CRISPR/Cas9 & Cas9 Variants

The enabling technology that changed how we study biology

As a company that was founded before CRISPR/Cas9 became a commercial genome editing tool—we were one of the first to license its use in cells and rodents in 2014—we can attest to the game-changing power of the technology. Suddenly, customizing genomes and engineering cell lines became a straightforward and reliable exercise that most molecular biology labs could do.

Now that we’re over a decade past its initial discovery, CRISPR/Cas9 continues to be an incredibly valuable tool, with new Cas9 variants and novel CRISPR-like systems expanding what the technology can accomplish.

Here, we review the basics of the CRISPR/Cas9 system and the Cas9 variants—CRISPRi and CRISPRa—that we use at Applied StemCell to enable promoter studies and cancer research.

CRISPR/Cas9 basics

Cas9 is a site-specific nuclease that uses a small guide RNA to target the Cas9 nuclease to a specific sequence in the genome. In bacteria, Cas9 uses two RNAs, a CRISPR RNA (crRNA) that targets the nucelase to a specific sequence and a transactivating CRISPR RNA (tracrRNA) that binds to the crRNA and activates the CAS9 nuclease. For convenience in the lab, the crRNA and tracrRNAs are fused by a linker into a single guide RNA (gRNA).

Wild-type Cas9 creates double stranded breaks in the genome that can be repaired using non-homologous end joining (NHEJ)—creating an indel (insertion/deletion)—or via homologous recombination with a donor plasmid (homology directed repair (HDR)) to create knock-ins or point mutations.

When and when not to use CRISPR/Cas9

CRISPR/Cas9 is great for:

• Making or correcting point mutations
• Knocking out genes through frameshifts, indels, and point mutations
• Knocking in less than 5 kb of DNA
• Basic research where you can tolerate off-target effects
• Genome editing dividing cells

CRISPR/Cas9 is challenging for:

• Knocking in DNA larger than 5 kb
• Efficiently knocking in DNA
• Non-dividing cells, because it relies on homologous recombination
• Therapeutic use and other applications where safety is critical and off-target events cannot be tolerated
• Commercial development due to the complex IP landscape and the need for licenses from multiple organizations

Cas9 variants—expanding the power of CRISPR

By modifying the Cas9 protein, scientists have turned an already powerful technology into one that works better and does more. At Applied StemCell, we leverage a number of Cas9 variants to ensure that we achieve your goals. Furthermore, we will always recommend the most appropriate technology for your application based on our extensive cell line engineering experience.

Improving efficiency and accuracy with Cas9 variants

Off-target events and low edit efficiency are two of the biggest challenges when working with CRISPR/Cas9.

Fusion proteins can help.

Enhanced Knock-in Efficiency

• Cas9-POLD3, a fusion of Cas9 to the POLD3 protein, a subunit of the DNA Polymerase Delta 3, increases knock-in efficiency by speeding up the initiation of DNA repair.¹
• Cas9-CtIP, a fusion of Cas9 to the CtIP protein, which plays a role in HDR initiation, increases efficiency by stimulating HDR.²

Reduced off-target events

By stimulating HDR, both fusion proteins reduce NHEJ, thus reducing the occurrence of random repair and off-target edits.

Controlling gene expression with Cas9 variants

The ability to direct an enzyme to a specific genomic location by simply creating a guide RNA is what makes the CRISPR/Cas9 system so powerful.

Knocking out Cas9’s nuclease activity and fusing the protein to activators and repressors turns a gene editing enzyme into a site-directed transcription control agent.

Repressing transcription

CRISPRi fuses a catalytically inactive Cas9 to transcriptional repressors, such as KRAB (Krüppel-associated box) to provide site-specific silencing of gene expression.³

Activating transcription

CRISPRa fuses a catalytically inactive Cas9 to transcriptional activators, such as the VPR system (VP64-p65-Rta), to assemble gene transcription machinery at a specific site.⁴

Applications of Cas9 variants

Using Cas9 variants expands what the Applied StemCell team can help you accomplish. Here are just a few examples:

Cancer Research

• Explore gene function
• Study promoters
• Map genetic pathways and networks
• Build cell-based disease models with confidence

Drug Discovery

• Engineer cell lines for high-throughput screening
• Validate drug targets
• Explore disease physiology
• Elucidate a drug’s mechanism of action

Cell-based Models of Disease

Study disease in relevant cell types with full confidence that the phenotype you observe is not due to an off-target mutation, whether you are knocking in a functional gene, knocking out a gene, or exploring mutations in non-coding regions.

Functional Genomics

Explore gene function and map out gene interactions, genetic pathways, and complex gene networks with clarity and confidence.