PCR-based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopietic Cell Lines

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This paper describes how the PCR is a Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines. Hematopoietic cell lines are responsible for producing all types of blood cells, including red blood cells (erythrocytes), white blood cells (leukocytes), and platelets, by differentiating from a single “hematopoietic stem cell” which has the ability to self-renew and generate new blood cells throughout a person’s life to maintain a healthy blood supply; essentially, they are the foundation for the body’s blood cell production process. CRISPR is a gene editing technology that is also found in the Covid Vaccines. Also, this paper mentions the use of HEK293T Cells that possess the SV40 large T antigen, allowing them to generate recombinant proteins using plasmid vectors that carry the SV40 promoter. SV40 is known to cause Cancer. This paper states that NO INFORMED CONSENT IS NEEDED FOR THIS TECHNOLOGY TO BE USED.

(Summary Below)

PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines

https://www.mdpi.com/2072-6694/15/17/4263

• This study presents a PCR-based method for creating CRISPR constructs, successfully used to edit IDH2 and MYBL2 genes in hard-to-transfect leukemia cells.

• This technique offers a simple, viral-free alternative for gene modification in these cells, achieving comparable efficiency to ribonucleoprotein methods without off-target effects.

• This study develops a new method for efficiently introducing CRISPR/Cas9 technology into difficult-to-transfect hematopoietic cells.

• A fusion PCR protocol creates constructs expressing single guide RNAs with an EGFP reporter.

• The method’s effectiveness is demonstrated by targeting IDH2 and MYBL2 genes in NB4 and HL60 cell lines, and its efficiency is compared to ribonucleoprotein complexes.

• The approach offers a cost-effective gene editing strategy.

• CRISPR/Cas9 gene editing in hematological research, using lentiviral delivery, shows high NHEJ mutation rates but low HDR efficiency.

• A new “hit-and-run” strategy improves gene targeting efficiency, particularly in challenging cell lines like NB4 and HL60, by using a streamlined PCR protocol and achieving higher transfection rates than standard CRISPR plasmids.

• A cost-effective CRISPR/Cas9 construct generation method using fusion PCR was developed and tested in HEK293, NB4, and HL60 cells.

• Mutagenesis frequencies ranged from 2% to 6.3%, primarily due to NHEJ.

• No off-target effects were detected.

• The method’s versatility allows application in various cell types.

• This research used two ISCIII grants (CP22/00028 and PI22/01371), co-funded by the EU.

• Data is publicly available on the European Nucleotide Archive (PRJEB54077).

• No IRB statement or informed consent is needed, and there are no conflicts of interest..

If you want to learn more about CRISPR/Cas9 gene editting technology here’s a search result on YouTube…several short videos available: https://www.youtube.com/results?search_query=crispr+cas9+explained