User:Cchoates/sandbox

CRISPR

Article is fairly neutral-- ignores taking sides on current legal and political entrenchment of CRISPR technology

Article is listed as a B-class "top priority" for computational biology. I hope to improve this section of CRISPR knowledge on Wiki. I believe the best way I can do that is by expanding on the current "Identification" subheading:

"CRISPRs are widely distributed among bacteria and archaea and show some sequence similarities. Their most notable characteristic is their repeating spacers and direct repeats. This characteristic makes CRISPRs easily identifiable in long sequences of DNA, since the number of repeats decreases the likelihood of a false positive match. Three programs used for CRISPR repeat identification search for regularly interspaced repeats in long sequences: CRT, PILER-CR and CRISPRfinder.

Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci do not typically assemble, due to their repetitive nature or through strain variation, which confuses assembly algorithms. Where many reference genomes are available, polymerase chain reaction (PCR) can be used to amplify CRISPR arrays and analyse spacer content. However, this approach yields information only for specifically targeted CRISPRs and for organisms with sufficient representation in public databases to design reliable polymerase chain reaction (PCR) primers.

The alternative is to extract and reconstruct CRISPR arrays from shotgun metagenomic data. This is computationally more difficult, particularly with second generation sequencing technologies (e.g. 454, Illumina), as the short read lengths prevent more than two or three repeat units appearing in a single read. CRISPR identification in raw reads has been achieved using purely de novo identification or by using direct repeat sequences in partially assembled CRISPR arrays from contigs (overlapping DNA segments that together represent a consensus region of DNA) and direct repeat sequences from published genomes as a hook for identifying direct repeats in individual reads."

Knockdown/activation:

"Using "dead" versions of Cas9 (dCas9) eliminates CRISPR's DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dCas9s, enabling them to turn almost any gene on or off or adjust its level of activity. Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. Conversely, CRISPR activation (CRISPRa) finds these enhancer regions to promote gene activation. The targeted site is methylated, epigenetically modifying the gene. This modification inhibits or activates transcription. Cas9 is an effective way of targeting and silencing specific genes at the DNA level. In bacteria, the presence of Cas9 alone is enough to block transcription. For mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA sequences called promoters that immediately precede the target gene.

Cas9 was used to carry synthetic transcription factors that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter. This method allows for the rapid identification of potential location of gene enhancers-- a previously difficult task."

I also hope to improve the understanding of the future medical impacts CRISPR can have beyond just "designer babies"; specifically, I hope to improve the understanding and current discussion behind gene drives and xenotransplantation. I added an edit on 9/5 to the 'biomedicine' subheading in 'Applications' (edits include underlined section with a verifiable citation, and an updated link to 'xenotransplantation' instead of the less accurate 'transplantation'):

CRISPR may revive the concept of xenotransplantation,or the transfer of animal organs into people. Retroviruses present in animal genomes could harm transplant recipients. In 2015 a team eliminated 62 copies of a retrovirus's DNA from the pig genome in a germline cell. A study published in August 2017 confirmed the potential to create pigs without these retroviruses present in their genome, in addition to demonstrating that these retroviruses have the potential to transfer from human cell to human cell in vitro.[1]