BIOL 3301: Chapter 17 Non-Coding RNAs

noncoding RNAs ncRNAs

• some genes don’t code polypeptides, but are transcribed into ?

  • est 1000s-10,000s ?’s in humans

• perform wide array of cellular fx’s in bacteria, archaea, eukaryotes

• in most cell types, ? are more abundant than mRNAs

  • in a typical human cell, only about 20% of transcription involves making mRNAs, whereas 80% focuses on making ?

ncRNAs

• in most cell types, ? are more abundant than mRNAs

  • in a typical human cell, only about 20% of transcription involves making mRNAs, whereas 80% focuses on making ?

CRISPR Cas system

• system in some prokaryotes that defends against foreign invaders

  • defense against bacteriophages, plasmids, transposons

  • ncRNAs play a key role

• 3 types exist

  • we focus on type II

CRISPR Cas system

• provides bacteria with defense against bacteriophages

• CRISPR locus recognized in 1993

  • site in prokaryotic chromosomes

  • contains a series of repeated sequences

• Francis Mojica, Giles Vergnaud, Alexander Bolotin independently proposed it provides protection against bacteriophage infection in 2005

  • based on sequence analysis revealing that the CRISPR locus contains segments derived from bacteriophage DNA

• Philippe Horvath and colleagues experimentally showed that the ? system provides defense against bacteriophage infection in 2007

CRISPR-Cas system organization

• ex., 5 genes

  • tracr

    • process pre-crRNA into usable crRNA

    • binds crRNA and guides Cas9 to target DNA

  • Cas9

    • “scissors”

    • cuts foreign DNA when guided by crRNA/tracrRNA

  • Cas1

    • work with Cas2 to recognize and insert new viral DNA pieces into CRISPR as new spacers (adaptation phase)

    • create genetic memory of new invaders

  • Cas2

    • “ “ but works with Cas 1

  • Crispr

    • group of Clustered, Regularly, Interspaced, Short, Palindromic Repeats

      • repeats

        • identical sequences

      • spaces

        • repeats are interspersed with spacers (short, unique sequences)

        • unique bits of viral DNA (memory of past infections)

CRISPR analogy

• CRISPR is a library of viral mugshorts

• Cas1 and Cas2 add new mugshots into CRISPR

  • insert new viral DNA pieces as spacers

• tracrRNA + crRNA guide Cas9 to arrest the invader (cut viral DNA)

adaptive defense system

• CRISPR Cas system is an ?

• bacterial cell must first be exposed to an agent (e.g., bacteriophage) to elicit a response

CRISPR adaptation phase

• (infection first happens, invading DNA is used to adapt the cell to future infections)

• Cas1 and Cas2 protein complex cleaves bacteriophage DNA

  • a piece of this bacteriophage DNA (typically 20-50 bp) is inserted into the Crispr locus as a new spacer

    • spacers in modern bacteria are from past infections

      • genetic “memories” of past infections

    • the spacer is passed to daughter cells, giving them immunity to same virus in future

CRISPR expression phase

• upon re-exposure, the Crispr, tracr, and Cas9 genes are transcribed

  • 2 noncoding RNAs (ncRNAs) are made

    • pre-crRNA

    • tracr-RNA

• region of tracrRNA base pairs with repeats in pre-crRNA

  • the pre-crRNA is then cleaved into many small crRNAs (each contains 1 spacer)

  • tracrRNA-crRNA complex binds Cas9 protein thru recognition site in tracrRNA

CRISPR interference phase

• during reinfection

  • each spacer in a crRNA is complementary to one strand of the bacteriophage DNA

  • crRNA acts as a guide that causes the tracrRNA-crRNA-Cas9 complex to bind to that strand

  • Cas9 protein functions as an endonuclease→makes DOUBLE-STRANDED BREAKS in the bacteriophage DNA

    • bacteriophage proliferation is inhibited

gene editing via CRISPR Cas

• experimentally altering a gene sequence

• analysis of mutations can provide important info about normal genetic processes

• mutations can rise spontaneously, or be induced by mutagens

• researchers have developed techniques to make mutations within cloned DNA and also within the DNA of living cells

crispr cas technology

• the system can also be used to edit genes in living cells

  • e.g., if you want to test the fx of a gene by “knocking it out” i.e., mutating the gene so that no functional protein is made

• tracrRNA and crRNA are fused into one molecule

  • called the single guide RNA (sgRNA); aka guide RNA gRNA

• the spacer region of the sgRNA is complementary to one of the strands of the gene to be edited (called the target gene)

• sgRNA binds to Cas9 and guides it to the target gene

• Cas9 then makes a double strand break in the gene

• after Cas9 cuts the DNA, 2 different repair events are possible

  • nonhomologous end joining NHEJ

    • region may incur a small deletion or insertion that inactivates gene (knockout)

  • homologous recombination repair

single guide sgRNA structure

• crRNA + tracrRNA joined by a linker

  • tracrRNA helps process pre-crRNA into crRNA

    • recruit/guide Cas9 to target

  • crRNA guides Cas9 to specific viral DNA to cut

  • contains “spacer” sequence matching viral DNA

• spacer region of sgRNA is complementary to target gene

• guides Cas9 to specific location in bacteriophage DNA

nonhomologous end joining

• possible repair event after cas9 cuts the DNA

• joins broken ends w/o template

• often causes small insertion/deletions (indels)

  • can inactivate gene

CRSPR Cas gene editing

• Cas9+sgRNA bind target DNA sequence

  • sgRNA= crRNA +tracrRNA joined by a linker

    • sgRNA spacer is complementary to target gene

• Cas9 cleaves both DNA strands, creating a double stranded break

  • 2 options for repair

    • nonhomologous end joining

      • fast, error prone

      • cause small insertions/deletions

      • may inactivate the gene

    • homologous recombination repair HRR

      • can introduce specific point mutation

CRISPR Cas tech

• can be used on different cell types and whole organisms

  • ex., inject a segment of DNA that encodes an sgRNA and Cas9 protein into a fertilized mouse oocyte

    • the 2 genes (sgRNA and Cas9 gene) are expressed in the oocyte

    • sgRNA-Cas9 complex would either cause a gene inactivation (NHEJ) or produce a point mutation (HRR)

  • ex., mouse embryos, zebrafish, frogs, adult mice, human cell lines, roundworms, variety of different plant cell species

CRISPR based therapeutics

• 2021: FDA approves CRISPR based treatment for sickle cell disease for clinical trials

• 2018: FDA approves clinical trial of CRISP based treatment for Leber congenital amaurosis (LCA), a vision loss disease

one, multiple

• thousands of genetic diseases afflict ppl

• many of these are the direct result of a mutation in ? gene

• many involve ? genes

  • complex pattern of inheritance

  • ex., diabetes, asthma, mental illness

genetic diseases

• study of this provides insight regarding our traits

• ex., analyzing ppl with hemophilia

  • researchers have ID’ed genes that participate in blood clotting

• thousands of human diseases have a genetic basis

  • we focus on those resulting from mutations in single genes

  • the occurrence of these diseases in family pedigrees often obey simple Mendelian inheritance patterns

family pedigrees

• genetic diseases here often obey simple Mendelian inheritance patterns

genes

• several observations are consistent w/ idea that a disease is caused, at least in part, by ?

  • when an individual exhibits a disease, the disorder is more likely to occur in genetic relatives than in the general poulation

  • identical twins share the disease more often than fraternal twins

    • concordance is 1 for identical twins

  • the disease does not spread to individuals sharing similar environmental situations

  • different populations tend to have different frequencies of the disease

  • the disease tends to develop at a characteristic age

    • many genetic disorders exhibit a specific age of onset

  • the human disorder resembles a genetic disorder that has a genetic basis in another mammal

  • a correlation is observed btw a disease and a mutant human gene or chromosomal alteration

monozygotic twins MZ

• formed from same sperm and egg

• identical twins

• concordance of disorder is 1

  • %age of twin pairs in which both twins exhibit the disorder or trait

dizygotic twins

• formed from separate pairs of sperm and egg

• fraternal twins

concordance

• the degree to which a disease is inherited

• refers to the percentage of twin pairs in which both twins exhibit the disorder or trait

  • 1 for identical twins

age of onset

• many genetic disorders exhibit a specific ?

  • disease tends to develop at a characteristic age

pedigree analysis

• inheritance patterns of human diseases may be determined via ?

• the pattern of inheritance of a human disorder that is caused by a mutation in a single gene can be deduced by analyzing human ?

• to use this method, a geneticist must obtain data from large ? with many affected individuals

autosomal recessive inheritance

• tay sachs disease TSD

  • affected individuals appear healthy at birth, but develop neurodegenerative symptoms at 4-6 months

    • cerebral degeneration, blindness, and loss of motor function

    • TSD patients typically die at 3-4 years of age

  • TSD is 100x more frequent in Ashkenazi (eastern Europe) Jewish populations than in others

  • result of a mutation in the gene that codes the enzyme hexosaminidase A (hexA) that breaks down certain lipids in neurons

  • an excessive accumulation of this lipid in cells of the CNS causes the neurodegenerative symptoms

autosomal recessive inheritance

• 4 common features

  • an affected offspring will have 2 unaffected parents

  • when 2 unaffected heterozygotes have children, the avg percentage of affected children is 25%

  • 2 affected individuals will have 100% affected children

  • the trait occurs with the same frequency in females and males

autosomal recessive

• typically disorders that involve defective enzymes

  • the heterozygote carrier has 50% of the functional enzyme

    • sufficient for a healthy (unaffected) phenotype

• hundreds of genetic diseases are inherited in this manner

  • in many cases, the mutant genes responsible have been ID’ed and characterized

• ex.,

  • albinism (tyrosinase)

  • cystic fibrosis (CFTR- a chloride transporter)

  • phenylketonuria PKU (phenylalanine hydroxylase)

  • sickle cell disease (beta globin)

  • tay sacs (hexosaminidase A)

autosomal recessive

• equal #s M and F affected

• affected kids have unaffected parents

• unaffected carriers have about 25% affected kids

• 2 affected parents have 100% affected kids

autosomal dominant inheritance

• huntington disease HD

  • major sx

    • degeneration of certain neurons in the brain

  • lads to personality changes, dementia, and early death (usually in middle age)

  • result of mutation in gene encoding protein huntingtin

    • mutation adds a polyglutamine tract to the protein

  • causes an aggregation of the protein in neurons

autosomal dominant inheritance

• affected offspring usually has one or both affected parents

  • can be altered by reduced penetrance

• affected individual with only 1 affected parent is expected to produce avg 50% affected offspring

• 2 affected, heterozygous individuals will have (on avg) 25% unaffected offspring

• trait occurs with the same frequency in both M and F

• often the homozygote is more severely affected with the disorder

autosomal dominant disorders

• 3 common explanations

  • haploinsufficiency

    • heterozygote has 50% of the functional protein

    • not sufficient for a healthy (unaffected) phenotype

  • gain of function mutations

    • mutation changes protein so it gains a new function

  • dominant negative mutations

    • mutant gene product acts antagonistically to the wild type gene product

autosomal dominant disorders

• aniridia

  • haploinsufficiency

• achondroplasia

  • gain of fx

• marfan syndrome

  • dominant negative

• familial hypercholesterolemia

autosomal dominant

• usually appears every generation

• usually affected individuals have about 50% affected offspring

• affected individuals have at least 1 affected parent

• same frequency of the trait in M and F

x linked recessive

• this type of inheritance poses a special problem for males

• males have only 1 copy of X linked genes (hemizygous)

• a female heterozygous for an X linked recessive allele will pass this trait to 50% of her male offspring

x linked recessive

• hemophilia

  • major sx

    • blood can’t clot properly when a wound occurs

  • for hemophiliacs, common accidental injuries pose a threat of severe internal or external bleeding

  • hemophilia A (classical hemophilia)

    • caused by defect in ? gene that encodes a clotting protein called factor VIII

  • “royal disease”

    • affected many members of european royal families

x linked recessive

• 3 common features

  • males more likely to exhibit the trait

  • females with affected male offspring often have male sibling or a male parent who are affected with the same trait

  • the female offspring of affected males will produce (on avg) 50% affected male offspring

x linked recessive

• duchenne muscular dystrophy

• hemophilia A

• hemophilia B

• androgen insensitivity syndrome

x linked recessive

• males way more affected

• mother usually unaffected

• females with affected fathers have about 50% affected offspring

• females with affected sons usually have affected brothers

x linked dominant

• rare

• males often more severely affected

  • females may be less affected due to WT copy of the other X chromosome

• F more likely to exhibit the trait when it is lethal to males

• affected F have a 50% chance of passing the trait to female offspring

x linked dominant

• vitamin D resistant rickets

• rett syndrome

• aicardi syndrome

• incontinentia pigmenti

x linked dominant

• rare

• males usually more severely affected

• if trait is lethal to males, then females show the trait more often (since there are not males that have the dominant allele)

• affected females pass the trait to 50% of female offspring

pre-birth genetic testing

• amniocentesis

  • fetal cells obtained from amniotic fluid

• chorionic villus sampling

  • fetal cells obtained from the chorion (fetal part of placenta)

  • can be performed earlier during the pregnancy than amniocentesis

  • poses a slightly greater risk of miscarriage

• noninvasive prenatal testing

  • collect blood sample from pregnant F and analyzing cell-free DNA

• preimplantation genetic diagnosis PGD

  • conducted before pregnancy even occurs

  • genetic testing of embryos hat have been produced by IVF

    • IVF combines sperm + egg outside mom’s body

    • 1 or 2 cells removed at 8 cell stage

    • tests conducted to check for problems

      • molecular tests can check for particular gene defects

      • chromosome composition can be checked

    • decision made whether or not to implant

  • raises ethical q’s

preimplantation genetic diagnosis PGD

• conducted before pregnancy even occurs

• genetic testing of embryos hat have been produced by IVF

  • IVF combines sperm + egg outside mom’s body

  • 1 or 2 cells removed at 8 cell stage

  • tests conducted to check for problems

    • molecular tests can check for particular gene defects

    • chromosome composition can be checked

  • decision made whether or not to implant

• raises ethical q’s

• method of genetic testing prior to birth

amniocentesis

• fetal cells obtained from amniotic fluid

• determine number of chromosomes per cell and whether changes in chrom structure have occurred

• method of genetic testing prior to birth

chorionic villus sampling

• fetal cells obtained from the chorion (fetal part of placenta)

• can be performed earlier during the pregnancy than amniocentesis

• poses a slightly greater risk of miscarriage

• method of genetic testing prior to birth