CRISPR is cheap, easy to do, and powerful… is it also dangerous? Anne B. Allison, Ph.D. Teaching Demonstration University of Richmond 3/20/17
Previously: natural genetic change mechanisms of genetic variation mobile genetic elements how viruses interact with genomes There is no natural mechanism for generating entirely new, long stretches of genetic sequence
Today: CRISPR how this new gene-editing tool works promises and limitations of this technology CRISPR provides a relatively easy and precise way to alter genomes Big questions: What specific measures may help policy keep pace with rapidly evolving technologies? To what extent should dual-use research be private, restricted, or prohibited?
What are some ways people alter genetic information? Let's take a few minutes to think-pair-share
Biologists learn about gene function by altering genetic information The past four decades have relied on techniques that are resource-intensive and/or lack precision: - targeted gene editing requires extensive training, cost, and time - dominant negative mutants often target more than one molecular pathway - RNA interference may have thousands of off-target effects By comparison, CRISPR is relatively easy and precise http://www.the-odin.com/diy-bacterial-gene-engineering-crispr-kit/
Cutting DNA can lead to: How CRISPR works Clustered Regularly Interspaced Short Palindromic Repeats discovered as adaptive immune system in bacteria and engineered as a genome-editing tool Cutting DNA can lead to: - disrupting gene - repairing a gene https://www.youtube.com/watch?v=avM1Yg5oEu0
CRISPR-Cas9 relies on extensive complementary basepairing to locate target DNA in a sea of chromatin 5’ ccntggtgcatctgactcctgtg 3’ 3’ ggnaccacguagacugaggacac 5’ CRISPR guide RNA Cas9 protein 3’ ggnaccacgtagactgaggacac 5’
Identifying on- and off-target sites
CRISPR may bind 20 or more off-target sites throughout the genome This low off-target frequency (~6.5x10-9) is modest and impressive, nevertheless, one off-target cleavage could have disastrous consequences Shengdar Q Tsai, et al. Nature Biotechnology (2015)
Components of the human genome T. Ryan Gregory Nature Reviews Genetics (2005)
Recommended sources of information Primary: Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096): 816-21. doi: 10.1126/science.1225829 http://science.sciencemag.org/content/337/6096/816.long Shengdar Q Tsai, et al. (2015). GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nature Biotechnology, 33: 187–197. doi: 10.1038/nbt.3117 http://www.nature.com/nbt/journal/v33/n2/full/nbt.3117.html News: ‘Rewriting the Code of Life’ by Michael Specter in New Yorker January 2, 2017 issue http://www.newyorker.com/magazine/2017/01/02/rewriting-the-code-of-life ‘Do CRISPR Enthusiasts have their head in the sand about the safety of gene editing?’ by Sharon Begley in STAT News July 18, 2016 https://www.statnews.com/2016/07/18/crispr-off-target-effects/ ‘Genetically Engineer Almost Anything’ by Tim De Chant and Eleanor Nelsen in NOVA Next July 17, 2014 http://www.pbs.org/wgbh/nova/next/evolution/crispr-gene-drives/ Video: Dr. Jennifer Doudna’s iBiology video series: https://www.ibiology.org/ibiomagazine/jennifer-doudna-genome-engineering-with-crispr-cas9-birth-of-a-breakthrough-technology.html
You may access these slides and related materials at https://osf.io/u2j26/
Gene drives engineer evolution
Genetic code
One letter alters hemoglobin protein function G A G G T G C T C C A C G A G G U G glutamic acid valine mutation DNA RNA protein normal protein is soluble mutant protein forms fibers which distort shape of red blood cells causing these cells to clog blood vessels leading to a range of serious symptoms
Malaria has exerted selective pressure for the sickle cell trait in human populations https://www.youtube.com/watch?v=Zsbhvl2nVNE MAPS SHOWING MALARIA DISTRIBUTION AND MATCHING DISTRIBUTION OF HUMANS CARRYING THE HbS (SICKLE CELL) GENE