crispr- lecture 6

Question 1

every human is born with around how many de novo mutations

Answer 1

70-80

Question 2

“simple” genetic traits

Answer 2

those that are determined by a single gene

Question 3

polygenic

Answer 3

have their roots in variation at many loci

Question 4

gene therapy

Answer 4

We attempt to address the problem at the level of its root cause: at the gene

Question 5

sickle cell disease (SC)

Answer 5

This was first identified in Chicago in 1910 when physician James Herrick observed weirdly “sickle shaped” red blood cells (RBCs) in the blood of
Walter Clement Noel, a dentistry student from Grenada in the West Indies. red blood cells are typically circular, donut shaped. On the basis of the difference between sickled RBCs and normal ones, SC was, in the late 1940’s, the first disease
to be attributed to a simple molecular difference, in this case between ‘normal’ hemoglobin, the oxygen-transporting molecule in RBCs, and sickling hemoglobin. Within ten years, scientists had identified the key difference, a single amino acid.

People with SC, have a single basepair mutation in their beta chains resulting in the substitution of a valine for a glutamic acid. It turns out that, under certain conditions, including a lack of oxygen, the presence of valine causes the globin molecules to stick together, to polymerize into long, relative rigid molecular cables. It’s these that cause the shape of the RBCs to distort. Sickle-shaped cells do not travel readily through capillaries, causing blockages and associated damage. People who are homozygotes for the SC allele experience many painfully debilitating symptoms and, despite improvements in therapies for containing or ameliorating the symptoms, a reduced life expectancy. It can be cured with a bone marrow transplant – red blood cells are produced by hematopoietic cells in the bone marrow – but this requires a close genetic match between recipient and donor, and is itself a risky procedure

Question 6

tetramer

Answer 6

hemoglobin is a tetramer, made up of four
protein molecules (plus the ‘heme’ group – the iron that is key to its functioning). The tetramer consists of two “alpha” globin and two “beta”
globin chains. These are derived from two very similar genes (indeed, the two genes are evolutionarily related, being derived from an ancient gene duplication of an ancestral globin gene)

Question 7

heterozygote advantage

Answer 7

Having an SC allele provides significant
protection against malaria

In a malarial region, the SA genotype is an at advantage over the SS and AA ones. Hence the surprisingly high frequency of S alleles,
especially in populations that either are currently, or historically have been, exposed to malaria. For people in the US derived ancestrally from African populations, the S allele may be an ever-present burden

Question 8

in vivo approach

Answer 8

most effective in situations in we can access the target cells in a highly constrained space

Question 9

examples of gene therapy

Answer 9

Targeting (in vivo/in vitro)
Germline genome modification (inject in embryo)
genome editing (change flawed sequence)

Question 10

how can targeting be bad

Question 11

how can embryonic genome editing not work for sickle cell

Answer 11

if someone is diagnosed at some age, you cant edit an “embryo”

Question 12

explain the assembly of the crrna/cas complex

Answer 12

the crispr locus is transcribed into a long crispr rna precursor

this precursor is then processed by cas enzymes and rnase 111 to produce short strands of crrnaa one for each spacer sequence

Question 13

the cut will only occur if the targer dna contains a

Answer 13

protospacer adjacent motif (pam),a 3 nucleotide long sequence a few bases downstream of the target dna

Question 14

once pam is found what happens

Answer 14

if a target dna sequence is present complementary to the crrna the crrna will anneal to the dna bringing the tracrrna and cas9 complex along with it

the cas9 then employs its nuclease activity to make a specific cut in the target dna

Question 15

two parts of cas9 complex

Answer 15

dna cutting protein cas9 enzyme and single guide rna

Question 16

steps of cas9

Answer 16

1) locate and bind to common sequence in the genome called a pam. once the pam is bound the guide rna unwinds part of the double helix

2) once the correct sequence is found cas9 can cut the dna- its two nuclease domains each make a nick leading to a double strand break (although the cell will try to repair the break the fixing process is error prone and could introduce mutations that disable the gene so crispr is useful for knocking out specific genes)

3) homology directed repair (use preexisting sequence as template to make repair)

Question 17

progeria

Answer 17

base pair substitution, synonymous mutation
changes splicing of the protein (exon intron junctions)

Question 18

examples of engineered nucleases for specific cut

Answer 18

Zinc Finger Nuclease (ZFN)
Transcription Activator-Like Endonucleases (TALENs)

Question 19

Zinc Finger Nuclease (ZFN)

Answer 19

Here the DNA binding domain is designed to bind three nucleotides at a time; multiple domains can be combined to recognize longer sequences. Typically, three domains are
used (ie a 9 basepair recognition site) and ZFN’s are used in pairs (yielding a recognition site of 18 bp). Once the ZFN binding domain hybridizes with the target site of interest, the cutting enzyme fused to the ZFN binding
domain then cleaves the DNA at the designated location. An 18bp sequence may be long enough to ensure only a single cut in a genome.
However, this method also came with its challenges: it is notoriously hard
to design and engineer the three-nucleotide ZFN binding domain, and has also shown a number of off target effects

Question 20

Transcription Activator-Like Endonucleases (TALENs)

Answer 20

TALENs are easier to design, as their protein-DNA binding domains only recognize one nucleotide at a time, and work in a similar manner to ZFNs. however, the development of individual nucleotide binding domains
makes TALENs very large and cumbersome – combining nucleotide after nucleotide to form a recognition site makes the entire protein too bulky for easy entry into cells

Question 21

Adaptive Immunity

Answer 21

a mechanism of remembering past
infections and mounting a fast and efficient immune response on reinfection (as opposed to innate immunity, which consists of an organism’s initial response to first-time infection). It’s the human adaptive immunity
system that vaccines capitalize on

Question 22

The natural CRISPR-Cas process has three stages:

Answer 22

1) Spacer Acquisition and Integration:
When a bacterium is first infected with a virus, it captures snippets of the virus’ genetic information and stores them in the CRISPR array, which serve as the bacterium’s “memory system.” The captured segment, called the protospacer because it fills the space between
the palindromic repeats of the CRISPR array, starts close to a short sequence in the viral genome (as few as three basepairs) called the
Protospacer Adjacent Motif (PAM). In the bacterial species in which CRISPR was first fully explored, the PAM sequence is NGG (N stands
for any nucleotide). The captured segment, the protospacer, a ~20-40 bp piece of the virus genome, is integrated in the bacterial
CRISPR locus between two CRISPR repeats

2) crRNA processing and assembly of the crRNA/Cas9 Complex
Once the spacer is integrated into the host cell genome, the CRISPR locus, containing different protospacers (i.e. genomic pieces from different viruses; 1-4 in the diagram above), is
transcribed into a long CRISPR-RNA (crRNA) precursor. This precursor is processed by Cas enzymes and RNaseIII to produce short strands of crRNAs, each with a single protospacer sequence. The palindromic sequences are then
transcribed into Trans-Activating CRISPR-RNA (tracrRNA). The 5’ end of the tracrRNA structure anneals to part of the crRNA structure. The palindrome allows for the formation of a
‘hairpin’, a type of structure in which strand can fold in on itself and hybridize
————-This hairpin then allows for the tracrRNA:crRNA structure to associate with the Cas enzyme. If a target DNA sequence is
present that is complementary to the crRNA, the crRNA will anneal to the DNA, bringing the tracrRNA and Cas complex along with it. The Cas then employs its nuclease activity to
make a double-stranded cut in the target DNA.
However, one more component is necessary. This cut will only occur if the target DNA contains a PAM. As we’ve seen, the PAM is usually a very short sequence (it varies from bacterial species to bacterial species). The PAM must be present in the intruding viral DNA in order for Cas cutting activity to be activated. If the PAM were not part of the system, the Cas-9
system would attack the bacteria’s own CRISPR genome region (because it harbors target viral DNA). The PAM sequence (eg NGG) is not in the palindromic sequences flanking the protospacer inserts in the CRISPR array, meaning that the Cas nuclease system will not attack the CRISPR domain.

3) Interception and destruction of newly invading virus by cleavage
If the virus strikes again, the bacteria’s crRNA, hybridized to a TransActivating CRISPR-RNA (tracrRNA) and complexed with Cas9, will
anneal to the viral sequence (since part of the crRNA sequence is complementary to the virus). If the PAM site is present in the viral
sequence, the Cas will digest the viral DNA, thus eliminating the threat of repeated infection, and furthering bacterial survival.

Question 23

the tracrRNA and the crDNA could be combined into just one manufactured molecule,

Answer 23

a single guide RNA (sgRNA)

Question 24

Cells have machinery for repairing double stranded breaks: explain Homology-directed repair vs non Homology-directed repair

Answer 24

1. Non-homologous end joining repair
   Here the cell basically attempts to glue together the two loose ends. This is an error-prone process so the repair is often imperfect, resulting in an inserted basepair, say, or a deleted one. (Either way, the system has just introduced a frameshift mutation into the sequence).
   Typically this results in the elimination of gene function, so CRISPR can be used in this way to shut down gene activity, creating gene knockouts (sometimes abbreviated KO). CRISPR evolved in bacteria as a search-&- destroy adaptive immune system: by cutting incoming viral DNA, it nullifies the viral threat. In many applications of the system, all we’re looking to do
   is just that – to nullify target gene function
2. Homology-directed repair
   The alternative is that DNA repair is carried out on the basis of an unbroken template molecule, which serves as a master reference in the repair process. This template would typically be the second copy of the targeted chromosome in the cell. However, researchers can use this system to their advantage: by adding a manufactured template consisting of the desired (ie changed) sequence, they can induce the repair system to incorporate the new sequence into the cut DNA
   molecule

Question 25

CRISPR-Cas Applications

Answer 25

1. De-extinction: the return of the mammoth?
   One particularly spectacular CRISPR project is being carried out in
   George Church’s lab at Harvard Medical School. There, researchers are
   doing what was thought to be impossible: bringing back extinct organisms
   (i.e. de-extinction)! By using ancient DNA techniques to sequence the
   genome of the woolly mammoth (extinct 4,000 years ago), scientists
   compared the genome of the mammoth to its closest living relative, the
   Asian elephant. The two are surprisingly closely related. Initially, they are
   focusing on a handful of key differences in genes that they believe are
   important for physiology (i.e. adaptation to cold temperatures). Using
   CRISPR, they are editing the mammoth variants into the extant Asian
   elephants’ genome in the hope of resurrecting a “proxy species” – a kind
   of wooly, cold-resistant Indian elephant – for the extinct woolly mammoth.
2. Progeria: gene therapy
   Progeria, a genetic disease that results in what looks like
   accelerating aging (and early) death, is – mercifully – extremely rare, but
   it is utterly devastating when it occurs. Hutchinson-Gilford progeria
   syndrome, to give it its full name, results from a single-base change in the
   14
   gene for a protein called lamin A that helps support the membrane
   forming the nucleus in cells. That it performs such a fundamental function
   in all cells is one of the reasons the condition is so devastating.
   Remarkably, the causal mutation is synonymous; its impact is to cause missplicing of the mRNA in a way that results in an abnormal protein, called
   progerin, that disrupts the nuclear membrane and is toxic to cells in many
   tissues. Toddlers soon become bald and have stunted growth, body fat
   loss, stiff joints, wrinkled skin, osteoporosis, and atherosclerosis. People with
   progeria die on average around age 14 from a heart attack or stroke.