lect 24 crispr

CRISPR-Cas System in bacteria function

naturally occurring bacterial defense system; adaptive immune system against viruses

all CRISPR-cas systems contain:

1. CRISPR array

2. Genes encoding Cas proteins

The CRISPR array

repeated palindromic sequence (24-48bp long) that has stretches of protospacers between the repeats

Protospacers

segments of foreign DNA (like from viruses) that bacteria capture and incorporate into their CRISPR arrays

The CRISPR Locus

contains spacer DNA and repeated DNA

contains genes for Cas9 and tracrRNA

Cas9

endonuclease) destroys nucleic acid in the middle of the molecules (not the end)○Makes double-stranded cuts in DNA

TracrRNA

trans-activating CRISPR RNA

MAIN IDEA of crispr system

the bacteria wants Cas9 to be targeted to viral DNA toc ut it into pieces & destroy it.

Steps of natural crispr

1. Bacteria transcribe long RNA that includes repeats & protospacres

2. Cas endonuclease cuts longRNA into pieces calledCRISPR RNAs (crRNAs)

3. Each crRNA hybridizes totransactivating CRISPR RNA(tracrRNA

4. Each RNA hybrid binds Cas9 nuclease to form an effector complex

5. Effector complex targets viral DNA sequence for destruction

PAM Sequence

stops effector complex from binding to CRISPR array in genome &cleaving genome into pieces

What is the PAM sequence needed for

for the effector complex to find a target

How does PAM work

Provides an initial binding site for effector complex (then unwinds virus andsees if DNA is complementary); if a nearby sequence is complementary tocrRNA, Cas9 cuts DNA

What is the role of the tracrRNA in the CRISPR system?

It binds to both the crRNA and Cas9, thereby facilitating the assembly of the effectorcomplex

What is one difference between the naturallyoccurring CRISPR system and the engineeredCRISPR system used in the lab?

There is a single guide RNA that interacts with Cas9 in the engineered system

Engineered crispr

• Single guide RNA (sgRNA) binds to Cas9 to form an effector complex

• Researchers target any site in the genome by creating sgRNA with the correct complementary sequence

After the Cas protein cuts the target DNA…

DNA repair mechanism mustfix the break

NHEJ= Nonhomologous End Joining

ligating the broken DNA ends together without a homologous template

HDR= Homology-Directed Repair

fixes double-strand breaks (DSBs) by using a homologous DNA template, like a sister chromatid or an introduced donor DNA

DNA editing requires…

homology-directed repair

DNA repair involves

using DNA as a template to fix the break made by the Cas protein

crispr summary

1. Cas9 protein binds to sgRNA to create an effector complex; Contains target genomic sequence

2. Effector complex scans DNA &stops at each PAM

3. DNA unwound; if sgRNA binds its complement, Cas9 will cut DNA

4. Cellular DNA repair proteins can fix cuts

Benefits of crispr

1. Cuts can be made anywhere in the genome.

2. It can be used to edit the genome.

3. The function of Cas9 can be modified

crispr challeneges

1. Unanticipated off-target modifications

2. Mosaicism

3. Delivering CRISPR components to cells