CRISPR System Biology PDF

Biology of CRISPR system 11.12 Preserving Genome Integrity: CRISPR Interference CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats): A Prokaryotic “immune system” that evades viral destruction and maintains genome stability https://schaechter.a...

Biology of CRISPR system 11.12 Preserving Genome Integrity: CRISPR Interference CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats): A Prokaryotic “immune system” that evades viral destruction and maintains genome stability https://schaechter.asmblog.org/.a/6a00d8341c5e1453ef0147e3de9490970b-popup  The Discovery of CRISPR 1987 — Yoshizumi Ishino et al. discovered palindromic repeats in E. coli iap gene 1993 - 2005 — Francisco Mojica, University of Alicante, Spain: Francisco Mojica was the first researcher to characterize what is now called a CRISPR locus, reported in 1993. He coined the term CRISPR through correspondence with Ruud Jansen, who first used the term in print in 2002. In 2005 he reported that these sequences matched snippets from the genomes of bacteriophage (Mojica et al., 2005). CRISPR timeline: https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-timeline Biological significance of the spacer sequences Francisco Mojica This finding led him to hypothesize, correctly, that CRISPR is an adaptive immune system.  11.12 Preserving Genome Integrity: CRISPR Interference mechanism memory bank of incoming nucleic acid sequences for surveillance against foreign DNA consists of segments of foreign DNA (spacers) that previously invaded cell alternating with identical repeated sequences (Figure 11.33) Key is transcription of long RNA molecule cleaved in the middle of repeated sequences by nuclease activity of CRISPR- associated (Cas) proteins. ∅ _ generates CRISPR RNAs (crRNAs) that base-pair with invading nucleic acids, resulting in destruction ^ CRISPR RNAs include crRNA and a “Trans-activating crRNA” – “trascrRNA” θ 아 ④ Figure 11.33  EXAM Cthe texther) CRISPR mechanism I Four protein-coding genes (cas9, cas1, cas2, and cns2) and the tracrRNA. Repeat regions (black diamonds) separated by spacer regions (colored rectangles) Two RNAs are produced: crRNA and trascrRNA Processing into short RNAs by RNase III Recognition of DNA target by both a pattern sequence (PAM) and a specific sequence recognition Induction of double-stranded DNA break; thus interrupt the functions of plasmid and DNA phage https://www.broadinstitute.org/files/news/pdfs/PIIS0092867415017055.pdf  Distribution of CRISPR Widely distributed (~90 percent of Archaea and 70 percent of Bacteria) Bacteriophages can overcome recognition by genome mutation. 12.12 Genome Editing and CRISPRs Charpentier and Doudna contributed to the biochemical characterization of Cas9-mediated cleavage of genome and reported that the crRNA and the tracrRNA could be fused together to create a single, synthetic guide RNA. (2012) Virginijus Siksnys also discovered the function of Cas9 but published slightly later. (2012) Feng Zhang and George Church groups first reported CRISPR genome editing in human cells. (2013) Then there was a flood of papers on CRISPR in different species since 2013.  Genome editing using Cas9 Sequence targeting by the Cas9 protein Cas proteins of CRISPR systems function as En endonucleases when guided to nucleic acids. Synthetic RNA (synthetic guide RNA [sgRNA]) → - that recruits Streptococcus Cas9 and binds to target DNA enables cutting in genome of almost any cell; DNA can be ligated or used to insert new DNA. (Figure 12.36) also requires protospacer adjacent motif (PAM) DNA break repair process can induce insertions and/or deletions (indels), resulting in gene mutation. Homologous recombination can be used to incorporate new DNA.  Homologous recombination with a template DNA sequence can induce DNA insertion Double cleavage sites can delete a large piece of DNA Figure 12.36  CRISPR editing in practice Example: Tomato mutation leads to needle-like or wiry leaves. (Figure 12.37) mutation of gene SlAGO7 gene encoding an argonaute homolog  CRISPR baby and ethical issue Controversial application: human babies (Lulu and Nana) with HIV-1 receptor CCR5 mutation created by He Jiankui in China. We do not fully understand the risk of genome editing yet and there are profound ethical consequences. https://theness.com/neurologicablog/index.php/new- information-on-the-crispr-babies/  Cosmetics made from microbe SK-II: annual revenue estimated at $51m “Pitera is the broth that's made by the yeast. It is rich in amino acids, proteins, organics, acids.” “The story of PITERA™ began in the 1970s with a team of scientists, a search for a game-changing ingredient that can engender beautiful skin, and elderly Japanese sake brewers. In the course of their research, the scientists noticed that the hands of sake brewers remained extraordinarily soft and youthful in spite of their age and wrinkled visage. After dedicating 10 years to studying this phenomenon, they eventually isolated the one single yeast strain within 350 that created healthy, smooth and luminous skin. “ From a Saccharomycopsis yeast strain https://vogue.sg/sk-ii-late-night-portraits/  Saccharomycopsis fibuligera cell × morphology. b Pseudohyphae Saccharomycopsis fibuligera, also known Saccharomycopsis fibuligera as Endomyces fibuliger or Saccharomyces colony morphology fibuligera, is a yeast that produces ascospores and is widely found in all types of fermentation starters. https://www.researchgate.net/figure/Saccharomycopsis-fibuligera-cell-morphology-1040-magnification-a-Cell-morphology-b_fig1_350976586