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R&D Tools for Biotech Startups and Scaleups: How to Streamline your Crispr-Cas9 Workflows

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R&D Tools for Biotech Startups and Scaleups: How to Streamline your Crispr-Cas9 Workflows

Biotech startups and scaleups are at the forefront of innovation, using the latest technologies to create new life-saving products. In the constantly evolving field of molecular biology, new techniques and technologies are revolutionizing the way that TechBio companies develop new diagnostics and therapeutics. From gene editing to single-cell analysis, the past few decades have seen a rapid advancement in molecular biology, making it easier to uncover the mechanisms underlying disease. This is the first in our regular series of articles highlighting game-changing molecular biology techniques used in biotech R&D.

CRISPR-Cas9: Revolutionizing Gene Editing in Biotech

CRISPR-Cas9 is a revolutionary gene-editing technology that has taken the scientific community by storm. Biotech companies can use CRISPR-Cas9 to precisely target and manipulate DNA sequences with incredible ease and accuracy. The technology has completely changed the way scientists approach genetic research and has opened up new possibilities for treating genetic diseases, improving crop yields, and much more.

How CRISPR-Cas9 Edits Genes

CRISPR-Cas9 uses a guide RNA to direct the Cas9 nuclease enzyme to a specific DNA sequence. Once the Cas9 enzyme reaches its target DNA, it makes a double-stranded break in the DNA, allowing scientists to make precise modifications to the genetic material. The DNA can then be repaired using the cell's natural repair mechanisms, resulting in deletions that disrupt functional genes or insertions that introduce new sequences.

Challenges Biotech Companies Face When Using CRISPR-Cas9 

Though the CRISPR/Cas9 system holds great promise for treating cancer and genetic disorders, there are several major challenges that must be overcome. One of the biggest limitations is off-target modifications, where the Cas9 protein cleaves DNA at unintended sites. This can result in unwanted mutations and potentially lead to autoimmune diseases. Another challenge is the lack of techniques to safely and effectively deliver the CRISPR-Cas9 system to target cells and tissues. Researchers are actively working to find solutions to these challenges, such as improving guide RNA selection. There are some key tools and strategies that biotech companies can use to optimize the specificity of their gene editing system. 

How Biotech Companies Can Improve the Accuracy of CRISPR-Cas9 Gene Editing Workflows

Better Guide RNA Specificity

Improving guide RNA specificity can reduce off-target effects. By choosing target DNA with a unique sequence, biotechs are more likely to be able to design guide RNAs that bind specifically to that DNA sequence and not to off-target sequences. As the number of mismatches between a guide RNA and the target DNA increases, the total number of potential off-target sites also dramatically increases. Off-target sites with three or fewer mismatches are significantly more likely to be cleaved than sites with more mismatches. Therefore, selecting the most unique target site possible is a valuable strategy to improve guide RNA specificity. 

Using a Cas9 Engineering

Biotech labs can also reduce off-target effects by replacing the Cas9 nuclease, which causes double-stranded DNA breaks, with a Cas9 nickase, which causes single-stranded DNA breaks. Cas9 nickases cause less damage in target DNA than Cas9 nucleases and also result in less off-target DNA damage. 

Startups can also use another recent protein engineering approach, Cas9 fusion proteins, which are much more specific than standard Cas9 nucleases. For example, SpCas9MT-pDBD fusion proteins have a mutated Cas9 with attenuated PAM-binding (SpCas9MT) linked to a programmable DNA-binding domain (pDBD) such as zinc finger or transcription activator-like effector nuclease (TALEN) that target nearby genomic DNA. SpCas9MT-pDBD uses two independent DNA-binding events, and is, therefore, up to 150-fold more specific than standard Cas9. 

Another successful approach has been to alter the PAM recognition sequence of Cas9. Since the PAM is perhaps the most stringent determinant of Cas9 specificity, using a longer or less abundant PAM can improve genome-wide specificity.  

Overcoming Delivery Challenges

The delivery of CRISPR-Cas9 technology into target cells and tissues is challenging. Biotechs can overcome the packaging challenge by splitting the Cas9 protein into two smaller AAV vectors (AAV-split-Cas9), which can reduce the risk of off-target effects and increase efficiency of delivery. Another option is using ribonucleoprotein (RNP) complexes, like recombinant CRISPR-Cpf1 Ribonucleoprotein (CRISPR-Cpf1-RNP), which has been shown to suppress off-target activity in mouse cells.

How Scispot Can Help Biotechs Improve CRISPR-Cas9 Workflows 

Online tools and applications have been developed for in silico design of guide RNAs, delivery vectors and prediction of their off-target sites. Scispot’s platform includes Sequence Designer, which biotech startups and scaleups can use to efficiently design RNA for CRISPR workflows. Scispot can also help biotechs create templates for protein engineering or vector development, and streamline workflows by recording all results, modifications, and deviations. With Scispot’s no-code, fully configurable digital platform, biotechs can track progress, assign tasks, and collaborate with team members. You can manage every step of your CRISPR-Cas9 workflow in Scispot’s flexible, regulatory-compliant platform. 

Request a demo to learn more about how Scispot can help you leverage the power of CRISPR-Cas9 gene editing. 

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