CRISPR

DSB: Explain homology-dependent repair (HDR).

- What cells are selected to do (over NHEJ) - Use homologous sequences from the other chromosome to repair mutation without error in sequence at break site - Based on recombination - Based on source - can repair with no change, or repair break with whatever allelic variant was on other chromosome (if heterozygous for this gene)

CRISPR was first identified in _____. The two main parts of CRISPR DNA are _____, which are short segments of DNA that are palindromes, meaning transcribed RNA can form ______. These are all ___ and interspaced, and used to mark off and help bacteria find ____. The other parts are ______, which are between repeats. Each segment between repeats is ____, not identical; each piece of spacer DNA matches with viral DNA from _____. Also apart of this system are ___-____ genes (___) genes, which code for ___ proteins. The two main types function as ___ that unwind DNA and ___ that cut DNA.

CRISPR was first identified in E COLI. The two main parts of CRISPR DNA are REPEATS, which are short segments of DNA that are palindromes, meaning transcribed RNA can form HAIRPINS. These are all IDENTICAL and interspaced, and used to mark off and help bacteria find SPACERS. The other parts are SPACERS, which are between repeats. Each segment between repeats is UNIQUE, not identical; each piece of spacer DNA matches with viral DNA from BACTERIOPHAGES. Also apart of this system are CRISPR-ASSOCIATED genes (CAS) genes, which code for CAS proteins. The two main types function as HELICASES that unwind DNA and NUCLEASES that cut DNA.

What are the two main limitations of gene drives?

For a gene drive to spread through a population it must be used on a sexually reproducing species with a relatively short generation time For the gene drive to convert all of the population, it needs to be 100% effective - NHEJ occurring at a low percentage, disrupting the recognition site for CRISPR and rendering that gene copy immune to conversion - NHEJ occurrences prevent the integration of the HDR containing cas9 and gRNA into the second chromosome

How does the CRISPR/Cas9 system work in general? How can it be used to insert a new gene?

In general: 1. Create a gRNA with corresponding RNA to the section of DNA you want to cut 2. gRNA/Cas9 complex - DNA feeds in, unwinds, and binds at corresponding section to gRNA to be cut 3. Cas9 cuts DNA at section corresponding to gRNA on both strands - this inactivates gene 4. Cell usually responds by mutations (insertions/deletions) to splice DNA back together Inserting a new gene: 1. Need Cas9, gRNA, and DNA to be inserted into genome 2. Follow steps as in normal system 3. After genomic DNA is cut, the DNA will be inserted at the genomic DNA cut, and host DNA repair system will fix by adding the matching bases

How do gene drives work?

Normally - Cas9 and guide RNA genes are on a vector that doesn't replicate and therefore is not passed to offspring These two genes can be put into the homologous DNA that is being added for HDR along with what gene is targeted (payload gene) Then, the cas9 gene and gRNA are copied into each new cut by HDR - First - one chromosome is converted to the gene drive allele - Second - the second chromosome is cut by Cas9 expressed from the first chromosome - Third - the second chromosome is converted by recombination with the first This converts the individual to a homozygous with both copies having the gene drive allele When this individual mates with a WT individual, gamete formation and fertilization occur as normal Now, though, the new zygote expresses Cas9 and gRNA - can be used to copy cas9 and gRNA into the other chromosome Converts the second chromosome to the gene drive allele by recombination with the first

T or F: Bacteria and archaea can have the ability to acquire long-term immunity to threats they experience. T or F: Widely spread throughout bacteria (almost half of all species examined have CRISPR) - perhaps greatest horizontal gene transfer found. T or F: Provides selective disadvantage. T or F: Mechanism is the same throughout all species CRISPR is found in. T or F: Similarities to RNAi (almost certainly due to convergent evolution) might suggest possible uses in the lab .

TRUE TRUE FALSE - selective advantage FALSE - many variants with differing mechanisms TRUE

The CRISPR/Cas9 system is an ___ _____ ____. The ability to break DNA at any sequence indicates the ability to ___ DNA at any sequence. A nucleic acid can direct a nuclease to almost any sequence in any genome. This is similar to the function of ___ ____, although those enzymes are specific to sequences. This system is guided by the RNA (not the other way around), so it can be ___ to be more flexible to change ANY sequence. It is basically a ___ ___ guided by RNA.

The CRISPR/Cas9 system is an RNA-GUIDED NUCLEASE. The ability to break DNA at any sequence indicates the ability to CHANGE DNA at any sequence. A nucleic acid can direct a nuclease to almost any sequence in any genome. This is similar to the function of RESTRICTION ENZYMES, although those enzymes are specific to sequences. This system is guided by the RNA (not the other way around), so it can be ENGINEERED to be more flexible to change ANY sequence. It is basically a RESTRICTION ENDONUCLEASE guided by RNA.

Through gene drives, all offspring are converted to ____ and all of their offspring in subsequent generations will be ____. This will spread the engineered allele through the ____ as a ____ allele, as long as the population is fast-____ and ____-reproducing. Gene drives could be potentially used to _______.

Through gene drives, all offspring are converted to HOMOZYGOUS and all of their offspring in subsequent generations will be HOMOZYGOUS. This will spread the engineered allele through the POPULATION as a DOMINANT allele, as long as the population is fast-BREEDING and SEXUALLY-reproducing. Gene drives could be potentially used to WIPE OUT A WHOLE SPECIES.

How does CRISPR hold a history of a bacterial cell's past infections?

each spacer in the system is DNA integrated by Cas proteins from past infections this way, when the same bacteria injects the same DNA into the cell, this spacer can be transcribed and excised to base pair with the viral DNA in the Cas complex to break down the viral DNA

What is the evolutionary function of the CRISPR system in bacteria?

immune system defense against bacteriophages

What is the PAM sequence?

in gRNA, a section of DNA bases in the spacer that can't base pair to target DNA

What are the two things that can happen when DNA undergoes a double strand break?

non-homologous end-joining (NHEJ) homology-dependent repair (HDR)

What is guide RNA?

tracrRNA + crRNA instead of base paired to each other, one long RNA molecule

True or False: Both CRISPR and Cas genes are a part of the bacterial genome.

true

True or False: CRISPR is a hereditary, acquired immunity system in bacteria.

true

What happens in a viral infection if the bacteriophage injects DNA that the bacterial cell does not have a matching spacer for?

Cas1 and Cas2 proteins form a complex, find and bind the invading viral DNA Complex binds the viral DNA, breaks it apart, and then copies it into the CRISPR system of the bacterial genome at the ends of repeats as a spacer This viral DNA becomes a new spacer in the CRISPR system

DSB: Explain non-homologous end-joining (NHEJ).

- Enzymes stick ends back together without regard for sequence - Mistakes are made; insertions and deletions randomly occur (indels) - Last-ditch repair effort - can lead to frameshifts and cause the allele of the gene to become non-functional (knocks out gene) - Happens at a certain frequency, more so when double stranded breaks stay broken

A ____ ____ not only makes modifications to the individual organism being engineered but also to all of its descendants. The offspring don't just inherit the change, DNA inherited from their other parent is subsequently ____ as well. These changes are much more rapid than ___ genetics.

A GENE DRIVE not only makes modifications to the individual organism being engineered but also to all of its descendants. The offspring don't just inherit the change, DNA inherited from their other parent is subsequently CHANGED as well. These changes are much more rapid than MENDELIAN genetics.

After the CRISPR system functions on a targeted gene, HDR may occur. To make sure the targeted gene is knocked out, you could add a lot of ____ into the cell so the targeted gene is cut ____ until NHEJ causes an ____ and an inactivated, knocked out gene. What is better solution to make sure the gene is knocked out and even insert other genes in?

After the CRISPR system functions on a targeted gene, HDR may occur. To make sure the targeted gene is knocked out, you could add a lot of CRISPR/CAS9 into the cell so the targeted gene is cut A LOT until NHEJ causes an INDEL and an inactivated, knocked out gene. What is better solution to make sure the gene is knocked out and even insert other genes in? - put in an engineered copy of homologous DNA and let this be used for HDR instead of the matching chromosome - We can use ANY DNA, meaning we can insert genes between homologous ends that match to cut genomic DNA - At the same time, can inactivate gene by breaking it apart and insert genes at break to be expressed for some function - Don't have to inactivate a gene, can be done in other regions too

After the CRISPR system functions on a targeted gene, the gene has a ____. If NHEJ occurs after this, what happens? If HDR occurs, what happens?

After the CRISPR system functions on a targeted gene, the gene has a DSB. If NHEJ occurs after this, what happens? -can lead to frameshift mutation and gene may already be knocked out If HDR occurs, what happens? -gene may not be knocked out

How would a bacteriophage infection play out if bacteria did not have the CRIPSR system?

Bacteriophage inserts its DNA, hijacks the cells Viral DNA becomes imbedded in bacterial genome Transcribed viral DNA by bacterium makes a bunch of new phages that kill the cell (lytic cycle)

Effects of CRISPR Gene Editing: All DNA matching the guide RNA will be cut (all copies in a diploid/polyploid organism) with high efficiency, creating _____ directly. Repair mechanisms will allow most cells to survive; what repair occurs determines result. Efficiency is high enough that ____ isn't always required, the desired result can be screened for; multiple ___ can be made at once. (This means that you don't always need an ____ ___ gene; screening can be used) If germ line cells are affected, the changes are _____ (Mendelian).

Effects of CRISPR Gene Editing: All DNA matching the guide RNA will be cut (all copies in a diploid/polyploid organism) with high efficiency, creating HOMOZYGOTES directly. Repair mechanisms will allow most cells to survive; what repair occurs determines result. Efficiency is high enough that SELECTION isn't always required, the desired result can be screened for; multiple CHANGES can be made at once. (This means that you don't always need an ANTIBIOTIC RESISTANCE gene; screening can be used) If germ line cells are affected, the changes are _____ (Mendelian).

True or False: The Cas9 complex integrates new phage DNA into genome (CRISPR system), and then Cas1/Cas2 uses CRISPR system to cut phage DNA and end infection.

FALSE Cas9 - cuts phage DNA using CRISPR system Cas1/Cas2 - integrates new phage DNA into genome

How can you instigate the CRISPR system in a cell? What are the reagents needed and the two options for methods?

Reagents: Cas9 protein and a guide RNA, also need to insert HDR template as well if goal is to insert new genes Option A: Microinjection of these elements into a cell using a very fine capillary pipette Option B: Use gene entry mechanism of your choice to get the genes coding for Cas9 and your guide RNA (and HDR template DNA) into the cell on a plasmid - Transformation, electroporation - The cell itself transcribes the cas9 and guide RNA genes and translates the cas9 mRNA

The CRISPR/Cas9 system involves using the CRISPR system in bacteria as a ___ ____ tool. it was first engineered from ___ ____. Cas9 is a nuclease that has two ___ ____ ___. In this system, two types of RNA are used: ____, which comes from spacer segments in the CRISPR system of genome, and _______, which holds crRNA in place and has its own promoter from the rest of the crRNA. Instead of using two separate molecules, biologists can create a tracrRNA-crRNA ____ by combining these two types of RNA; this is also called ______.

The CRISPR/Cas9 system involves using the CRISPR system in bacteria as a MOLECULAR ENGINEERING tool. it was first engineered from STREPTOCOCCUS PYOGENES. Cas9 is a nuclease that has two DNA-CUTTING DOMAINS. In this system, two types of RNA are used: crRNA, which comes from spacer segments in the CRISPR system of genome, and tracrRNA, which holds crRNA in place and has its own promoter from the rest of the crRNA. Instead of using two separate molecules, biologists can create a tracrRNA-crRNA CHIMERA by combining these two types of RNA; this is also called GUIDE RNA (gRNA).

To function, Cas9 needs crRNA. crRNA is transcribed from ___ and ___, resulting in a long ____ molecule that contains the entire system. ____ is processed and cut up into individual ___ elements. The Cas9 protein will form a complex with the matching crRNA to the DNA from the invading phage. These spacer elements contain the spacer and the ____ (this is ____ crRNA). Mature, matching crRNA combines with ____, which is also coded for in the CRISPR system but has its own ____. crRNA base pairs with _____ for part of the length in ____ section of crRNA. The spacer itself remains single-stranded and not base paired together, only the ___ and ____ are based paired within Cas9. This allows the spacer to be open and can bind to phage DNA by invading the closed ____. Cas9 used crRNA as a ____ to cut phage DNA. When phage DNA base pairs with spacer, Cas9 cuts both strands: this is called a ____ ___ ____. This means that Cas9 can be directed to cut any sequence of DNA as long as it ______.

To function, Cas9 needs crRNA. crRNA is transcribed from REPEATS and SPACERS, resulting in a long PRE-crRNA molecule that contains the entire system. PRE-crRNA is processed and cut up into individual SPACER elements. The Cas9 protein will form a complex with the matching crRNA to the DNA from the invading phage. These spacer elements contain the spacer and the REPEAT (this is MATURE crRNA). Mature, matching crRNA combines with tracrRNA, which is also coded for in the CRISPR system but has its own PROMOTERS. crRNA base pairs with tracrRNA for part of the length in REPEAT section of crRNA. The spacer itself remains single-stranded and not base paired together, only the REPEAT and tracrRNA are based paired within Cas9. This allows the spacer to be open and can bind to phage DNA by invading the closed dsDNA. Cas9 used crRNA as a GUIDE to cut phage DNA. When phage DNA base pairs with spacer, Cas9 cuts both strands: this is called a DOUBLE STRAND BREAK. This means that Cas9 can be directed to cut any sequence of DNA as long as it iS INTEGRATED AS A SPACER IN crRNA.

What CRISPR and Cas do in the event of a bacteriophage infection: - Bacteriophage inserts its DNA into bacterial cell - ___ genes are transcribed/translated to form ___ protein complex made of ___ and ___ - CRIPSR DNA (repeats and spacers) translated as ____ (___ RNA), which fits inside the ___ ____ ____ - If the DNA injected by the phage matches one of the ____ on the crRNA, the viral DNA will enter the protein complex, base pair with the ___ and be ____ ___ the complex - ends and prevents the infection - this is performed by ___

What CRISPR and Cas do in the event of a bacteriophage infection: - Bacteriophage inserts its DNA into bacterial cell - CAS genes are transcribed/translated to form CAS protein complex - CRIPSR DNA (repeats and spacers) translated as crRNA (CRISPR RNA), which fits inside the CAS PROTEIN COMPLEX - If the DNA injected by the phage matches one of the SPACERS on the crRNA, the viral DNA will enter the protein complex, base pair with the crRNA and be BROKEN UP the complex - ends and prevents the infection - this is performed by CAS9

True or False: Using an engineered piece of DNA for HDR competes with other methods (NHEJ and HDR using other chromosome), but eventually after multiple cutting events the engineered copy with insert genes will be used in HDR to fix the break.

true