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Method Article
Here, we present a protocol that integrates CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to identify candidate genes that are crucial for the abnormal proliferative capacity of hematopoietic stem and progenitor cells.
Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.
Hematopoiesis, which produces all lineages of blood cells throughout life, is maintained by a small population called hematopoietic stem cells (HSCs). HSCs maintain themselves by self-renewal and produce various committed progenitors that give rise to fully differentiated functional blood cells1,2,3. This process is tightly regulated. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) impedes normal differentiation, confers abnormal proliferative ability, and ultimately transforms HSPCs into leukemia-initiating cells (LICs)4,5,6,7. Identifying and confirming leukemogenic mutations still remains a big challenge for cancer research.
Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been developed and become an efficient and precise gene-editing method, consisting of the Cas9 nuclease and single-guide RNA (sgRNA)8. The sgRNA guides the Cas9-sgRNA complex to a specific genomic location through RNA-DNA complementarity, where the Cas9 nuclease induces double-strand breaks. The broken DNA is subjected to endogenous gene editing mechanisms either through precise homology-directed repair in the presence of homology templates or mismatch-prone, non-homologous end-joining, which leads to small insertions or deletions9. Currently, the CRISPR/Cas9 system has become a powerful tool for targeted gene editing and transcriptional regulation10.
Here, we utilized CRISPR/Cas9 technology to introduce genetic alterations in HSPCs and then performed the Cell Counting Kit-8 (CCK-8) assay to investigate whether the introduced mutations were critical for the abnormal proliferation of HSPCs. Specifically, bone marrow cells enriched for HSPCs were harvested from Cas9 mice followed by viral transfection with sgRNAs targeting candidate genes. Then, the CCK-8 assay was performed to determine whether the mutations conferred an increased proliferative ability to the transfected HSPCs. Finally, the mutations introduced by CRISPR/Cas9 were confirmed by Tracking of Indels by Decomposition (TIDE)11 or targeted deep sequence12. This protocol provides a powerful tool for assessing cancerous mutations and potential therapeutic targets for leukemia.
All animal experiments conducted under this protocol were approved by the Animal Care and Welfare Committee of Beijing University of Chinese Medicine.
1. Construction of sgRNA plasmids targeting candidate genes (4 - 5 days)
2. Lentiviral packaging (2 - 3 days)
3. Bone marrow cell extraction and culture (12 - 13 days)
NOTE: All mouse lines were maintained in a pure C57BL/6 genetic background. LSL-Cas9 mice (T002249) were crossed with Mx1-Cre mice13 to generate LSL-Cas9/+; Mx1-Cre/+ mice. Here, we collected bone marrow cells from Mx1-Cre/+; LSL-Cas9/+ mice and their littermates.
4. Lentiviral transfection of bone marrow cells (2 - 3 days)
5. Perform in vitro CCK-8 assay (3 - 4 days)
6. Gene editing verification (5 - 7 days)
Effect of Trp53 knockout on the proliferative capacity of mouse HSPCs
Here, we used Trp53 knockout as an example to demonstrate this protocol. To investigate the effect of Trp53 knockout on the proliferative capacity of mouse HSPCs, we employed lentiviral transfection to introduce Trp53 sgRNA into Cas9-expressing bone marrow cells enriched for HSPCs after 5-FU treatment. Simultaneously, unrelated sgRNAs were transfected into bone...
CRISPR/Cas9 technology has been widely used in biomedical research due to its user-friendly nature and high efficiency, facilitating application such as gene function screening18, disease modeling19, immunotherapy20, and live cell labeling and imaging21. Recent studies have conducted large-scale CRISPR screening to identify novel therapeutic targets across various cancer types, including leukemia22...
The authors have no conflicts of interest to disclose.
We would like to thank Beijing University of Chinese Medicine for use of its shared services to complete this research. This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (No. 31701280 to J. Zhang).
Name | Company | Catalog Number | Comments |
0.45 μm microfiltration membrane | Sartoriu | 16533K-1 | |
1.5 mL EP tube | LABLEAD | MCT-150-CLD-1 | |
10 cm dish | Corning | 430167 | |
15 mL Tube | LABLEAD | LXG1500 | |
1 mL sterile syringe | |||
2x Accurate Taq Master Mix (dye plus) | Accurate Biology | AG11010 | For colony PCR |
50 mL Tube | LABLEAD | LXG5000 | |
5-Fluorouracil | Sigma | F6627-1 | |
6-well plate | Corning | 3516 | |
70 µm cell sieve | Falcon® | 352350 | |
96-well plates | Corning | 3599 | |
ACK Lysis Buffer | Leagene | CS0001 | |
Agar | BIODEE | DE0010 | |
Ampicillin | Solarbio | A8180 | |
Applied Biosystems Thermal Cyclers for PCR | Thermofisher | ||
Cell Counting Kit-8 | Bimake | B34304 | |
Centrifuge | eppendorf | Centrifuge 5810R | |
CO2 Incubator | Thermo SCIENTIFIC | ||
DMEM | Gibco | C11995500BT | |
DNA Clean&Concentrator-5 | ZYMO | D4014 | |
Dneasy Blood & Tissure Kit | Qiagen | 69504 | |
Esp3I (BsmbI) | NEB | R0580L | |
Fetal Bovine Serum | ExCell | FSP500 | |
GraphPad Prism 9.3 | |||
HEK-293T | ATCC;Virginia,USA | ||
HEPEs | Sigma | V900477 | |
KOD-Plus-Neo | TOYOBO | KOD-401 | |
lentiGuide-Puro | Addgene | 52963 | backbone vector |
LSL-Cas9 mice | Model Animal Research Center of NANJING UNIVERSITY | T002249 | |
microplate absorbance spectrophotometer | BIO-RAD | xMark™ | |
Nacl | Rhawn | R019772 | |
PBS | Solarbio | P1003 | |
PEI | Proteintech | B600070 | |
Penicillin-Streptomycin Solution, 100x | Solarbio | P1400 | |
pMD2.G plasmid | Addgene | 12259 | |
polybrene | Beyotime | C0351 | |
Polyinosinic-polycytidylic acid sodium salt | Sigma | P1530 | |
psPAX2 | Addgene | 12260 | |
QIAquick PCR Purification Kit | Qiagen | 28104 | |
RecombinantMurineIL-3 | Peprotech | 213-13-10ug | |
RecombinantMurineIL-6 | Peprotech | 216-16-2ug | |
RecombinantMurineSCF | Peprotech | 250-03-10ug | |
Regular agarose | BIOWEST | BY-R0100 | |
Shrimp Alkaline Phosphatase (rSAP) | NEB | M0371S | |
SpectraMax QuickDrop Ultra-Micro Spectrophotometer | MOLECULAR DIVICES | ||
Stbl3 Competent cells | BioMed | BC108-01 | |
T4 Polynucleotide Kinase | NEB | M0201L | |
T4-Ligase | Takara | 2011B | |
TIANprep Rapid Mini Plasmid Kit | TIANGEN | DP105-03 | |
Trypan Blue solution | LABLEAD | C0040-100ml | |
Trypsin-EDTA | Gibco | 25200072 | |
Tryptone | OXOID | LP0042 | |
WEHI-CM | Nanjing Fuksai Biotechnology | CBP50079 | |
YEAST EXTRACT | OXOID | LP0021 |
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