 Centromere-Associated Protein-E CENP-E-/- Knockout 세포주 생성

Genetic deletion of Kinesin-7 and CENP usually results in cell cycle arrest and cell death. The construction of stable CENP knockout cell lines is an important unresolved issue in CENP biology. In this study, we generated CENP knockout HeLa cells using a CRISPR/Cas9 system, and established three optimized screening strategies.Recently, several genome editing technologies, including zinc-finger nucleases, transcription activator-like effector nucleases, and how we make our nucleases, has been engineered to cleave genomes at a specific size. The CRISPR/Cas9 gene knockout technology has revolutionized basic biology, biotechnology, and medicine. CENP is essential for attaching kinetochores to microtubules and aligning chromosomes.Disrupting CENP, whether through antibody microinjection, sgRNA deletion, chemical inhibition, or genetic deletion, result in chromosome misalignment, mitotic defects, and often cell death, complicating the study of CENP protein mechanisms. In this study, three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment of phenotypes, and the fluorescent intensities of CENP proteins, which improved the screening efficiency and the experimental success rate. Importantly, CENP deletion results in chromosome misalignment, the abnormal location of BubR1 proteins, and mitotic defects.The interaction modes and the molecular mechanisms of CENP and its inhibitors are important research fields in cell biology and cancer research. In this study, the CENP knockout HeLa cell line has been established as a valuable tool for the validation of the specificity and the toxicity of CENP inhibitors.

CENP-E Knockout HeLa 세포의 형질주입, 분리 및 스크리닝

먼저 0.25%Trypsin-EDTA 용액을 HeLa 세포에 넣고 섭씨 37도에서 2-3분 동안 배양합니다. 피펫팅을 통해 세포를 부드럽게 해리하고 세포 통과를 위해 1:4의 비율로 새 플레이트에 파종합니다. 플라스미드를 HeLa 세포에 transfection하려면 검증된 PX458 sgRNA 플라스미드 1마이크로그램을 튜브 A.In 튜브 B에 있는 50 마이크로리터의 환원 혈청 배지와 혼합하고 2 마이크로리터의 transfection 시약과 50 마이크로리터의 환원 혈청 배지를 부드럽게 혼합하고 실온에서 5분 동안 배양합니다.그런 다음 튜브 A와 B의 내용물을 결합하고 실온에서 5분 더 배양합니다. 혼합물을 12웰 플레이트에 넣고 섭씨 37도에서 6시간 동안 세포를 배양합니다. 배양이 끝나면 배지를 새 DMEM 배지로 교체합니다.24시간 또는 48시간 후 형광 현미경으로 HeLa 세포를 검사하여 transfection 효율을 평가합니다. 0.25%트립신 EDTA를 사용하여 형질주입된 HeLa 세포를 완전히 해리하고 섭씨 37도에서 3-5분 동안 배양합니다. 그런 다음 Neubauer 챔버 또는 자동 세포 계수기를 사용하여 세포를 계수합니다.연속 희석 방법에 따라 각 transfection된 집단에 대해 세포를 3개의 개별 96웰 플레이트로 패팅합니다. 세포를 가습 인큐베이터에 1-2주 동안 다시 넣습니다. CENP-E Knockout의 표현형 기반 스크리닝 및 검증을 위해 5-7일 동안 24-웰 또는 12-웰 플레이트에서 세포를 해리하고 확장합니다.10배 및 20배 배율의 대물 렌즈가 있는 도립 현미경을 사용하여 더 작은 콜로니 직경을 가진 돌연변이 세포를 스크리닝합니다. 다음으로, 제조업체의 지침에 따라 Column Animal Genomic DNA Extraction Kit를 사용하여 DNA 추출을 위한 세포를 수확하고 중합효소 연쇄 반응을 설정합니다. 제조업체의 프로토콜에 따라 표적 DNA를 pMD18-T 벡터에 연결합니다.ligated plasmid를 유능한 DH5-Alpha 세포에 transfection하고 클론 선택을 위한 배양을 진행합니다. CENP-E Knockout의 유형을 식별하려면 제조업체의 지침에 따라 Plasmid Extraction Kit를 사용하여 plasmid DNA를 분리합니다. 다음으로, M13 forward 및 reverse primer를 사용하여 각 colony의 plasmid DNA에 대한 Sanger sequencing을 수행합니다.야생형 및 CENP-E 돌연변이 HeLa 세포를 24웰 플레이트의 12mm 유리 커버 슬립에 파종합니다. 전체 DMEM 배지를 제거하고 4% 파라포름알데히드와 PBS 용액을 사용하여 실온에서 10분 동안 세포를 고정합니다. 그런 다음 실온에서 5분 동안 DAPI로 핵을 염색합니다.형광 현미경을 사용하여 염색체 정렬 표현형을 기반으로 CENP-E 돌연변이 세포를 관찰하고 검증합니다. 콜로니의 표현형 스크리닝은 CENP-E Knockout 세포가 대조군에 비해 염색체 정렬 불량이 증가했음을 보여주었습니다.

Research

JoVE Journal

Biology

Transfection, Isolation, and Screening of the CENP-E Knockout HeLa Cells

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...

연구

생물학

Immunofluorescence Staining and High-Resolution Confocal Microscopy of CRISPR/Cas9-Mediated CENP-E Knockout HeLa Cells

CRISPR/Cas9 매개 CENP-E Knockout HeLa 세포의 면역형광 염색 및 고분해능 공초점 현미경

Chromosome Preparation and Karyotype Analysis of CRISPR/Cas9-Mediated CENP-E Knockout HeLa Cells

CRISPR/Cas9 매개 CENP-E Knockout HeLa 세포의 염색체 준비 및 핵형 분석