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Targeted nucleases are effective methods for mediating genome changes with high precision. By simply specifying a 20-nt targeting sequence within its guide RNA, the RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to promote efficient genome engineering in eukaryotic cells. We present a collection of tools for Cas9-mediated genome editing in mammalian cells using nonhomologous end joining (NHEJ) or homology-directed repair (HDR), as well as the generation of modified cell lines for functional studies later on. We also define a double-nicking strategy using the Cas9 nickase mutant and paired guide RNAs to reduce off-target cleavage. This protocol provides experimentally derived instructions for target site selection, cleavage efficiency assessment, and off-target behavior analysis. Gene modifications can be completed in as little as 1–2 weeks after target design, and improved clonal cell lines can be derived in 2–3 weeks.
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Dr. Zhang’s protocol Dr. Zhang is a Chinese physician who graduated from Shanghai Second Medical University in 1962 with a degree in clinical research combining Chinese and Western medicine. He won a prize from the World Health Organization in 1980. In addition to his work as a researcher at the Oriental Healing Arts Institute in Long Beach, California, where he studied the treatment of AIDS with Chinese medicine, he also published two books. In 1990, he founded his own practice, first in California and then in New York. Dr. Zhang has been focusing on the treatment of chronic viral infections with Chinese kruiden since 1987. Consider hepatitis and Aids, as well as infectious diseases such as Lyme and auto-immun ziekten.
“Een infectie is made up of two parts: the pathogen that binds to the lichaam and the lichaam’s reaction to the pathogen. In TCM (Traditional Chinese Medicine), an infection is viewed differently than it is in western medicine. It is widely accepted that the detection of a disease is aided by an internal oorzaak, the lichaamseigen immuunsysteem. In China, the pathogenese of a ziekten infection is known as “Zheng xu xie shi,” which means “reduced weerstand and increased pathogene factors.”
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After graduating from Shanghai Second Medical University in 1962, Dr. Zhang worked as a physician at Shanghai Second Medical University’s Reijing Hospital. His clinical practice and study focused on fusing the most beneficial aspects of TCM and modern medicine to improve clinical effectiveness and patient quality of life.
Dr. Zhang founded the Zhang Clinic in New York City in 1991. He was mainly interested in using Allitridi, a chemical precursor to Allicin, and other anti-microbial compounds to treat infections that were difficult to treat at the time.
The main antimicrobial compound present in garlic is allicin.
After ingestion, allitridi metabolizes into allicin, which is the more chemically stable pre-cursor. Dr. Zhang realized that allitridi’s main advantage was its much higher bioavailability, which made it an effective and stable antimicrobial agent.
Dr. Zhang discovered that allitridi was effective against many of the common opportunistic infections that threaten late-stage HIV/AIDS patients in the late 1980s and early 1990s. Many of these patients were able to obtain longer life spans and reasonably good Quality of Life by adding allitridi to their treatment regimen at a time when there were few treatment options available.
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Over the past 28 years, Dr. Richard Horowitz has been treating Lyme disease and other tick-borne infections. He had no idea he’d be landing right in the heart of tick territory when he moved from Bayside, New York, to the Hudson Valley. Dr. Horowitz started a practice in Hyde Park, New York, after specializing in internal medicine. He is the medical director of the Hudson Valley Healing Arts Center. Why Can’t I Get Better? is his book. In 2013, the book Solving the Mysteries of Lyme and Chronic Disease was published. Janet Jemmott, editor of the Lyme Connection, spoke with him about his thoughts on Lyme disease and chronic illness.
You’ve been interviewed on radio and television news shows since your book was published. What has been the reaction of the Lyme group and the general public? Has the Infectious Disease Society of America (IDSA) objected to your proposal?
The Lyme group has been fantastic and extremely helpful. The book is still selling well and has reached the New York Times Best Seller List, but in an epidemic where the CDC is admitting over 300,000 cases each year (it’s more likely one to two million cases), it doesn’t appear that many copies have been sold from that viewpoint! People don’t seem to know how bad the epidemic has become.