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RNA-directed Cleavage and Modification of DNA using CasY (CRISPR-CasY)

96 Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} The CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phage and viruses. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets.  Class 2 CRISPR-Cas are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation.  Current CRISPR Cas technologies are based on systems from cultured bacteria, leaving untapped the vast majority of organisms that have not been isolated.  There is a need in the art for additional Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations).     UC Berkeley researchers discovered a new type of Cas protein, CasY.  CasY is short compared to previously identified CRISPR-Cas endonucleases, and thus use of this protein as an alternative provides the advantage that the nucleotide sequence encoding the protein is relatively short.  CasY utilizes a guide RNA to perform double stranded cleavage of DNA. The researchers introduced CRISPR-CasY into E. coli, finding that they could block genetic material introduced into the cell.  Further research results indicated that CRISPR-CasY operates in a manner analogous to CRISPR-Cas9, but utilizing an entirely distinct protein architecture containing different catalytic domains.   CasY is also expected to function under different conditions (e.g., temperature) given the environment of the organisms that CasY was expressed in.  Similar to CRISPR Cas9, CasY enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.   

RNA-directed Cleavage and Modification of DNA using CasX (CRISPR-CasX)

96 Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} The CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phage and viruses. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets.  Class 2 CRISPR-Cas are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation.  Current CRISPR Cas technologies are based on systems from cultured bacteria, leaving untapped the vast majority of organisms that have not been isolated.  There is a need in the art for additional Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations).   UC Berkeley researchers discovered a new type of Cas protein, CasX, from groundwater samples. CasX is short compared to previously identified CRISPR-Cas endonucleases, and thus use of this protein as an alternative provides the advantage that the nucleotide sequence encoding the protein is relatively short.  CasX utilizes a tracrRNA and a guide RNA to perform double stranded cleavage of DNA. The researchers introduced CRISPR-CasX into E. coli, finding that they could block genetic material introduced into the cell.  Further research results indicated that CRISPR-CasX operates in a manner analogous to CRISPR-Cas9, but utilizing an entirely distinct protein architecture containing different catalytic domains.   CasX is also expected to function under different conditions (e.g., temperature) given the environment of the organisms that CasX was expressed in.  Similar to CRISPR Cas9, CasX enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation. 

Mobile Molecular Diagnostics System

There is a growing interest in point-of-care testing (POCT) where testing is done at or near the site of patient care, since POCT has a short therapeutic turnaround time, decreased process steps where errors can occur and only a small sample volume is required to perform a test.    UC Berkeley researchers have developed a mobile molecular diagnostics system that leverages efficient and dependable blood sampling, automated sample preparation, rapid optical detection of multi-analyte nucleic acids and proteins, and user-friendly systems integration with wireless communication.  The system includes a hand-held automated device with an adaptive sample control module, an optical signal transduction module, and an interface to a smartphone making this a reliable and field-applicable system for point-of-care and on-demand diagnostics. 

Optical Cavity PCR

Outbreaks of infectious diseases especially require diagnostic tools that can be used at the point-of-care (POC). Polymerase chain reaction (PCR) is sensitive and allows accurate diagnoses, but developing simple and robust PCR methods that can be used at POC remains a challenge. In particular, slow thermal cycling capability and high power consumption continue to be barriers.  Researchers at UC Berkeley have developed optical cavity PCR to address these challenges. This technology allows ultrafast cycling with low power consumption, high amplification efficiency and a simple fabrication process, enabling its use as a POC device. 

Hemolysis-Free Blood Plasma Separation Device

Blood plasma separation is often the first step in blood-based clinical diagnostic procedures. Although centrifugation is the traditional method for blood plasma separation, it is time consuming, labor intensive, and therefore not suitable for point-of-care testing. Centrifugation can also lead to hemolysis (the rupture of red blood cells) which further results in plasma contamination and hinders effective protein and nucleic acid analysis in diagnostic testing. Researchers at UC Berkeley have developed a simple and robust on-chip blood plasma separation device that addresses the problems found with traditional centrifugation. The novel hemolyis-free microfluidic blood plasma separation device reduced clogging of red blood cells (the hemoglobin concentration in the separated plasma was reduced about 90% compared to conventional devices), yet provided comparable target molecule recovery.

Portable PCR Thermal Cycler

Polymerase chain reaction (PCR) is a standard diagnostic method used for a variety of applications, including medical diagnostics, food safety, and environmental monitoring. PCR is conventionally done in bench-top thermocyclers, which are heavy, can be complex to operate, and have high power consumption. Furthermore, PCR is time-intensive, requiring up to 40 thermal cycles consisting of multiple temperature steps.   Researchers at UC Berkeley have addressed these issues by developing a system to rapidly heat thin Au films, allowing fast thermal cycling. This system can be used as a simple and portable PCR thermal cycler for point-of-care diagnostics. 

Compositions and Methods for the Treatment of Respiratory Conditions via NKG2D Inhibition

Respiratory conditions such as chronic obstructive pulmonary disease (COPD) and asthma afflict millions of people each year.  COPD is characterized by peribronchial and perivascular inflammation and largely irreversible airflow obstruction. Asthma is characterized by infiltration of leukocytes subsets including eosinophils, macrophages and lymphocytes including natural killer (NK) cells that contribute to the sustained inflammation.  There is no cure for these conditions and individuals experience varied success with current treatment modalities. Investigators at UC Berkeley have revealed important roles for NK cells in the development and progression of COPD and asthma, which indicates that these mechanisms are controlled by the presence and function of NKG2D.  A therapeutic effect is achieved by administering agents that inhibit NKG2D-mediated activation of leukocytes, such as antibodies or soluble ligands or receptors that block NKG2D or its ligands. 

Live Imaging of Corneal Lymphatic Vessels

Lymphatic research is an explosive field of new discovery in recent years. Lymphatic dysfunction has been found in a wide array of disorders which include but are not limited to cancers and tumors, inflammation, infection, autoimmune diseases, dry eye, chemical burn, and tissue or organ transplant rejection, etc. The cornea provides an optimal site for lymphatic research due to its accessible location, transparent nature, and lymphatic-free but inducible features. Because there are no pre-existing vessels to consider in this unique tissue, it is exceptionally straightforward and accurate to assess lymphatic events (from formation to maturation and regression) in the cornea. Since lymphatic vessels are not easily visible as blood vessels, previous studies using the cornea have relied on traditional immunohistochemistry assays with dead tissues. Currently, there are no means of direct and harmless visualization of lymphatic vessels within live cornea. Investigators at University of California at Berkeley have addressed this challenge by developing the first live imaging of corneal lymphatic vessels. Lymphatic specific dye is injected into the subconjunctival space to visualize lymphatic vessels at various stages in the cornea under a fluorescence stereo, confocal, or two-photon microscope. Moreover, lymphatic vessels can be visualized in different colors to produce two, three, and four-dimensional images or live videos at a molecular level. The investigators have demonstrated a proof of principle in live mouse cornea. The technique allows time course tracking of dynamic lymphatic processes within the same tissue or subject over a short or long period of time, and can be ideally used to assess the progression of disease development and the effect of drug treatment. Live imaging of corneal lymphatic vessels allows visualization of lymphatic vessels in their natural morphology, state, and interactions with the local environment. This noninvasive method of live imaging of corneal lymphatic vessels is readily applicable to patient examination and the lymphatic dye of dextran is bio-degradable and harmless to human health.

Non-Thermal Irreversible Electroporation And Immune System Enabled Tissue Engineering

Treatment of disease and trauma to the coronary arteries and the peripheral vessels often includes the use of bypass grafting. Autologous grafts are most often used and are typically taken from the saphenous vein, internal mammary artery, or the radial artery; however, this method is not an option in patients without a vein that is suitable to use. Also, the costs for harvesting autologous vessels are considerable, and there is a significant level of morbidity associated with the procedure.  Also, the use of synthetic grafts in small diameter vessels tends to lead to poor compliance and low patency, often resulting in thrombogenicity.  Furthermore, although recent tissue engineering methods have focused on a variety of tissue decellularization methods, these methods risk damage to the extracellular membrane (ECM), which acts as a scaffold for tissue repair and regeneration, compromising the scaffold's further development and integration into the recipient's body. UC Scientists have developed an alternative technology that does not have the disadvantages and shortcomings seen in both autologous and synthetic grafts.  Such technology includes a method of treatment comprising subjecting a target area of tissue in a mammal to non-thermal irreversible electroporation (NTIRE) in order to kill cells at the target site without the use of any chemical agents, toxins, enzymes or use of physical devices beyond the NTIRE devices. After the immune system has removed cells killed with the NTIRE, and before there is substantial growth of new cells the tissue is removed from the mammal and transplanted to a repair site, all of which is carried out in the absence of any immunosuppressant drugs.

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