CRISPR Cas9: The Art of Genome Editing

Cas9, an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, has the faculty to memorize, interrogate and cleave foreign DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking whether it is complementary to the 20 basepair spacer region of the guide RNA. As an engineering tool inducing site-directed double strand breaks in DNA, the Cas9 protein is becoming prominent in the field of genome editing.

A wide array of spinal cord injuries

A wide array of spinal cord injuries

A wide array of spinal cord injuries

A wide array of spinal cord injuries

The term spinal cord injury (SCI) produces images in the untrained, non-scientific mind of a back problem, perhaps a spine surgery gone wrong or a celebrity paralyzed in a horse riding accident. The reality is that those with SCI have a brain that can’t fully communicate with their bodies. The extent of the disruption in this response system along the human back dictates the extent of the impaired function, from minimized skin sensation to full body paralysis.

Recent research outcomes, addressed in a recent article written by Nisha Kaul Cooch, Ph.D. for the Michelson Medical Research Foundation, demonstrate progress in finding new modes of addressing the injury as well as its effects. That is, scientists are finding ways of both reversing the actual spinal cord injury, and improving bodily functions impaired by the injury, such as muscle function.

CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]The level of Spinal Cord Injury (SCI) determines what parts of the body might be affected by paralysis and loss of function. The level of injury refers to the lowest point on the spinal cord where there is a decrease or absence of motor and/or sensory function. Generally speaking, the higher the spinal cord injury, the more effect the injury has on movement and/or feeling. For example, an injury of the cervical spinal cord may result in full paralysis and make it impossible to breathe without a respirator, while an injury of the lumbar spinal cord may result in paralysis or weakness in the legs and cause some loss of body function in the lower extremities. 
 
 

CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the cervical spinal cord (levels C1-C8) causes quadriplegia (also called tetraplegia), which refers to paralysis or weakness in both arms and legs. All parts of the body located below the neck may be affected. Involuntary functioning, such as breathing, regulating body temperature, and sweating may be impaired, necessitating a respirator and other mechanical devices. A person with quadriplegia may not be able to sense touch (or other sensations) may lose bladder and bowel control, and may experience sexual dysfunction. 
 
 
 
 
 

CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the thoracic spinal cord (levels T1-T12) causes paraplegia, which means paralysis or weakness in the legs. Depending upon where the injury is located on the thoracic spinal cord, an individual with this level of SCI may also experience weakness in their torso, although will generally possess good control of their hands. These injuries may also result in loss of sensation, loss of bladder and bowel control, as well as sexual dysfunction. Due to the rib cage, thoracic spinal cord injuries occur less often, as the rib cage offers protection from such injuries. 
 
 
 
 
 

CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the lumbar or sacral spinal cord (L1-L5) causes paraplegia, again referring to paralysis or weakness of the legs. Because of the lower location of this injury, upper body functions are usually not affected. However, a person with a lumbar SCI may experience the loss of many of the sensory functions associated with thoracic spinal cord injuries. 
 
 
 
 
 
 
 
 
 
 

CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the sacral spinal cord (S1-S4) is rare and generally causes loss of bladder and bowel function as well as sexual dysfunction. Some sacral injuries can result in weakness or paralysis of the hips and legs. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]The level of Spinal Cord Injury (SCI) determines what parts of the body might be affected by paralysis and loss of function. The level of injury refers to the lowest point on the spinal cord where there is a decrease or absence of motor and/or sensory function. Generally speaking, the higher the spinal cord injury, the more effect the injury has on movement and/or feeling. For example, an injury of the cervical spinal cord may result in full paralysis and make it impossible to breathe without a respirator, while an injury of the lumbar spinal cord may result in paralysis or weakness in the legs and cause some loss of body function in the lower extremities.

CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the cervical spinal cord (levels C1-C8) causes quadriplegia (also called tetraplegia), which refers to paralysis or weakness in both arms and legs. All parts of the body located below the neck may be affected. Involuntary functioning, such as breathing, regulating body temperature, and sweating may be impaired, necessitating a respirator and other mechanical devices. A person with quadriplegia may not be able to sense touch (or other sensations) may lose bladder and bowel control, and may experience sexual dysfunction. 

CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the thoracic spinal cord (levels T1-T12) causes paraplegia, which means paralysis or weakness in the legs. Depending upon where the injury is located on the thoracic spinal cord, an individual with this level of SCI may also experience weakness in their torso, although will generally possess good control of their hands. These injuries may also result in loss of sensation, loss of bladder and bowel control, as well as sexual dysfunction. Due to the rib cage, thoracic spinal cord injuries occur less often, as the rib cage offers protection from such injuries.

CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the lumbar or sacral spinal cord (L1-L5) causes paraplegia, again referring to paralysis or weakness of the legs. Because of the lower location of this injury, upper body functions are usually not affected. However, a person with a lumbar SCI may experience the loss of many of the sensory functions associated with thoracic spinal cord injuries. 
 
 
 
 
 

CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the sacral spinal cord (S1-S4) is rare and generally causes loss of bladder and bowel function as well as sexual dysfunction. Some sacral injuries can result in weakness or paralysis of the hips and legs. 
 
 
 
 
 
 
 
 
 
 

The level of Spinal Cord Injury (SCI) determines what parts of the body might be affected by paralysis and loss of function. The level of injury refers to the lowest point on the spinal cord where there is a decrease or absence of motor and/or sensory function. Generally speaking, the higher the spinal cord injury, the more effect the injury has on movement and/or feeling. For example, an injury of the cervical spinal cord may result in full paralysis and make it impossible to breathe without a respirator, while an injury of the lumbar spinal cord may result in paralysis or weakness in the legs and cause some loss of body function in the lower extremities.CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the cervical spinal cord (levels C1-C8) causes quadriplegia (also called tetraplegia), which refers to paralysis or weakness in both arms and legs. All parts of the body located below the neck may be affected. Involuntary functioning, such as breathing, regulating body temperature, and sweating may be impaired, necessitating a respirator and other mechanical devices. A person with quadriplegia may not be able to sense touch (or other sensations) may lose bladder and bowel control, and may experience sexual dysfunction.CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the thoracic spinal cord (levels T1-T12) causes paraplegia, which means paralysis or weakness in the legs. Depending upon where the injury is located on the thoracic spinal cord, an individual with this level of SCI may also experience weakness in their torso, although will generally possess good control of their hands. These injuries may also result in loss of sensation, loss of bladder and bowel control, as well as sexual dysfunction. Due to the rib cage, thoracic spinal cord injuries occur less often, as the rib cage offers protection from such injuries.CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the lumbar or sacral spinal cord (L1-L5) causes paraplegia, again referring to paralysis or weakness of the legs. Because of the lower location of this injury, upper body functions are usually not affected. However, a person with a lumbar SCI may experience the loss of many of the sensory functions associated with thoracic spinal cord injuries.CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the sacral spinal cord (S1-S4) is rare and generally causes loss of bladder and bowel function as well as sexual dysfunction. Some sacral injuries can result in weakness or paralysis of the hips and legs.CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

The level of Spinal Cord Injury (SCI) determines what parts of the body might be affected by paralysis and loss of function. The level of injury refers to the lowest point on the spinal cord where there is a decrease or absence of motor and/or sensory function. Generally speaking, the higher the spinal cord injury, the more effect the injury has on movement and/or feeling. For example, an injury of the cervical spinal cord may result in full paralysis and make it impossible to breathe without a respirator, while an injury of the lumbar spinal cord may result in paralysis or weakness in the legs and cause some loss of body function in the lower extremities.CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the cervical spinal cord (levels C1-C8) causes quadriplegia (also called tetraplegia), which refers to paralysis or weakness in both arms and legs. All parts of the body located below the neck may be affected. Involuntary functioning, such as breathing, regulating body temperature, and sweating may be impaired, necessitating a respirator and other mechanical devices. A person with quadriplegia may not be able to sense touch (or other sensations) may lose bladder and bowel control, and may experience sexual dysfunction.CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the thoracic spinal cord (levels T1-T12) causes paraplegia, which means paralysis or weakness in the legs. Depending upon where the injury is located on the thoracic spinal cord, an individual with this level of SCI may also experience weakness in their torso, although will generally possess good control of their hands. These injuries may also result in loss of sensation, loss of bladder and bowel control, as well as sexual dysfunction. Due to the rib cage, thoracic spinal cord injuries occur less often, as the rib cage offers protection from such injuries.CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the lumbar or sacral spinal cord (L1-L5) causes paraplegia, again referring to paralysis or weakness of the legs. Because of the lower location of this injury, upper body functions are usually not affected. However, a person with a lumbar SCI may experience the loss of many of the sensory functions associated with thoracic spinal cord injuries.CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

An injury of the sacral spinal cord (S1-S4) is rare and generally causes loss of bladder and bowel function as well as sexual dysfunction. Some sacral injuries can result in weakness or paralysis of the hips and legs.CRISPR Cas9 | Spinal Cord Injury [Geography & Symptoms Description]

The problem of targeting specific spinal cord injuries

The problem of targeting specific spinal cord injuries

The problem of targeting specific spinal cord injuries

The problem of targeting specific spinal cord injuries

But browsing recent literature also demonstrates the unique difficulty of SCI: because it is so central to so many bodily functions, and because it is an extended, interworking set of transmitters along one cord, isolating problems is a challenge. In research, attributing success to an experiment requires parsing many factors. Further, once the spinal cord is damaged, it cannot regenerate as can other cells, like on the skin’s surface. The spinal cord will create an inflexible scar, and with that, create a blockage in necessary signals to and from the brain. Put another way, scientists must understand several chain reactions within one extended, hyper-complex chain reaction. They haven’t had a way of just targeting and isolating one function. Until now.

CRISPR Cas9: the targeting and isolation believed impossible

CRISPR Cas9: the targeting and isolation believed impossible

CRISPR Cas9: the targeting and isolation believed impossible

CRISPR Cas9: the targeting and isolation believed impossible

A bacterial process called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) can produce targeted change by creating a DNA template. First identified and named in 2002, the concept has existed for decades, since scientists first described the phenomenon in in E. Coli bacteria in the late 1980s. CRISPR is a natural process used by bacteria to develop adaptive immunity to phages (viruses that specifically affect bacteria). Bacteria create an immunity by creating a CRISPR out of short segments of DNA, gained from previous exposures to a particular phage, between short repetitions of base sequences. The CRISPR, which grows following each encounter with a new phage, is then later used as a DNA template to detect and fight off the attack by cleaving the DNA of the same invading phage.

The more recently discovered Cas9 refers to CRISPR Associated Protein 9. Cas9 is a specific enzyme, which has recently become a popular subject of discussion in genetic engineering due to its relative ease of use and its target specificity. Its utility lies in its ability to target nearly any DNA sequence that is complementary to its associated guiding RNA sequence. The guiding RNA sequences that can be used by the Cas9 enzyme are stored by the CRISPR.

CRISPR Cas9 method for genome editing – a powerful new technology with many applications in biomedical research, including the potential to treat human genetic disease. Credit: Feng Zhang/MIT-McGovern Institute for Brain Research. [2014-11-05; McGovern Institute for Brain Research at MIT] [YouTube]

This animation depicts the CRISPR Cas9 method for genome editing – a powerful new technology with many applications in biomedical research, including the potential to treat human genetic disease. Feng Zhang, a leader in the development of this technology, is a faculty member at MIT, an investigator at the McGovern Institute for Brain Research, and a core member of the Broad Institute. Further information can be found on Prof. Zhang’s website at http://zlab.mit.edu [2014-11-05; McGovern Institute for Brain Research at MIT] [YouTube]

This animation depicts the CRISPR Cas9 method for genome editing – a powerful new technology with many applications in biomedical research, including the potential to treat human genetic disease. Feng Zhang, a leader in the development of this technology, is a faculty member at MIT, an investigator at the McGovern Institute for Brain Research, and a core member of the Broad Institute. Further information can be found on Prof. Zhang’s website at http://zlab.mit.edu [2014-11-05; McGovern Institute for Brain Research at MIT] [YouTube]

This animation depicts the CRISPR Cas9 method for genome editing – a powerful new technology with many applications in biomedical research, including the potential to treat human genetic disease. Feng Zhang, a leader in the development of this technology, is a faculty member at MIT, an investigator at the McGovern Institute for Brain Research, and a core member of the Broad Institute. Further information can be found on Prof. Zhang’s website at http://zlab.mit.edu [2014-11-05; McGovern Institute for Brain Research at MIT] [YouTube]

Breakthroughs using CRISPR Cas9

Breakthroughs using CRISPR Cas9

Breakthroughs using CRISPR Cas9

Breakthroughs using CRISPR Cas9

The CRISPR Cas9 combination has revolutionized the field of genetic engineering since its discovery. The combination of CRISPR and the Cas9 enzyme has been used to investigate everything from potential targets for cancer therapies (Shi), controlling genetically modified crops that have broken out of containment (Caliando), treatment of cardiovascular diseases with gene therapy (Rincon), and eradicating HIV infection of cells (Liao). Another exciting application uses CRISPR Cas9 to re-sensitize antibiotic-resistant bacteria such as MRSA (Methicillin-resistant Staphylococcus aureus), a skin disease, allowing the bacteria to once again be controlled with common antibiotics. A group at the Rockefeller University was able to re-sensitize a resistant strain of Staphylococcus Aureus bacteria, which is the bacteria responsible for MRSA, to tetracycline, potentially leading to improved treatments for this condition (Bikard). Another similar breakthrough was made by scientists at the Massachusetts Institute of Technology, who were able to genetically modify an antibiotic resistant strain of E. Coli to regain 99.9% sensitivity to another antibiotic (Citorik).

The next phase with CRISPR Cas9

The next phase with CRISPR Cas9

The next phase with CRISPR Cas9

The next phase with CRISPR Cas9

Not escaping controversy, CRISPR Cas9 has recently been used by Chinese scientists to investigate the ability to genetically modify early human embryos. Eventually the ability to modify humans’ genes could lead to the prevention or cure of diseases such as Down Syndrome, Cystic Fibrosis, and Muscular Dystrophy. While these investigators had some success in modifying human embryos, some encountered problems with the specificity and fidelity of the results. Ultimately, the investigators determined more work is needed before before any clinical applications in humans. However, the publication of their study added considerable kindling to the ethical fire surrounding the genetic modification of human embryos.

In November 2015, the New York Times Magazine published The Crispr Quandary, an in-depth article exploring a potential “ethical morass” in the CRISPR Cas9’s ability “to precisely snip out a piece of DNA at any point” and address a multitude of issues. Other social commentary has sprouted up around the seemingly limitless use of CRISPR Cas9’s functions.

CRISPR Cas9 | A History of CRISPR: 1987-2015 (Credit: The Noun Project)

A History of CRISPR: 1987-2015 (Credit: The Noun Project).

CRISPR Cas9 | A History of CRISPR: 1987-2015 (Credit: The Noun Project)

A History of CRISPR: 1987-2015 (Credit: The Noun Project).

CRISPR Cas9 | A History of CRISPR: 1987-2015 (Credit: The Noun Project)

A History of CRISPR: 1987-2015
(Credit: The Noun Project).

CRISPR Cas9 | A History of CRISPR: 1987-2015 (Credit: The Noun Project)

A History of CRISPR: 1987-2015
(Credit: The Noun Project).

Medical ethics are deeply important to the Michelson Medical Research Foundation. The exploration of medical ethics is a major initiative and, guided by those values, the Foundation will persist in the exploration of CRISPR Cas9 and its regenerative powers.

As Dr. Michelson put it when asked in Michelson Medical on July 28th, 2015 about the future of medicine, “I believe there is an answer. Frogs do it, starfish do it and we can too.” Dr. Michelson was referring to frogs’ and starfish’s ability to regrow parts of themselves, as humans may have the potential to regenerate damaged spinal cords (Note from the Editor- also see the regenerative process of SCIs in salamanders). So far, the research has focused on mechanical devices to ameliorate the functional challenges of SCI. Dr. Michelson continued in his explanation, “The greatest breakthrough applicable to this problem lies in understanding how to activate the already present genes, to address the injury to the cord. Research in the use of stem cells has been ongoing and understanding the full potential of CRISPR Cas9 has only just begun. Ultimately the solution to spinal cord injury will be biological, not mechanical.”

Dr. Gary K. Michelson

“I believe there is an answer. Frogs do it, starfish do it and we can too, The greatest breakthrough applicable to this problem lies in understanding how to activate the already present genes to address the injury to the cord. Research in the use of stem cells has been ongoing and understanding the full potential of CRISPR Cas9 has only just begun. Ultimately the solution to spinal cord injury will be biological, not mechanical”.

Dr. Gary K. Michelson

Dr. Gary K. Michelson

“I believe there is an answer. Frogs do it, starfish do it and we can too, The greatest breakthrough applicable to this problem lies in understanding how to activate the already present genes to address the injury to the cord. Research in the use of stem cells has been ongoing and understanding the full potential of CRISPR Cas9 has only just begun. Ultimately the solution to spinal cord injury will be biological, not mechanical”.

Dr. Gary K. Michelson

Dr. Gary K. Michelson

“I believe there is an answer. Frogs do it, starfish do it and we can too, The greatest breakthrough applicable to this problem lies in understanding how to activate the already present genes to address the injury to the cord. Research in the use of stem cells has been ongoing and understanding the full potential of CRISPR Cas9 has only just begun. Ultimately the solution to spinal cord injury will be biological, not mechanical”.

Dr. Gary K. Michelson

Dr. Gary K. Michelson

“I believe there is an answer. Frogs do it, starfish do it and we can too, The greatest breakthrough applicable to this problem lies in understanding how to activate the already present genes to address the injury to the cord. Research in the use of stem cells has been ongoing and understanding the full potential of CRISPR Cas9 has only just begun. Ultimately the solution to spinal cord injury will be biological, not mechanical”.

Dr. Gary K. Michelson

References

  1. Targeted DNA degradation using a CRISPR device stably carried in the host genome, Caliando BJ, Voigt CA, Nature Communications 6,, 2015
    Exploiting CRISPR-Cas9 nucleases to produce sequence-specific antimicrobials, Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, Fischetti VA, Marraffini LA, Nature Biotechnology 32, 1146-1150
  2. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells, Liao HK, Diaz A, Marlett J, Takahashi Y, Li M, Suzuki K, Xu R, Hishida T, Chang CJ, Esteban CR, Young J, & Belmonte JCI, Nature Communications 6, March 2015
    Gene therapy FOR cardiovascular disease: advances in vector development, targeting and delivery for clinical translation, Rincon MY, VendenDreissche T, Chuah MK, Cardiovascular Research, August 2015.
  3. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases, Citorik RJ, Mimee M, Lu TK, Nature Biotechnology 32, 1141-1145
  4. CRISPR-Mediated Genomic Deletion of Sox2 in the Axolotl Shows a Requirement in Spinal Cord Neural Stem Cell Amplification During Tail Regeneration, Fei JF, Schuez M, Tazaki A, Taniguchi Y, Roensch K, Tanaka EM, International Society for Stem Cell Research 3, 444-459, September 2014
  5. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains, Shi J, Wang, E, Milazzo JP, Wang Z, Kinney JB, Vakoc CR, Nature Biotechnology 33, 661-667, 2015
  6. The Crispr Quandary: A new gene-editing tool might create an ethical morass — or it might make revising nature seem natural. [2015-11-09; Jennifer Kahn, The New York Times Magazine]

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The Michelson Medical Research Foundation's Groundwork blog is brought to you thanks to the generous support of Dr. Gary K. Michelson and his wife, Alya Michelson.

The Michelson Medical Research Foundation's Groundwork blog is brought to you thanks to the generous support of Dr. Gary K. Michelson and his wife, Alya Michelson.

The Michelson Medical Research Foundation's Groundwork blog is brought to you thanks to the generous support of Dr. Gary K. Michelson and his wife, Alya Michelson.

The Michelson Medical Research Foundation's Groundwork blog is brought to you thanks to the generous support of Dr. Gary K. Michelson and his wife, Alya Michelson.