exam 2 adv lab tech

CRISPR function in bacteria

Adaptive immune system that stores viral DNA fragments to recognize and cut the same virus upon reinfection.

Cas1 and Cas2

Proteins that capture viral DNA spacers and insert them into the CRISPR array in bacterial genome.

CRISPR array structure

Repeats (palindromic) alternating with spacers (viral DNA). Transcription produces crRNA.

tracrRNA

Trans

PAM sequence

Short motif (NGG for SpCas9) adjacent to target DNA. Required for Cas9 to cut. Not present in bacterial CRISPR locus, preventing self- cutting

Why Cas9 doesn't cut its own CRISPR DNA

CRISPR locus lacks the PAM sequence adjacent to the spacer; Cas9 requires PAM for cleavage.

Two RNAs to sgRNA engineering

Combined tracrRNA and crRNA into single guide RNA (sgRNA) with 20 nt targeting sequence and a double-stranded cas9-binding scaffold

Cas9 cleavage mechanism

Creates double-strand break (DSB) 3-4 bases upstream of PAM. Both HNH and RuvC nuclease domains cut one DNA strand each.

HNH domain

Cas9 domain that cuts the DNA strand complementary to the guide RNA.

RuvC domain

Cas9 domain that cuts the non-complementary DNA strand.

NHEJ repair outcome

Non-homologous end joining creates insertions or deletions (indels) → frameshift → gene knockout.

HDR repair outcome

Homology-directed repair uses a donor template → precise knock-in or gene replacement.

Cas9 delivery methods

Plasmid DNA, mRNA + sgRNA (transient), ribonucleoprotein (RNP) complex, viral vectors (AAV, lentivirus).

CRISPR knockout in sea urchin (Oulhen et al. 2016)

Targeted PKS (polyketide synthase) pigmentation gene. Result: pigmentless larvae, confirming gene function.

dCas9

Catalytically dead Cas9 (mutated HNH and RuvC). Binds DNA without cutting. Used for CRISPRi or CRISPRa.

CRISPRi

dCas9 fused to repressor domain (e.g., KRAB) blocks transcription by steric hindrance or chromatin compaction.

CRISPRa

dCas9 fused to activator domain (e.g., VP64) recruits transcription machinery to enhance gene expression.

Epigenetic editing with dCas9

Fuse dCas9 to histone modifier (e.g., p300 for acetylation) or DNA methyltransferase (e.g., DNMT3A).

Mosaicism in CRISPR

Not all cells or alleles are edited. One cell may have wild-type, knockout, and heterozygous alleles simultaneously.

Off- target effects

Cas9 cuts sequences similar (but not identical) to the target, especially with mismatches in the PAM-distal region.

Mitigating off-target effects

Use high-fidelity Cas9 variants (eSpCas9, SpCas9-HF1), shorter sgRNAs, or truncated gRNAs.

Protein abundance problem in knockout

If wild-type protein is stable and abundant pre-editing, phenotype may be delayed until existing protein degrades.

CCR5 gene

Chemokine receptor; HIV uses it for entry. Natural CCR5-Δ32 mutation confers HIV resistance.

He Jiankui experiment

Edited CCR5 in twin embryos (Lulu and Nana) to mimic Δ32 mutation. Result: mosaicism, unknown off-targets, third baby (Amy) born later.

Consequences for He Jiankui

Fired from university, 3 years prison, fined 3 million RMB (2019).

Ethical concerns with germline editing

Heritable changes, unknown long-term effects, potential for eugenics/designer babies, lack of consent from future generations.

FDA-approved CRISPR trials

Ex vivo editing of patient cells (e.g., sickle cell disease, beta-thalassemia, certain cancers). Not germline.

Gene knockout definition

Make one or both alleles of a gene completely inoperative. Investigates gene function in vivo.

Gene knock-in definition

Replace an existing allele with a different sequence (e.g., specific mutation or reporter gene). Adds function.

Gene knockdown definition

Reduce gene expression (mRNA or protein) without eliminating it entirely. Reversible.

Conventional knockout in mice steps

1) Target ES cells 2) Targeting vector 3) Electroporation 4) Homologous recombination 5) Selection 6) Inject into blastocyst 7) Mosaic mouse 8) Breed for germline transmission.

Targeting vector components

Homologous arms (flank target gene), neoᵣ (positive selection), HSV-tk (negative selection).

Positive selection (neoᵣ)

Neomycin resistance gene. Cells without integration die in G418. Keeps cells with any vector integration.

Negative selection (HSV-tk)

Herpes simplex virus thymidine kinase. Converts ganciclovir to toxic product. Kills cells with random (non-homologous) integration.

Homologous recombination frequency in ES cells

Very rare (~10⁻⁶ to 10⁻⁷). Selection enriches for correct targeting.

Blastocyst injection

Modified ES cells injected into blastocoel. They mix with inner cell mass (ICM) cells to form chimeric embryo.

Chimera mouse

Contains cells from two origins: original blastocyst (wild-type) and modified ES cells (knockout).

Germline transmission

Breed chimera with wild-type. If modified ES cells contributed to germline, some offspring are fully heterozygous knockout.

Conditional knockout (Cre-lox)

Gene is floxed (loxP sites flanking exons). Cre recombinase excises the floxed region.

loxP site

34 bp sequence (13 bp inverted repeats + 8 bp spacer). Recognized by Cre recombinase.

Cre recombinase

Enzyme from bacteriophage P1 that catalyzes recombination between two loxP sites.

Cre-lox excision outcome

Two loxP sites in same orientation → Cre excises the intervening DNA as a circle (deletion).

Tissue-specific conditional knockout

Use Cre under a tissue-specific promoter (e.g., Alb-Cre for liver). Knockout only in that tissue.

Inducible conditional knockout

Use Cre-ERᵀ² (fused to estrogen receptor). Active only after tamoxifen treatment.

Floxed definition

Gene flanked by loxP sites. "Flanked by loxP."

Gene expression regulation stages

Chromatin accessibility, transcription, RNA processing (splicing/capping/polyA), RNA stability, translation, protein activity.

General transcription factors

Bind promoter and recruit RNA polymerase. Required for basal transcription of all genes.

Specific transcription factors

Bind enhancers or silencers. Activators increase transcription; repressors decrease it.

Enhancer

Distant DNA sequence (upstream, downstream, or intronic) bound by activators. Works via DNA looping.

DNA looping in transcription

Mediated by cohesin and mediator complex. Brings enhancer-bound activators close to promoter.

Alternative splicing regulation

Splicing factors bind pre-mRNA to include or exclude exons. Produces different protein isoforms from one gene.

RNA stability regulation

Lifespan of mRNA in cytoplasm. Determined by AU-rich elements, miRNA binding, and ribonucleases.

miRNA pathway

Endogenous small RNA (~22 nt) processed from hairpin precursor. Guides RISC to partially complementary mRNA → translational repression or degradation.

siRNA pathway

Exogenous double-stranded RNA processed into ~21 nt siRNAs. Perfect complementarity → RISC cleaves mRNA.

RISC complex

RNA-induced silencing complex. Core protein: Argonaute. Binds small RNA and targets complementary mRNA.

shRNA

Short hairpin RNA (engineered). Expressed from a vector, processed into siRNA in cell.

Translation regulation

RISC can block ribosome assembly or elongation. Also eIF2 phosphorylation (stress response) globally reduces translation.

Ubiquitination

Tags protein with ubiquitin chains (via E1, E2, E3 enzymes). Marks for proteasomal degradation.

Proteasome

Large protein complex that degrades ubiquitin-tagged proteins into peptides.

Mutagenesis definition

Process that creates mutations in genetic information. Used to study gene function or generate novel proteins.

Spontaneous mutation causes

Replication errors, hydrolysis (deamination, depurination), oxidation, alkylation, radiation, transposons.

Deamination of cytosine

C → U (pairs with T). After replication: C:G → T:A transition. CpG islands are hotspots.

Deamination of adenine

A → hypoxanthine (pairs with C). After replication: A:T → G:C transition.

Depurination

Loss of purine base (A or G). Leaves apurinic site. ~10,000 events per mammalian cell per 20 hours.

Depyrimidination

Loss of pyrimidine base (C or T). ~500 events per cell per day. Less frequent than depurination.

Alkylation damage (EMS)

Ethylmethane sulfonate adds ethyl group to guanine. G pairs with T → G:C to A:T transition.

Oxidative damage (8-oxoG)

Guanine oxidized to 8-oxoguanine. Pairs with A instead of C → G:C to T:A transversion.

UV radiation damage

Forms cyclobutane pyrimidine dimers (especially TT dimers). Blocks replication or causes mutations if bypassed.

Base analogs (5-bromouracil)

Incorporates like T but tautomerizes → mispairs with G. Causes transition mutations.

Intercalating agents (EtBr, proflavine)

Insert between stacked bases. Cause insertions or deletions during replication (frameshift).

Transposon Class 1 (retrotransposon)

Copy and paste: transcribed to RNA, reverse transcribed, integrates at new site.

Transposon Class 2 (DNA transposon)

Cut and paste: transposase excises element from original site, inserts elsewhere.

Global mutagenesis

Non-targeted. Expose organism to radiation or chemicals. Advantages: many mutations. Disadvantages: need to screen.

Site-directed mutagenesis

Introduce specific mutation at defined location. Uses oligonucleotide primers with mismatch.

Two-PCR method for linear DNA

Two separate PCRs each use one mutagenic primer. Mix products, denature, reanneal → heteroduplex with mutation.

Kunkel's method problem solved

Selects against wild-type template. Uses dut⁻ ung⁻ E. coli to incorporate uracil into template.

dut⁻ mutation

Lacks dUTPase. Causes high dUTP incorporation into DNA during replication.

ung⁻ mutation

Lacks uracil N-glycosylase. Cannot remove uracil from DNA. Uracil persists.

Kunkel's method steps

1) Grow phagemid in dut⁻ ung⁻ → uracil-containing DNA. 2) Anneal mutagenic primer, extend. 3) Transform into dut⁺ ung⁺ → wild-type (U) degraded, mutant survives.

Methylation selection method

Newly synthesized DNA unmethylated. DpnI cuts methylated (template) DNA. Only mutant plasmid survives.

DpnI

Restriction enzyme that cuts only methylated GATC sites (Dam methylation). Digests wild-type template, not mutant strand.

Gene shuffling purpose

Recombine fragments from homologous genes to create chimeric variants with novel functions.

Gene shuffling sources of variation

Natural polymorphisms, random in vitro mutagenesis, homologous genes from different species, protein family members.

Original gene shuffling (Stemmer)

DNase I fragmentation → pool fragments → denature, anneal, extend (no primers) → repeat → full-length chimeras.

StEP method

Very short annealing and extension cycles (5-10 sec). Promotes crossovers by incomplete extension.

RACHITT method

Fragments annealed to a scaffold strand. Gaps filled, uracil-containing fragments removed. High crossover frequency.

Illumina sequencing by synthesis (SBS)

Reversible dye terminators. Add one base at a time, image fluorescence, cleave terminator, repeat.

Flow cell

Glass slide with two types of oligos (P5 and P7) attached. Where cluster generation and sequencing occur.

Bridge amplification

DNA fragment hybridizes to P5 and P7 on flow cell. Forms bridge, polymerase copies, denatures. Repeated 1000x → cluster.

Cluster

~1000 identical copies of one original DNA fragment, all attached to flow cell at same location.

Read 1 primer binding site

Adapter sequence complementary to sequencing primer. After cluster generation, primer anneals to start sequencing.

Index (barcode) read

Short unique sequence (6-10 bp) per sample. Allows pooling multiple libraries in one run.

Paired-end sequencing

Sequence both ends of same fragment. Read 1 from one end, then read 2 from opposite end.

Paired-end advantage

Better assembly of repeats, detects structural variants (insertions, deletions, inversions, translocations).

Dual indexing

Two indices (one on each end). Increases multiplexing capacity (e.g., 384 × 384 samples possible).

Low clustering density problem

Too few clusters → low data output. Too many → overlapping clusters → poor base calling.

Low diversity in first bases problem

Same base added to all clusters → poor signal for phasing and focus. PhiX spike-in solves.

PhiX control library

Diverse genome (no bias). Spiked into low-diversity libraries to improve cluster identification and calibration.

RAD-seq

Restriction site-associated DNA sequencing. Cut with restriction enzyme → size select → sequence same loci across many individuals.

Infinium bead array for SNPs

Beads with allele-specific probes. Two colors: green (G/C), red (A/T). Ratio determines genotype.