Literature Learning Module

Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease 

Assessment of Biochemistry/Molecular Biology (BMB) Foundational Concepts

Henry Jakubowski, Ph.D., Professor, Chemistry Department, College of Saint Benedict/Saint John's University

 
05/30/2014

The questions below are based on data, graphs, and figures from the following article:

Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease.   Hongfan Chen, Jihoon Choi and Scott Bailey.The Journal of Biological Chemistry, 289, 13284-13294 (2014) . 

These guidelines apply to the reuse of articles, figures, charts and photos in the Journal of Biological Chemistry, Molecular & Cellular Proteomics and the Journal of Lipid Research.

These questions may be used to help assess student mastery of key foundational concepts in BMB developed by the American Society for Biochemistry and Molecular Biology (ASBMB) and tested in the new MCAT2015 developed by the American Association of Medical Colleges.  The particular foundational concepts and learning objectives relevant for this particular assessment are listed below.

ASBMB Biochemistry and Molecular Biology Foundational Concepts  -   Detailed Learning Objectives (Excel)

Foundational Concept 2:  Macromolecular structure determines function and regulation.

  • 2. Given a list of macromolecules, students should be able to devise an experiment on how they interact or interpret results of experiments on their interactions
  • 3.  Given structural changes of a macromolecule (or ligand), students should be able to predict the impact of structural substitution would have on macromolecule structure and function
  • 4. Given experimental data, students should be able to assess how enzymes facilitate biochemical reactions.

Foundational Concept 3:  Information storage and flow are dynamic and interactive

  • 2. Given an understanding of common mechanisms of gene regulation, students will be able to explain or predict changes in transcription in response to biologic variables.
  • 3. Given an understanding of genetic information transfer, students should be able to explain the role of RNA in the flow of genetic information 

Foundational Concept 4: . Discovery requires objective measurement, quantitative analysis, and clear communication. 

  •  2. Given a fundamental understanding of BMB concepts, students should be able to formulate experiments and assess the quality of experiments addressing molecular structure, assays of biological function, and isolation / separation of biomolecules.
  • 3. Given a data set, students should be able to assess the reliability of the data and draw appropriate conclusions.
  • 4. Given a set of data, students should be able to appropriately present and interpret the data.

MCAT 2015:  Foundational Concepts, Detailed Content and Topics (Excel)

Biological and Biochemical Foundations of Living Systems

Foundational Concept 1 : Biomolecules have unique properties that determine how they contribute to the structure and function of cells, and how they participate in the processes necessary to maintain life

  • 1A. Structure and function of proteins and their constituent amino acids

  • 1B. Transmission of genetic information from the gene to the protein

Chemical and Physical Foundations of Biological Systems

Foundational Concept 5: The principles that govern chemical interactions and reactions form the basis for a broader understanding of the molecular dynamics of living systems.

  • 5B. Nature of molecules and intermolecular interactions

  • 5D. Structure, function, and reactivity of biologically - relevant molecules

Scientific Inquiry and Reasoning Skills

  • 1.  Knowledge of Scientific Concepts and Principles; 

  • 2.  Scientific Reasoning and Problem;

  • 3.  Reasoning about the Design and Execution of Research;

  • 4.  Data-based and Statistical Reasoning

Background Reading:

Background Videos:

Background and Review

A promising tool for genome manipulation and regulation  in a wide variety of organisms has recently been identified in the RNA-guided DNA endonuclease activity of the CRISPR-Cas (clustered regularly interspaced short palindromic repeat–CRISPR-associated) system. CRISPR-Cas, an inheritable prokaryotic immune system, protects bacteria and archaea against mobile genetic elements via RNA-guided target silencing. CRISPR-Cas systems consist of an array of short direct repeats interspersed by variable invader-derived sequences (spacers)  and a cas operon. During invasion, small fragments of the invading DNA from phage or plasmids (protospacers) are incorporated into host CRISPR loci, transcribed, and processed to generate small CRISPR RNAs (crRNA). The invading nucleic acid is then recognized and silenced by Cas proteins guided by the crRNAs. There are three types of CRISPR-Cas system each characterized by the presence of a signature gene.

Programmed DNA cleavage requires the fewest components in the type II CRISPR-Cas system, requiring only crRNA, a trans-activating crRNA (tracrRNA), and the Cas9 endonuclease, the signature gene of the type II system. The system can be further simplified by fusing the mature crRNA and tracrRNA into a single guide RNA (sgRNA). In addition to its role in target cleavage, tracrRNA also mediates crRNA maturation by forming RNA hybrids with primary crRNA transcripts, leading to co-processing of both RNAs by endogenous RNase III. Cas9 contains two nuclease domains that together generate a double-strand (ds) break in target DNA. The HNH nuclease domain cleaves the complementary strand, and the RuvC-like nuclease domain cleaves the noncomplementary strand.

A short signature sequence, named the protospacer adjacent motif (PAM), is characteristic of the invading DNA targeted by the type I and type II CRISPR-Cas systems. The PAM serves two functions. It has been linked to the acquisition of new spacer sequences, and it is necessary for the subsequent recognition and silencing of target DNA. The sequence, length, and position of the PAM vary depending on the CRISPR-Cas type and organism. PAMs from type II systems are located downstream of the protospacer and contain 2–5 bp of conserved sequence. A variable sequence, of up to 4 bp, separates the conserved sequence of the PAM from the protospacer. This variable region is often included in the definition of the PAM sequence, but for simplicity, we refer to this variable region as the linker and the conserved sequence as the PAM. To date, Cas9 from Streptococcus pyogenes, Cas9 from Streptococcus thermophilus DGCC7710, and Cas9 from Neisseria meningitidis have been employed as tools for genome editing or regulation. For these Cas9 orthologs, the PAMs are GG, GGNG, and GATT, and the linkers are 1, 1, and 4 bp, respectively. 

The simplicity of sgRNA design and sequence-specific targeting means the RNA-guided Cas9 machinery has great potential for programmable genome engineering. Cas9 can be employed to generate mutations in cells by introducing dsDNA breaks. The capabilities of Cas9 can be expanded to various genome engineering purposes, such as transcription repression or activation, with its nickase (generated by inactivating one of its two nuclease domains) or nuclease null variants. Another appealing possibility for the Cas9 system is to target different Cas9-mediated activities to multiple target sites, for example transcriptional repression of one gene but activation of another. To achieve this, multiple Cas9 orthologs will need to be employed as a single ortholog cannot concurrently mediate different activities at multiple sites. Therefore to broaden our understanding of Cas9 proteins, we have characterized the Cas9 ortholog from S. thermophilus LMG18311, which we refer to as LMG18311 Cas9.

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