106 Matching Annotations
  1. Oct 2015
    1. it is likely that the target locus's underlying chromatin structure and epigenetic state will also affect the efficiency of genome editing in eukaryotic cells (13), although we suspect that Cas9's helicase activity may render it more robust to these factors, but this remains to be evaluated

      This is another area for possible future research.

    2. We also incorporated these target sequences into a 200-bp format compatible with multiplex synthesis on DNA arrays (14) (fig. S11 and tables S2 and S3)

      These tables are available for future researchers who wish to use the method described here.

    3. To facilitate this process, we bioinformatically generated ~190,000 specific gRNA-targetable sequences targeting ~40.5% exons of genes in the human genome

      The authors ran a computer simulation to see how much of the human genome could possibly be targeted using this system.

    4. These results demonstrate that this approach enables efficient integration of foreign DNA at endogenous loci in human cells

      The authors had previously inserted DNA into an artificially inserted target. Now they targeted a natural site.

    5. NHEJ-mediated deletions for T1 and T2 were centered around the target site positions,

      The deletions were not always exactly on target, but they tended to center around the target thus providing evidence that the process itself targets specific DNA sequences.

    6. NHEJ events

      In non-homologous end joining splices in DNA are repaired by splicing rather than by referring to an intact complementary strand of DNA. This method of repair is less accurate than homologous recombination.

    7. PGP1 human induced pluripotent stem (iPS) cells

      Induced pluripotent stem cells come from adult cells which have been artificially changed to exhibit stem cell properties. These properties are immortality and the ability to develop into various different cell types. These cells are thus unspecialized. https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/generation-human-induced-pluripotent-stem-cells-fibroblasts.html

    8. integrated reporter

      The integrated reporter was the green fluorescent DNA sequence which was integrated into the target DNA. The green fluorescent sequence could then be manipulated, and the results could be measured.

    9. these results confirm that RNA-guided genome targeting in human cells is simple to execute and induces robust HR across multiple target sites

      The authors demonstrated that the system can edit various different genes in the human genome.

    10. HR but lower nonhomologous end joining (NHEJ) rates

      By increasing the proportion of homologous recombination the researchers have better control over the process. Nonhomologous end joining is a less exact process and leads to more spontaneous errors.

    11. GFP+ cells appearing ~20 hours post transfection compared with ~40 hours for the AAVS1 TALENs.

      The results of the editing process appeared sooner in the case of the authors' engineered guide RNAs as compared with the earlier method using TALENs

    12. resource of ~190 K unique gRNAs targeting ~40.5%

      The authors generated a database of about 190,000 guide RNAs which can potentially target about 40.5% of the human genome. This will be a resource for future experiments.

    13. is sequence-specific

      The authors had to show that the results follow the addition of specific components to the system. By demonstrating this, they support their explanation for what is happening on the microbiological level.

    14. renders the expressed protein fragment nonfluorescent

      By introducing the green fluorescent protein sequence and then disrupting it the authors were able to measure how well their genetic editing worked. They could measure this by determining how many of the daughter cells were fluorescent.

    15. 68-bp

      Since DNA consists of a double strand, the bases form into pairs such that cytosine always pairs with guanine and adenine always pairs with thymine. A 68 bp sequence would contain 68 of these pairs.

    16. PAM (protospacer-adjacent motif)

      The PAM is a short DNA sequence close to the targeted DNA sequence of the invading organism. The PAM is crucial for the system to recognize whether or not the DNA sequence is self or not self. If the PAM is missing the system will not be activated

    17. guide RNAs

      In the natural CRISPR system guide RNAs take fragments of the viral or plasmid DNA and guide them to specific locations in the CRISPR locus. The idea here is that these artificial guide RNAs can similarly guide a short strand of nucleic acids into a specific place in the target genome.

    18. RNA

      In contrast to DNA, which is a double strand of linked amino acids, RNA is a single strand. The four "letters" in the DNA alphabet are adenine, thymine, cytosine, and guanine. RNA is made up of adenine, thymine, and cytosine, but in place of guanine it has uracil.

    19. reconstitution of the Streptococcus pyogenes type II CRISPR system demonstrated that crRNA fused to a normally trans-encoded tracrRNA is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA

      The cited article describes an experiment which demonstrated DNA cleavage by Cas9, principal protein in type II CRISPR systems. Cas9 protein was purified from a preparation of Streptococcus pyogenes.

      The researchers found that addition of crRNA alone was not able to guide cleavage of target plasmid DNA, but with the addition of tracrRNA the cleavage occurred.

      By comparing these results with similar results using short double sequence DNA, the authors demonstrated that the functions of tracrRNA are triggering the enzyme RNase III and activating cleavage of target DNA by means of cas9 under the guidance of crRNA.

      The authors fused the two essential elements: crRNA and tracrRNA to provide greater convenience for targeted genetic editing.

      Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science (Washington D C), 337(6096), 816-821.

    20. DNA

      Deoxyribonucleic acid (DNA) is a double stranded helix (coiled in a spiral) of proteins which form a sort of "alphabet" to encode genetic information. The "letters" of this alphabet consist of adenine, thymine, cytosine, and guanine.

      Adenine always pairs with thymine, and cytosine always pairs with guanine, thus, when the two strands are divided the complete DNA molecule can be exactly reproduced. http://ghr.nlm.nih.gov/handbook/basics/dna

    21. viral

      Viruses are submicroscopic parasites with a simple structure. Their main parts are a strand of nucleic acid (DNA or RNA) and an surrounding protein shell called a capsid. Some viruses also have an outer viral envelope surrounding their capsid. Viruses are not capable of reproducing on their own, but rather must invade a cell and commandeer that cell's resources to reproduce themselves http://www.ncbi.nlm.nih.gov/books/NBK21523/

    22. clustered regularly interspaced short palindromic repeats

      The scientists who first discovered these sequences had no understanding of their possible function. Later it was observed that some of the spacers correspond to the DNA of invading viruses.

      This led to the discovery that CRISPRs play a protective role defending bacteria and archaea from other invasive organisms.

      There is speculation that this immune function is only one of the functions of CRISPRs. They may be involved in other functions such as regulating the genetic expression of the barcterial or archaeal organism's own DNA. https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/

    23. clustered regularly interspaced short palindromic repeats

      Clustered regularly interspaced short palindromic repeats (CRISPRs) are strands of DNA with repeating sequences (repeats) interspersed with other sequences which don't repeat (spacers).

    24. CRISPR-associated (Cas) systems

      CRISPRs do not act in isolation. They are part of a complete system which includes the CRISPR array, associated genes, and an upstream leader sequence. The CRISPR array is the strand of DNA with repeating and unique segments interspersed.

      Many, but not all, of the repeating segments are palindromes (sequences that read the same from right to left as the do from left to right). Many of the spacers have DNA which matches that of invading viruses (phages) or plasmids. CRISPR associated genes often code for proteins which can cleave or bind DNA or RNA sequences. The leader sequence of the system occurs before the first repeat and is important for transcription as well as for obtaining new spacers. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497052/

    25. adaptive immune defenses

      Adaptive immunity is immunity that one gets in response to challenges from the environment. This is in contrast to innate immunity, which is the immunity one is born with.

      Adaptive immunity is not something exclusive to higher animals. Bacteria and archaea also have immune responses to foreign organisms which threaten them (mainly viruses and plasmids) http://www.ncbi.nlm.nih.gov/pubmed/23495939

  2. Sep 2015