Download 26. The CRISPR-Cas System of Bacterial Immunity

BIMM 122 Lecture Notes #26
The CRISPR-Cas
System of Bacterial Immunity
Dr. Milton Saier
“Man's mind, once stretched by a new idea, never regains its original dimensions.”
– Oliver Wendell Holmes
1. Clustered regulatory interspaced short palindromic repeats (CRISPRs) target
invading phage and plasmids.
2. They are found in many bacteria and most archaea.
3. They deal with environmental stressors including invading viruses and plasmids
4. These systems are described as the “bacterial immune system.”
5. The CRISPR repeats incorporate “spacers” homologous to fragments of the alien
viral or plasmid genomes.
6. Possibly, the host incorporates short sequences from invading genetic elements
(DNA) (virus or plasmid) into the CRISPR region.
7. The mechanism might comprise a constantly changing, co-evolutionary “arms
race” between the host and invading agents.
8. The CRISPR and cas genes appear to evolve rapidly, accounting for their
tremendous sequence divergence.
9. The process is sequence-specific, involving a variable cassette of CRISPRassociated (cas) genes.
10. There are at least four cas genes that are unique to and may be conserved in
microorganisms having the CRISPR system.
11. The 4 Cas proteins of Yersinia (Csy 1-4) assemble into a 350 kDa
ribonucleoprotein complex; some other bacteria/archaea have more cas genes.
12. Cas proteins from various organisms may be homologous, having common
origins and structures.
13. Sequence similarity between these systems is meager, but they have conserved
structural features.
14. There appear to be three phylogenetically distinct groups of these systems, all
possibly related.
15. These complexes facilitate target nucleic acid recognition by the CRISPR-Cas
system.
16. This occurs because of sequence-specific hybridization between the CRISPRRNA and the target nucleic acid.
17. Target recognition is enthalpically driven (e.g., uses hydrophilic forces including
ionic and polar bonds).
18. Target recognition is localized to a “seed sequence” at the 5’ end of the CRISPR
RNA spacer.
19. The Cas complex is a crescent shaped particle in many bacteria including E. coli.
20. When these sequences are transcribed and processed with small (s)RNAs, they
guide the Cas system to recognize, bind to and cleave the invading genetic
material.
21. This “adaptive immunity system” (at the nucleic acid rather than the protein
levels) uses a library of small non-coding RNAs as a potential weapon against
fast-evolving viruses and plasmids.
22. The system reveals the increasingly dynamic nature of prokaryotic genomes.
23. There are CRISPR polymerases, and “Repeat-associated Mysterious Proteins”
(RAMPs), containing RNA recognition motif (RRM) domains.
24. CRISPR-Cas promotors are regulated, among other regulators, by H-NS, which is
involved in induction, and by a chaperone protein, HtpG (High temperature
protein G).
25. It has been suggested that these systems utilize a (poorly defined) Lamarckian
mechanism.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated (Cas) systems protect prokaryotes from invading viruses and plasmids. These
defense systems rely on small RNAs for sequence-specific detection and silencing of
foreign nucleic acids. CRISPR/Cas systems are composed of cas genes organized in
operon(s) and CRISPR array(s) consisting of genome-targeting sequences (called
spacers) interspersed with identical repeats. CRISPR/Cas-mediated immunity occurs in
three steps. In the adaptive phase, bacteria and archaea harboring one or more CRISPR
loci respond to viral or plasmid challenge by integrating short fragments of foreign
sequence (protospacers) into the host chromosome at the proximal end of the CRISPR
array. In the expression and interference phases, transcription of the repeat-spacer
element into precursor CRISPR RNA (pre-crRNA) molecules followed by enzymatic
cleavage yields the short crRNAs that can pair with complementary protospacer
sequences of invading viral or plasmid targets. Target recognition by crRNAs directs the
silencing of the foreign sequences by means of Cas proteins that function in complex
with the crRNAs.
There are three types of CRISPR/Cas systems. The type I and III systems share
some overarching features: specialized Cas endonucleases process the pre-crRNAs, and
once mature, each crRNA assembles into a large multi-Cas protein complex capable of
recognizing and cleaving nucleic acids complementary to the crRNA. In contrast, type II
systems process pre-crRNAs by a different mechanism in which a trans-activating
crRNA (tracrRNA) complementary to the repeat sequences in pre-crRNA triggers
processing by the double-stranded (ds) RNA-specific ribonuclease RNase III in the
presence of the Cas9 protein. Cas9 is thought to be the sole protein responsible for
crRNA-guided silencing of foreign DNA.
In type II systems, Cas9 proteins constitute a family of enzymes that require a
base-paired structure formed between the activating tracrRNA and the targeting crRNA
to cleave target dsDNA. Site-specific cleavage occurs at locations determined by both
base-pairing complementarity between the crRNA and the target protospacer DNA and a
short motif [referred to as the protospacer adjacent motif (PAM)] juxtaposed to the
complementary region in the target DNA. The Cas9 endonuclease family proteins can be
programmed with single RNA molecules to cleave specific DNA sites, thereby raising
the possibility of developing a simple and versatile RNA-directed system to generate
dsDNA breaks for genome targeting and editing (Jinek et al. 2012)).
“Evolution, like the tinker, does not produce innovations from scratch. It works on what
already exists, transforming a system to give a new function, or combining several
systems to produce a more complex one.”
- F. Jacob from “The Possible and the Actual”
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