CRISPR Technology Lecture Notes

Discovery and Origins of CRISPR

• The CRISPR field emerged in the mid-2000s with the discovery of repetitive DNA sequences in bacteria.
• These sequences were termed CRISPRs, an acronym for "clusters of regularly interspaced short palindromic repeats."
• CRISPRs are a distinctive feature in bacterial chromosomes, consisting of repetitive sequences (black diamonds) flanking unique sequences (colored boxes).
• Three research teams discovered that the unique sequences in CRISPR arrays often originate from viral DNA, suggesting an acquired immune system in bacteria.
• CRISPR-associated (Cas) genes, typically located near CRISPR arrays, encode proteins that are part of this adaptive immune system.
• Jillian Banfield's research at Berkeley revealed the abundance of CRISPR elements in diverse environmental bacteria, indicating their active use in viral defense.

Mechanism of CRISPR Immunity

• Viruses inject DNA into bacteria.
• Bacteria can acquire viral DNA fragments and integrate them into CRISPR arrays, maintaining the pattern of repeats.
• The cell copies these sequences into RNA molecules, which are then broken into smaller units, each containing a virus-derived sequence and a repeat sequence.
• These RNAs are marked as CRISPR molecules by the repeat sequence and combine with a second RNA called tracrRNA to form a structure that binds to the Cas9 protein.
• The resulting RNA-protein complex surveys the cell for DNA sequences matching the RNA sequence.
• Upon finding a match, the complex unwinds the DNA to allow RNA-DNA hybridization, inducing a double-stranded break in the DNA.

Harnessing CRISPR for Gene Editing

• In bacteria, double-stranded breaks lead to viral DNA degradation, protecting the cell.
• In eukaryotic cells (plant or animal cells), the machinery repairs double-stranded breaks.
• This repair can introduce small changes at the break site or integrate new DNA.
• Introducing targeted double-stranded breaks in plant and animal genomes is an effective method for genome engineering.
• This technology has spread across various areas of biology, enabling research and applications previously difficult or impossible.

Further Research Directions

• Further understanding the mechanism of RNA-guided DNA cutting to improve applications.
• Investigating new CRISPR pathways and their interactions with other bacterial proteins.
• Studying anti-CRISPR proteins, found in cells and viruses, that inhibit CRISPR pathways.

Societal Implications

• CRISPR technology grants humans significant power to control the evolution of organisms and potentially our own evolution.
• This power raises ethical considerations and the need for caution and respect when using this technology.

Adaptive Immunity in Bacteria

• Bacteria possess an adaptive immune system, analogous to antibodies in more complex organisms.
• This system works differently, using acquired sequences integrated into CRISPR arrays.
• These sequences become templates for RNA molecules that include copies of integrated viral DNA and associated repeat sequences.
• The repeats are often palindromic and can form hairpin structures recognized by CRISPR Cas proteins.
• The cell labels these RNAs as CRISPR RNAs using unique, unstructured sequence tags.
• Processed RNA molecules combine with Cas proteins to form surveillance complexes that search for matching DNA or RNA sequences, leading to the degradation of foreign nucleic acids.

Classification of CRISPR Systems

• CRISPR systems are classified into two broad categories: class one and class two.
• Class one systems include multiple Cas proteins that assemble with CRISPR RNAs to form surveillance complexes.
• Class two systems include a single gene encoding a large protein that combines with CRISPR RNAs to provide protection.
• Class two systems are simpler and easier to harness for gene editing purposes.

Cas9 Function

• Cas9 is a protein that recognizes double-stranded DNA at positions matching a 20-nucleotide sequence in the guiding RNA.
• The guide RNA is often in a single guide form, combining the natural CRISPR RNA and tracer RNA.
• Cas9 makes a blunt double-stranded break at a precise location in the DNA.

Challenges and Questions

• How does Cas9 unwind DNA without an external energy source?
• How does it deal with chromatin and find the correct sequences in eukaryotic cells?
• What happens when it binds to the wrong site?
• Can we prevent it from accessing the wrong sites and make it more accurate?
• Can we harness its DNA recognition activities for other purposes, such as DNA imaging or regulating gene expression?

Conformational Changes in Cas9

• Cas9 undergoes conformational changes upon binding to nucleic acids.
• Crystallographic structures show Cas9 rotating to open a channel for the guide RNA.
• Additional structural changes occur when the protein binds to a DNA molecule, accommodating the RNA-DNA hybrid.
• The HNH domain, which cuts the DNA strand that base pairs with the guide RNA, must be properly positioned to make a cut.

Structure of Cas9 Bound to Double-Stranded DNA

• Cryo-electron microscopy captured a structure of Cas9 bound to its true substrate, a double-stranded DNA molecule.
• The protein opens up the DNA, forming a duplex with the guide RNA.
• The non-target strand is located on the surface of the protein.
• The HNH domain is positioned near where it needs to be to cut the DNA.

Sensing DNA Complementarity

• Cas9 acts as a sensor, detecting the degree of match between the target DNA and the guide RNA.
• This sensing is conveyed through conformational changes in the protein.
• The protein goes through a series of steps to reach an active state, starting in an inactive form, transitioning through an intermediate state, and finally reaching a fully docked state before cutting the DNA.
• This process is used to design versions of Cas9 that are more accurate sensors of target DNA.

Anti-CRISPR Proteins

• Anti-CRISPR proteins inhibit CRISPR pathways and are encoded by host organisms.
• These proteins are typically small (under 100 amino acids) and inhibit differently.

Mechanism of Anti-CRISPR Proteins

• One anti-CRISPR protein (C1) blocks DNA cutting.
• Experiments show that C1 does not prevent Cas9 from binding to the DNA substrate but prevents it from being cut.
• The HNH domain is responsible for protein-protein association.
• The anti-CRISPR protein physically blocks the chemically important residues in the HNH domain, preventing them from interacting with DNA.
• This inhibitor prevents the HNH domain from swinging into place and cutting the DNA.

Technological Implications of Anti-CRISPR

• This mechanism is an example of a natural inhibitor that can trap the protein and its guide RNA on a DNA target.
• Blocking DNA cutting by Cas9 allows it to function in a regulatory fashion, like effector proteins that repress or activate transcription.
• This allows bacteria and phages to expand the function of Cas9 naturally in cells by blocking its ability to cut DNA but retaining its ability to bind in an RNA-guided fashion.

Responsible Progress

• It is important to consider the societal implications of this technology moving forward.