CRISPR Cas9 Cassettes

BioInnovatise CRISPR Team

Updated January 8, 2024

What Is A CRISPR Cassette?

A CRISPR cassette refers to a specific DNA sequence in the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system. The CRISPR cassette typically consists of repeated and spacer sequences.

Repeated sequences are short DNA segments that are identical or nearly identical and are interspersed with unique spacer sequences derived from the DNA of viruses or other foreign invaders that the organism has encountered.
Spacer sequences serve as a memory of past infections.

The CRISPR cassette, with its repeated and spacer sequences, plays a crucial role in guiding the Cas9 protein to the target DNA for editing.

What Are The Components Of A CRISPR Cassette?

A CRISPR cassette vector typically includes several components to facilitate the targeted modification of genes, known as the gene of interest (GOI). The main components of a CRISPR cassette vector include:

Promoter: A promoter initiates the transcription of the target gene. In the CRISPR system, a promoter is often used to drive the expression of the Cas9 gene, ensuring that the Cas9 protein is produced within the cell.
gRNA (guide RNA) expression cassette: The guide RNA is a crucial component that directs the Cas9 protein to the specific target DNA sequence for editing. The gRNA sequence is designed to be complementary to the target DNA sequence, guiding Cas9 to the precise location for editing.
Cas9 gene: Cas9 is a protein that cuts the DNA at the targeted site specified by the guide RNA.
Selectable marker: A selectable marker, such as an antibiotic resistance gene, is included to help identify cells that have successfully taken up the CRISPR cassette.
Origin of replication: The origin of replication is a DNA sequence that allows the CRISPR cassette vector to replicate within the host organism’s cells. This is important for maintaining and propagating the vector during cell division.
Polyadenylation signal (PolyA): A polyadenylation signal is included to terminate the transcription of the Cas9 gene or other components, ensuring proper processing of RNA molecules.
Backbone sequence: The backbone of the vector consists of additional DNA sequences that provide stability and structure to the vector. It may include plasmid elements necessary for cloning, propagation, and maintenance in a bacterial host.

Our CRISPR team is excited to bring your desired CRISPR cassette vector to life to advance your CRISPR Cas9 based research. Our team offers three different CRISPR cassettes to choose from, and we can always create a custom CRISPR cassette vector to match your research requirements. Learn more about our CRISPR Cas9 services.

Does My Choice of CRISPR Cassette Vector Affect My Plasmid DNA Integrity?

The CRISPR cassette vector itself does not inherently affect plasmid DNA integrity. However, the construction, handling, and storage of plasmid DNA, including CRISPR cassette vectors, can impact its integrity. Our team has compiled a brief list of factors that can affect your plasmid DNA construct:

Construction and cloning: The process of constructing a CRISPR cassette vector involves molecular biology techniques such as PCR, restriction enzyme digestion, and ligation. Learn more about our molecular cloning services.
Sequence accuracy: The accuracy of the cloned sequences, including the Cas9 gene, gRNA, and other elements, is essential. Errors or mutations in these sequences can affect the functionality of the CRISPR system and may impact plasmid integrity. Our cloning team performs sequencing QC tests to ensure your plasmid construct maintains high integrity.
Quality of DNA polymerase: The choice of DNA polymerase during PCR amplification can influence the fidelity of DNA replication.
Plasmid propagation: Plasmid DNA is typically propagated in bacterial hosts. Using appropriate bacterial strains and growth conditions, as well as ensuring the presence of the necessary antibiotic selection, helps maintain plasmid integrity during propagation. Our team has completed plasmid midi prep, plasmid maxi prep, and large scale plasmid prep to ensure researchers have enough high quality plasmid DNA for their research. Learn more about our plasmid prep services.
Endotoxin contamination: Plasmid preparations can sometimes contain endotoxins from bacterial hosts. However, our plasmid DNA constructs will always have a 1.8 Nucleic Acid 260/280 Ratio to ensure high quality endotoxin free plasmid DNA.

Can A Cassette Vector Be Introduced To A Lentiviral Vector?

Sure! Lentiviral vectors are commonly used as delivery vehicles for introducing genetic material into a wide range of cells. By incorporating the CRISPR components into a lentiviral vector, you can leverage the efficient delivery and integration capabilities of lentiviruses for CRISPR Cas9 genome editing. Learn more about our lentiviral packaging services. Our team would be happy to discuss your lentivirus based CRISPR research if you have any questions.

Let’s get started! Our CRISPR team is excited to bring your project to life. Learn more about our quick turnaround CRISPR Cas9 services. Our team performs CRISPR vector productions, sgRNA construct cloning services, cassette and donor vector construction design services, and CRISPR knock out and CRISPR knock in services.

Want to learn more about the latest in CRISPR genome-editing based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News continuously collect and publish the latest information on CRISPR based research.

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