CRISPR Technology Updates

CRISPR: The New Tool In The Gene Editing Revolution Explained

CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, is an integral part of a bacterial defense system. It is also the basis of the CRISPR-Cas9 system.

The CRISPR molecule is made up of short palindromic DNA sequences that are repeated along the molecule and are regularly-spaced. Between these sequences are “spacers”, foreign DNA sequences from organisms that have previously attacked the bacteria. The CRISPR molecule also includes CRISPR-associated genes, or Cas genes. These encode proteins that unwind DNA, and cut DNA, called helicases and nucleases, respectively.

The CRISPR immune system protects the bacteria from repeated virus attacks thru three steps:

1. Adaptation – When DNA from a virus invades the bacteria, the viral DNA is processed into short segments and is made into a new spacer between the repeats. These will serve as genetic memory of previous infections.

2. Production of CRISPR RNA – The CRISPR sequence undergoes transcription, including spacers and Cas genes, creating a single-stranded RNA. The resulting single-stranded RNA is called CRISPR RNA, which contains copies of the invading viral DNA sequence in its spacers.

3. Targeting – The CRISPR RNAs will identify viral DNA and guide the CRISPR-associated proteins to them. The protein then cleaves and destroys the targeted viral material.

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CRISPR-Cas9 allows researchers to perform the following:

Gene Knock-Out

Gene silencing using CRISPR starts with the use of a single guide RNA (sgRNA) to target genes and initiate a double stranded break using the Cas9 endonuclease. These breaks are then repaired by an innate DNA repair mechanisms, the non-homologous end-joining (NHEJ). However, NHEJ is error-prone and results in genomic deletions or insertions, which then translates into permanent silencing of the target gene.

DNA-Free Gene Editing

CRISPR can be used for DNA-free gene editing without the use of DNA vectors, requiring only RNA or protein components. A DNA-free gene editing system can be a good choice to avoid the possibility of undesirable genetic alterations due to the plasmid DNA integrating at the cut site or random vector integrations.

Gene Insertions or “Knock-ins”

The CRISPR-induced double-strand break can also be used to create a gene “knock-ins” by exploiting the cells’ homology-directed repair. The precise insertion of a donor template can alter the coding region of a gene. Previous studies have demonstrated that single-stranded DNA can be used to create precise insertions using CRISPR-Cas9 system.

Transient Gene Silencing

By modifying the Cas9 protein so it cannot cut DNA, transient gene silencing or transcriptional repression can also be done. The modified Cas9, led by a guide RNA, targets the promoter region of a gene and reduces transcriptional activity and gene expression. Transient activation or upregulation of specific genes can be effectively done.

For more info, get sample copy of this study here

References:

• isaaa.org
• Image Source : curethefuture.org
• theinsightpartners.com

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats

Repetitive DNA sequences, called CRISPR, were observed in bacteria with “spacer” DNA sequences in between the repeats that exactly match viral sequences. It was subsequently discovered that bacteria transcribe these DNA elements to RNA upon viral infection. The RNA guides a nuclease (a protein that cleaves DNA) to the viral DNA to cut it, providing protection against the virus. The nucleases are named “Cas,” for “CRISPR-associated.”

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Genome editing

In 2012, researchers demonstrated that RNAs could be constructed to guide a Cas nuclease (Cas9 was the first used) to any DNA sequence. The so-called guide RNA can also be made so that it will be specific to only that one sequence, improving the chances that the DNA will be cut at that site and nowhere else in the genome. Further testing revealed that the system works quite well in all types of cells, including human cells.

Implications

With CRISPR/Cas, it’s easy to disrupt a targeted gene, or, if a DNA template is added to the mix, insert a new sequence at the precise spot desired. The method has profoundly changed biomedical research, as it greatly reduces the time and expense of developing animal models with specific genomic changes. JAX scientists now routinely use the CRISPR/Cas system for this purpose in mice. And for human diseases with a known mutation, such as cystic fibrosis, it’s theoretically possible to insert DNA that corrects the mutation. There are clinical applications in human trials now, including for engineering T cells outside of the body for CAR-T cancer therapy and for editing retinal cells for leber’s congenital amaurosis 10, an inherited form of blindness.

Limitations

CRISPR/Cas is an extremely powerful tool, but it has important limitations. It is:

• difficult to deliver the CRISPR/Cas material to mature cells in large numbers, which remains a problem for many clinical applications. Viral vectors are the most common delivery method.
• not 100% efficient, so even the cells that take in CRISPR/Cas may not have genome editing activity.
• not 100% accurate, and “off-target” edits, while rare, may have severe consequences, particularly in clinical applications.

For more info, get sample copy of this study here

References:

• theinsightpartners.com
• jax.org

A simple guide to CRISPR, one of the biggest science stories of the decade

It could revolutionize everything from medicine to agriculture. Better read up now...

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology is a simple but powerful tool for genome editing. This tool enables life science researchers to easily edit DNA sequences and modify gene function. It has many potential applications include correcting genetic defects, treating and preventing the spread of diseases and improving crops. By delivering the CRISPR enzyme Cas9 nuclease coupled with synthetic guide RNA (gRNA) into a cell, the cell’s genome can be cut at a desired location, that allows existing genes to be removed or add new ones.

Increasing usage of CRISPR systems in microbiology, growing government and private investments on research and development of genome editing, rising prevalence of genetic disorders, and increases application of CRISPR/Cas9 technology to improve crop production drives the global CRISPR technology market. However, ethical issues associated with CRISPR and lack of skilled personnel restrain the global CRISPR technology market over the forecast period.

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The future of CRISPR

Just because recent cutting edge medical technologies have turned out to be much more difficult than initially hoped, that does not mean that CRISPR will not meet or exceed expectations. It is, however, a reminder that hype needs to be tempered with caution.

CRISPR is an amazingly powerful technology, and all the hype about its potential to cure genetic diseases, fight cancer, genetically modify organisms, and aid in genetics research are actually reasonable and well-founded. However, we also have to remember that organisms are complex machines, and genetics are also horrifically complex.

So while the technology itself works and continues to advance rapidly, that applications of that technology, especially in humans, are going to take time to carefully research. There are clear safety issues, and there is the possibility of unintended consequences. These are all solvable issues, but we need to proceed with care. So far it seems that we are.

We also need to be patient. I suspect CRISPR will deliver on much of its promise, and perhaps more quickly than most similar new technologies, but that is still likely to take longer than the public’s attention span. For now we have to wait and see how the research pans out.

For more info, get sample copy of this study here

References:

• sciencebasedmedicine.org
• vox.com
• theinsightpartners.com