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3.5 Identify and Annotate an Actual CRISPR-Cas System

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    • This is an optional problem for those wishing to test out working with the free, cloud-based software called Benchling, a platform to analyze and design DNA sequences.

      Lesson 1.4 CRISPR-Cas and Bacterial Adaptive Immunity described the basic genetic architecture of CRISPR-Cas systems, and in the lab, we analyze CRISPR arrays and CRISPR-associated (Cas) genes from new bacterial genomes all the time. Now, with the help of Benchling, you can explore and inspect the genes and CRISPR array of a ‘real’ CRISPR-Cas system—at the DNA level—taken from a biomedically relevant bacterial pathogen, namely, Streptococcus pyogenes. (The Cas9 protein from this bacterium, often called “SpyCas9”, is the very same Cas9 that has revolutionized genome editing worldwide.)

      Here’s a Benchling link to a portion of the S. pyogenes genome, where Professor Sternberg has annotated the CRISPR components. Please watch this brief video tutorial presented by Sanne Klompe, a molecular biologist and member of the Sternberg Lab, that will introduce you to Benchling and outline how the various DNA sequences and genes can be visualized. 

      Below are some questions that will help you think through the significance and interpretation of various aspects of this real-life CRISPR-Cas system. You may use the discussion board to pose questions about the problem and discuss the software. You can also upload answers to the questions to share with other World Science U users.

      1. How many different repeats and spacers does this CRISPR-Cas system from Streptococcus pyogenes have? What does that tell us, about the number of distinct viral pathogens this strain might have immunity to? (The word “might” is used purposefully; why might this bacterium be immune to more or less viruses?)
      2. The CRISPR array is flanked by CRISPR-associated (Cas) genes. The gene sequence of “Cas1” is “translated” in Benchling, so that each set of three DNA letters (aka each “codon”) is shown above the amino acid that it codes for, during translation of the messenger RNA into protein by the ribosome. Now, using a standard table of the genetic code for help (for more background, read here), list the first ten amino acids that would be “translated” by the ribosome during expression of the Cas9 protein from the Cas9 gene.
      3. Accurate gene expression depends on proteins being synthesized of a defined amino acid sequence and length. How long is the Cas9 protein, and what “signal” tells ribosomes when to stop, or terminate, production of the Cas9 protein during mRNA translation?
      4. Cas9 uses the guide RNA to target matching viral DNA sequences for degradation, thereby protecting the cell from infection. The Cas1 protein is completely dispensable for this RNA-guided DNA cutting reaction, and indeed, is also not used for gene editing. However, Cas1 is essential to CRISPR-Cas adaptive immunity, and is almost universally conserved in all CRISPR-Cas systems. Based on the way that CRISPR-Cas systems evolve to provide adaptive immunity, can you guess what Cas1 might be important for? (Hint: Google can help.)
    • July 8, 2021 at 3:51 pm

      Really interesting stuff, I bookmarked it for further study.

    • October 31, 2021 at 7:08 am

      Here is my shot at this:
      1. Repeats: 6+1 mutant; Spacers: 6; Pathogen immunity : ~6
      2. MDKKY SIGLD
      3. 1369 amino acids; TGA opal stop codon
      4. Four Cas1’s, together with two Cas2’s, form a stable heterohexameric complex that catalyzes spacer integration (via two transesterification reactions) and thus are responsible for the ability of the CRISPR immune system to adapt to new viral infections. Integrase that inserts the short DNA spacer fragments into the CRISPR array.

    • February 24, 2022 at 8:51 am

      Very cool. Thanks for sharing how some of the work is done.

    • September 18, 2022 at 12:54 am

      Optional problem.

    • January 28, 2023 at 3:53 am

      1. Repeats: 6 (+1 considering last repeat that has mutation), Spacers: 6
      2. MDKKYSIGLD
      3. Length of Cas9 gene: 4106bp or 1369 amino acids long, Stop codon: TGA
      4. Cas1 and Cas2 form a heterohexameric complex that catalyzes spacer integration via two transesterification reactions on each strand of a double-stranded prespacer substrate at the phosphodiester backbone within the CRISPR array.

    • October 30, 2023 at 9:09 am

      cool

    • August 29, 2024 at 12:15 am

      1. Repeats and Spacers:
      Count the spacers in the CRISPR array; each spacer represents a potential virus the bacterium might have immunity to. However, immunity may vary due to viral mutations.

      2. First 10 Amino Acids of Cas9:
      Use Benchling and a genetic code table to translate the first 10 codons of the Cas9 gene into amino acids.

      3. Cas9 Length and Stop Signal:
      The Cas9 protein is around 1,368 amino acids long. Translation stops at a stop codon (UAG, UAA, or UGA).

      4. Role of Cas1:
      Cas1 is crucial for integrating new viral DNA into the CRISPR array, enabling adaptive immunity against new viruses.

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