FPM Exam 1

Histone structure and modification

- Histones are octamers of 2x H4, H3, H2A, H2B. - Histone tails are flexible and accessible for modification. - Euchromatin=open. Heterochromatin=closed/inaccessible. - SITE OF MOD MATTERS: PTM at different sites can have different function.

si/shRNA or CRISPR/Cas functions in perturbing RNAs

1. Can prevent transcription of small RNAs with CRISPR-dCas9-KRAB binding to promoter. 2. Can impede promoter function by CRISPR/Cas9 to KO promoter for small RNA. 3. Can use CRISPR/Cas13 to mutate site on target RNA that small RNA would normally bind to; impede small RNA binding ability and therefore targeting. 4. Can use si/shRNA to degrade mRNAs or lncRNAs to test their function in a knockdown model. Quick and easy experiment.

ChIP-seq workup

1. Crosslink chromatin (DNA + histones + anything bound to DNA or protein) with UV. 2. Lyse cells. 3. Isolate and fragment DNA. 4. Immunoprecipitate with beads conjugated to an antibody for your PTM of interest. 5. Strip proteins. 6. Purify DNA. 7. Sequence DNA or run qPCR. 8. Results tell you which GENOMIC LOCI have this specific PTM.

Sanger Sequencing workup and expected results

1. Extract and purify DNA. 2. PCR amplify this incubation reaction: DNA, forward and reverse primers, a DNA polymerase, dNTPs, and ddNTPs attached to a fluorescent probes. As ddNTPs get incorporated into daughter strands, elongation will stop. 3. PCR generates many fragments of the gene of all different sizes depending on when the ddNTP stopped elongation. Run your amplified DNA on a gel. 4. Visualize sequence by seeing the order in which fluorescent probes ran out on the gel. First nucleotide is farthest on the gel since its fragment is the smallest.

Illumina-sequencing workup

1. Fragment DNA. 2. Attach universal adaptors to ends of fragment. 3. Separate DNA strands and hybridize adaptors to oligonucleotides on a lawn. 4. DNA polymerase makes daughter strand, incorporating fluorescent dNTPs. 5. Readout is sequence of fluorescent flashes.

Necessary components of vectors

1. Gene of interest 2. Promoter and transcription terminator 3. ORI-- so plasmid replication occurs 4. MCS-- so gene can be excised from plasmid to be ligated into genome 5. Antibiotic resistance-- for positive selection 6. Reporter

PCR primer design considerations

1. High (but not too high) GC content: 3 H-bonds between C-G more stable than A-T. 40-60% is good. 2. F/R annealing temperatures should be about the same so both primers hybridize with DNA with same efficacy. 3. AVOID: lots of base repeats, intra- or inter-primer homology (primers will bind to itself/each other-- primer dimers)

Off-target effects of miRNAs

1. Off-target effects since you only need 6-8bp of complementarity. 2. miRNA seed families have similar seeds (similar 6-8nt sequence for base pairing) so can usually compensate if one miRNA is inhibited

3 modes of miRNA target binding (to mRNA or other ncRNA) -- details

1. Perfect pairing when miRNA is bound to Ago2: exact base-pairing of miRNA to target. Target slicing and degradation. 2. Seed pairing when miRNA is bound to Ago: only 6-8 base pairs need to be made between miRNA and target. miRNA recruits other proteins, causes target RNA is de-capped, de-adenylated, and sequestered to P-bodies. 3. Extensive pairing when miRNA is bound to Ago: not quite perfect pairing and target bulges out in the middle of the miRNA/target RNA complex. UNO REVERSE! TARGET-DIRECTED MIRNA DEGRADATION. Ago gets poly-ubiquitinated and degraded, leaving miRNA vulnerable to degradation since it's not protected by embedding in Ago and has no 5' cap or 3' poly-A tail. miRNA is degraded.

Quick! Name some covalent PTMs and their general effect & function. I thought of 6 :)

1. Poly-ubiquitination: changing protein half-life by targeting protein for degradation. 2. Mono-ubiquitination: protein localization: receptor endocytosis and translocation between membranes. 3. Acetylation on lysines: in histones, this promotes decompaction of chromatin. 4. Methylation on lysines or arginines: in histones, this promotes compaction of chromatin and transcriptional repression. 5. Di-sulfide bonds: stabilize protein folding (tertiary structure). ***6. PHOSPHORYLATION: allosteric protein regulation via changing the conformation of the active site, which will change the protein's binding partners.

siRNA/shRNA off-target effects and solutions

1. Short base pairing requirements can mean it binds to many RNAs you didn't intend for it to bind. If off-target targets even have >50% complementarity, si/shRNA can still bind: many paralogs are off-target RNAs that are knocked down. SOLUTION: use multiple si/shRNAs intended to target same RNA at different points and increase specificity of knockdown. 2. Adding too much siRNA can outcompete endogenous miRNA binding of Ago or Ago2, cause off-target reduction of miRNA functions. SOLUTION: less is more. Minimize amount of si/shRNA used at lowest effective dose.

Examples of vectors in genetic methods

1. Using vector to deliver Cre in Cre/Lox system: temporal and spatial (if you used cell-type specific promoter) control of Cre recombination 2. Use vector to deliver sgRNA in a mouse that has constitutive Cas9 expression

siRNAs vs. shRNAs

1. siRNAs can be endogenous or exogenous. shRNAs are only exogenous tools that take advantage of siRNA biology principles. 2. siRNA generally used for transient knockdowns, shRNA is also transient by itself but can be packaged into a lentivirus for stable knockdown. 3. shRNAs usually bigger than siRNAs. 4. shRNAs are always synthesized in ds-hairpin structure; siRNAs can come from ds-hairpin OR from longer dsRNA.

Why do inteins exist? Evolutionary benefit?

2 hypotheses (no real answer) 1. Inteins help to stabilize a protein to protect it from degradation since they are close to the active site and are so well-folded themselves, act as internal chaperones 2. (more likely) inteins are selfish genes: since they were first incorporated into the genome so early in evolution, they stick around in the protein because if the intein gets degraded, the essential proteins also get degraded.

How to link enhancers to target genes

4C: genes in contact with single enhancer

siRNA function

ALWAYS slicing and degradation of targets with help from Ago2. Unlike miRNAs, siRNAs don't bind Ago and don't sequester target RNAs.

Why do organisms use histone PTMs to regulate chromatin function?

Active and dynamic changes in chromatin accessibility/transcription?????

Long PCR

Amplify DNA up to 25kb

What's the point of transcriptional regulation?

Basis of genetic variability from the same DNA sequence

SILAC use and general workup

Best for comparing PTMs in two different conditions, e.g. control vs. drug-treated or healthy vs. disease. Grow Group 1 cells in media with light carbon isotopes. Group 2 cells in media with heavy carbon isotopes. Any newly synthesized proteins in cells will include light or heavy carbon. Helpful in combination with mass spec to determine abundance of modified protein in light (control) vs. heavy (experimental) conditions.

Enhancer/silencers

Bind TFs which recruit co-activators/repressors to promote/silence transcription

How to find enhancers

Binding of TFs and/or co-activators (detected by chip-seq/cut&run)

How to validate enhancers' function

CRISPR/Cas9 to KO enhancer, CRISPR/dCas9-KRAB to repress enhancer function

Structural Variants definition

Changes to the sequence of the DNA that have the potential to alter RNA, and later, protein products.

3C vs. 4C vs. HiC

Chromatin capture assays. All tell you if two loci on genome interact. 1. Crosslink chromatin. 2. Digest chromatin. 3. Ligate close chromatin loci together. 4. Purify DNA. 5. Sequence DNA. 3C: do two specific loci interact? Design primers against two loci of interest. 4C: How does this one locus interact with the rest of the genome? Use primers against one locus of interest and see what else is attached. HiC: unbiased genome-wide sequencing of how loci interact (more expensive)

C-value paradox

Chromosome number does not necessarily correspond with genome size or organism complexity

Cis vs. trans inteins

Cis: NATIVE (EXISTS IRL) in single protein. Intein is spliced out of protein via reaction with a cysteine. Trans: A TOOL. 2 proteins or fragments come together to generate semi-synthetic proteins. One fragment has the N-terminal half of an intein, other fragment has C-terminal half of intein. IntN and IntC click together (high binding affinity and high specificity) and splice out of protein, leaving the two fragments to be spliced together.

Coding sequences vs. Non-coding RNA vs. Non-coding regulatory elements

Coding sequences encode for protein products. Non-coding RNA is made from DNA that is transcribed but not translated and makes a functional RNA product but no protein. Non-coding regulatory elements are part of the DNA sequence that is never transcribed.

Structural genomics vs. functional genomics vs. comparative

Comparative = comparing two species Structural = studying genome structure (e.g. position of genes) and structural variations Functional = studying functions of genomic elements and interactions

CNVs

Copy number variations; change the gene load (more or less copies than normal) which has the potential to change the amount of RNA products made from the gene

Bulk RNA-seq general use, workup, and major caveat

Detecting RNA expression across all cells in a sample without accounting for cellular hetereogeneity. One major caveat is that RNA-seq is expression dependent so may be inaccurate for lowly expressed genes. A method to amplify lowly expressed genes before sequencing (e.g. with capture probes in capture-seq) can mitigate this. 1. Dissect tissue. 2. Isolate cells and generate a single cell suspension. 3. Use an antibody-based approach (e.g. FACS) to sort for a cell type of interest you're studying (e.g. microglia). 4. Extract mRNA from your sorted microglia and make cDNA via reverse transcription. 5. Make probes to hybridize with various lowly expressed immune mRNA molecules you want to study and its isoforms. 6. Using PCR, enrich expression of these captured target genes to have enough sample for analysis. 7. Sequence sample (via NGS).

Why does an organism need PTMs?

Diversification of the proteome comes from many things like mRNA splicing but also PTMs! Can change structure, function, and binding partners of protein usually dynamically.

Differential looping

Enhancer can communicate with different genes at different times in life OR enhancer looping can be changed due to change in gene expression

Enhancer looping

Enhancers control distal genes by DNA looping: forming physical loops onto promoters; indirectly bind promoters by mediator proteins and TFs

DNA methylation

Epigenetic modification of linear genome. Methyl group binds CpG sequences in genome. Methylation is repressive/pro-heterochromatin because it can: 1. Block recognition/binding of TF to enhancer 2. Serve as a docking site for co-repressors

Cysteine alkylation general principles

Exploit the fact that histones have no natural cysteines by mutating histones to express a cysteine at a site you want to modify, then cysteines can be alkylized to produce a pseudo-PTM at your site of interest.

PCR principles

Exponentially amplify a specific DNA sequence: 2^n copies, n=cycles. Used for genotyping, sequencing, disease diagnosis

Long non-coding RNAs: general facts and vibes

Extremely diverse functions and there are almost as many lncRNAs in the human genome as there are coding genes so they are probably important regulators of gene and RNA expression... however they are HARD TO STUDY because they are lowly expressed.

GWAS

Genome wide association studies; can provide information about correlation of structure (i.e.

Topological associated domains (TADs)

Heat map of 3D interactions. Linear genome is horizontal line at the bottom. Insulators block interactions between 2 loci (see lighter color). Enhancers enhance interaction between two loci (see darker color).

Capture-seq general use and workup

High throughput sequencing and annotation of lowly expressed transcripts, e.g. long non-coding RNAs. (paper from Hagen's discussion) Design specific probes that will bind specific sequences of interests you want to amplify. Similar assay as illumina-seq but can be done for lncRNAs. Goal is to PCR amplify lowly expressed transcripts before you sequence them to improve accuracy of sequencing.

Cysteine

Highly reactive amino acid; used frequently in protein modification methods

Validating siRNA/shRNA knockdown

Immunoblot/WB to see protein level to show a functional kd (i.e. less protein so less potential activity/action by protein globally), can also RNA-seq to see if you have other off-target effects but hard to conclude from these results cuz genes might be changing in response to changes in downstream signaling of shRNA target

Nested PCR use and workup

Increases probability of amplifying only your target DNA: 2 consecutive reactions to increase specificity of amplification if you're worried about off-target primer hybridization. 1. First reaction: primers are slightly external to target site, amplify longer sequence that mostly includes your target DNA but some extra on each side. 2. Second reaction: primers are for target sequence and are much more likely to specifically bind to target since aren't just put in the mix with your entire genomic DNA, only given your short first sequence as input

Inversions and indels

Indels are most likely to result in a phenotype since they are able to cause frameshift mutations that change the coding reading frame of the gene, resulting in a non-functional protein during translation.

Detecting structural variants

Just sequence it lol

Insulators

Keep enhancer/promoter pairs together and separate from other enhancer/promoter pairs

ChIP-seq function

LOCALIZATION of protein (or protein modified by a PTM) on genome. e.g. localization of a TF on genome, or localization of H3k4me3 on histones in genome.

What is an intein?

Large, well-folded globular domains that are EMBEDDED IN ESSENTIAL PROTEINS usually near active site. Unlike introns, they get translated.

Express Protein Ligation general principles

Ligating two proteins together based on hyperreactivity of C-terminal thioester on one protein and N-terminal cysteine on the other protein. Use recombinant expression of SPPS to synthetically create these two recombinant proteins, then allow the reactivity to do the work and ligate the proteins together.

Problems with TAD boundaries

Likely arose from a mutation in an insulator

FPKM

Method of normalizing RNA-seq reads to the size of the gene and relative amount of input sample to make quantitatively accurate predictions about relative expression of that particular transcript in a sample

What is a genome?

More than just genes; entire information of genetic material and its modifications

Pyro-sequencing and ion semi-conductor sequencing

NGS (short-read) Pyro: readout is light generated by luciferase Ion semi-conductor: readout is pH (generated by proton release as nucleotides integrated)

Cis regulatory elements

Non-coding parts of gene on DNA, e.g. promoter, enhancer, insulator

Aligning RNA-seq reads

Not trivial! mRNA fragments can originate from multiple exons and therefore align to a genome with a chunk taken out in the middle where the intron between the exons was. You can tell code of an aligner (e.g. STAR RNA-seq Aligner) to only count mRNAs that span x (e.g. x=6) amount of nucleotides on each exon at such junctions to preserve real fragments and exclude reads that are just incorrectly mapped.

2D genome

Nucleosome position: which binding motifs are accessible density: how close are nucleosomes, do they interact composition: DNA + histones + modifications

Phosphorylation

Occurs at serine/threonine/tyrosine Kinase = writer Phosphatase = eraser

Homolog

One gene related to another in the same organism by common ancestral DNA

siRNA biogenesis

Originate from a DOUBLE-STRANDED RNA molecule, either in long ds form or shorter hairpin form. 1. Dicer cleaves long dsRNA or ds-hairpin RNA to make an siRNA duplex (unlike miRNA, siRNA is NOT processed by a microprocessor). 2. Duplex is split and one strand is loaded onto Ago2.

miRNA biogenesis

Originate from a ssRNA molecule that is folded on itself in a hairpin structure with some mismatched base pairs. 1. Microprocessor cleaves off cap + poly-A. Now is pre-miRNA now in short hairpin form. 2. Dicer cleaves off hairpin to make a miRNA duplex. 3. Duplex is unzipped and one strand (the guide) is loaded into an Argonaute protein.

Studying PTMs in vivo

Peptides in a dish don't recapitulate PTM in endogenous cells; readers/writers/erasers need nucleosome context to fully understand their function. 1. UAA in vivo: problem, low yield. 2. EPL inside cell with intein halves, one half is attached to a PTM, one half attached to loci where you want PTM to be integrated. 3. CRISPR/dCas9 to bring PTM to specific locus on histone.

Sanger Sequencing controls

Positive = run unknown sequence alongside known sequence of that gene; align new sequencing read to reference genome to confirm you amplified what you wanted Negative = run reaction with no DNA, just water. No reaction should occur and gel should be blank.

Quantitative mass spectrometry use and general workup

Precise quantification of amount of PTM in genome. Proteins are fragmented and ionized, blasted into mass spectrometry machine and plotted by mass to charge ratio. Larger molecules will be further to right (higher mass/charge ratio). Peak suggests abundance of that fragment.

Processing PTMs vs. covalent PTMs

Processing: Cleavage of polypeptide backbone involved; IS TERMINAL / NOT REVERSIBLE! Covalent: dynamic (reversible) additions of chemical compounds onto available amino acids of a protein, e.g. on accessible side chains.

ChIP-seq pros/cons

Pros: get information about where PTM is localized on the genome Cons: can only assess one PTM at a time, epitope occlusion may occur when you linearize DNA

Express Protein Ligation pros/cons

Pros: high yield, can install multiple PTMs at a time, chemical flexibility Cons: technically very complex (SPPS especially difficult), limited to alterations at the ends of proteins

Sanger Sequencing pros/cons

Pros: relatively high-throughput, cheaper and faster than NGS, good for validation/confirmation of DNA sequence you already know Cons: not as high-throughput as NGS, harder to sequence novel DNA

UAA incorporation pros/cons

Pros: site-specific PTM, can be done in vivo. Cons: restricted to one PTM type at a time, can only incorporate something that can be loaded onto tRNA, also need to generate synthetic tRNA and tRNA synthetase AND mutate mRNA or DNA, low yield.

Click chemistry pros/cons

Pros: site-specific modifications Cons: azide/alkyne reaction must take place in presence of copper but too much copper is toxic to cells. A version of click chemistry has been developed to modify the chemical reaction so copper is not needed to overcome this challenge.

Cysteine alkylation pros/cons

Pros: site-specific, can have PTM at multiple sites depending on how many cysteine-creating mutations in histone you generate. Cons: Not all PTMs can be developed from cysteine modification, can't introduce more than one PTM at a time

Long-read sequencing pros/cons

Pros: useful for identifying complex structural variations and de novo assembly (no reference genome) and resolve splicing events Cons: lower accuracy than short-read sequencing, higher cost

Q-RT-PCR

Quantitative PCR of complementary DNA (cDNA) made from RNA via reverse transcriptase. Provides quantitative information about RNA expression. High cycle threshold (Ct) value = amount of cDNA took longer to reach the threshold so you started with a lower amount vs. low Ct value: reached threshold quickly.

Readers/writers/erasers

Reader = detect PTM Writer = add PTM Eraser = remove PTM

piRNAs (basics)

Regulate transposons and gene expression in germ cells during development. Binds to Piwi instead of Ago proteins.

Short-read RNA-seq output / analysis

Requires reference genome to align reads to! Peaks analysis = how many distinct reads aligned at this part of the genome. Lack of peak indicates an intron (since mature mRNA would have spliced out introns) Fastq output = for each read, contains sequence, machine info, and likelihood of mistake at sequencing each nucleotide in the sequence. Mistake prediction data can be used to exclude reads with a high probability of inaccuracy.

SYBR Green vs. TaqMan q-RT-PCR

SYBR: Non-specific integration and release of fluorescent probe into dsDNA as sequences get amplified. TaqMan: specific probes for your target sequence bound to a reporter which is quenched by a quencher. Probe binds to DNA and when sequence is being amplified, R gets knocked off by Q so R is released and no longer quenched; fluorescence is measured. CAN MULTIPLEX MANY TARGETS IN SAME WELL WITH DIFFERENT FLUORESCENT PROBE/PRIMER PAIRS.

Classes of non-coding RNAs

Short (<45nt): miRNAs, piRNAs, siRNAs, tRFs Intermediate size: tRNAs, snRNAs Long (>200nt): rRNAs, lncRNAs, circRNAs

Benefits of short-read vs. long-read sequencing

Short-read sequencing is more accurate and better for studying gene expression. Long-read sequencing is less accurate but better at reconstructing unknown transcripts so is useful in studying isoforms.

10X Genomics scRNA-seq workup (probably don't need to know specifics but know general principles)

Single cells are attached to gel beads in a lipid droplet. Each gel bead contains many probes with a poly-dT sequence, a UMI, and a common barcode specific to that gel bead. Probe binds to mRNA molecules in the cell because the poly-dT tail hybridizes with the poly-A tail on the mature mRNA. Make from mRNA in this manner. Each cDNA will have a UMI and barcode. Every UMI will be different: how to tell two tagged molecules apart, and to tell whether two distinct reads are just PCR-amplified copies of the same molecule or distinct copies of the molecule physiologically present in the cell. Every mRNA from that cell will receive the same barcode: how to tell mRNA is from one cell vs. another.

SNPs

Single nucleotide polymorphisms; likely to be problem in coding genes as they may code for a different amino acid to be added in the polypeptide at that location.

Click chemistry general principles

Takes advantage of intein chemistry to click together a PTM with a target protein, based on azide+alkyne bonding.

Balance between writers and erasers?

There are generally more writers than erasers in the genome. Why? Writers are very SPECIFIC to what unmodified sites they have to recognize and add a PTM to, while erasers need to act QUICKLY and ROBUSTLY to stop writing pathways from staying on too long: zoom to remove any of the PTM it can find, doesn't matter where it's bound.

What are isoforms and why do they matter?

Through ALTERNATIVE SPLICING, different RNA products (and by extension, proteins for coding mRNAs) can be produced from one common DNA sequence depending on what exons/parts of the DNA sequence are kept in the mature RNA molecule after splicing. They help produce genetic diversity as they can change structure and function of RNAs and proteins! ******if something is funky/unexpected about your gene expression: it could be the result of a spliceoform!!!!! Different antibodies or treatments may also target different spliceoforms differently and thus have different levels of efficacy...

Single cell RNA-seq general use

To account for a hetereogeneous sample and see cell-type specific mRNA expression. Helpful for understanding differential gene expression in smaller cell populations whose expression patterns might get drowned out in bulk-seq.

Transfection and some example methods

Transfection = non-viral vector delivery Electroporation: electric pulse applied to membranes briefly destabilizes it so plasmid can pass through to nucleus. Low efficiency, transient. Lipofection: plasmid is enveloped in a lipid droplet so it can come into nucleus through the hydrophobic membrane. Low efficiency, transient.

Transient vs. stable expression of vector

Transient = foreign DNA does not get incorporated into genome. E.g. AAV, electroporation, lipofection. Stable = foreign DNA DOES get incorporated into genome. E.g. lentivirus.

Ortholog

Type of homolog: genes in different species related to each other by a common ancestor, usually similar function

Paralog

Type of homolog: one gene that arose from another by duplication that has a slightly different function; same organism

UAA incorporation general principles

UAG = "amber" stop codon: least frequent in genome and rarely terminates genes. 1. Use Cas13 to edit mRNA to have amber stop codon at one or more loci of interest. 2. Special tRNA synthetase fuses a tRNA with an AUC anti-codon with an UAA tagged with your PTM. 3. During translation, when ribosome encounters amber stop codon, special tRNA will be recruited (competes with regular tRNA with AUC anti-codon) to integrate your UAA/PTM into the polypeptide.

Unbiased vs. biased RNA-seq analysis

Unbiased: genes are not subgrouped from the start. Unbiased checks to see if sample groups show patterns, i.e. what cells cluster together based on similar gene expression (e.g. UMAP, t-SNE plots) Biased: functionally similar genes plotted into specific subgroups with the goal of finding patterns of up/downregulation of genes with similar functions in various sample clusters (e.g. pathway analysis)

tRNA fragments (tRFs) (basics)

Unlike miRNAs/siRNAs, can bind directly to RNA without help from protein. But, can also be loaded onto Ago and function like a miRNA if has similar structure to miRNA.

ATAC-seq

Used for assessing chromatin density and accessibility of chromatin to binding partners/modification.

ATAC-seq general use

Useful in determining chromatin accessibility/openness. Generally, transposases act to insert reporter sequences wherever chromatin is open; where you don't see the reporter is heterochromatin/inaccessible.

Nanopore-seq

Version of long-read sequencing. As voltage applied to membrane, Negatively charged DNA passes to positively charged side of a membrane through a pore. The current changes measured as the DNA passes through the pore indicates what nucleotide is passing through as they are all different sizes. NO DNA POLYMERASE NEEDED.

Transduction and some example methods

Viral vector delivery 1. Adenovirus: carries dsDNA into cell. Pros: higher package size than AAV. Cons: highly immunogenic so may have off-target immune responses to general virus delivery. 2. AAV: carries ssDNA into cell. Pros: Not immunogenic, safe for experimenters. Cons: low package size. 3. Lentivirus: stable integration of vector into genome via reverse transcription. Pros: permanent if you want that, can affect dividing and non-dividing cells (e.g. neurons). Cons: highly dangerous for experimenters!

Promoter

Where RNA polymerase and some TFs (basal and others) bind to initiate transcription

ChIP-seq and CUT&RUN general use

and IP for Crosslink chromatin (DNA and histones) to see where on the genome proteins (e.g. TFs) or modifications (e.g. PTMs) are bound

3D genome organization

long range interactions, e.g. between enhancers and promoters of genes

si/shRNA vs. CRISPR/Cas13 vs. CRISPR/Cas9

si/shRNA inteferes with RNA only, not DNA, so it's only a KNOCKDOWN (repression of translation). DNA still exists and is still being transcribed. Less efficient than CRISPR/Cas13 but easy to use: only need to give cells/organism your si/shRNA construct. CRISPR/Cas13 is also effectively a knockdown since it targets RNA and not DNA. It's more efficient than si/shRNAs but has disadvantage of needing to provide both a Cas13 and guide, and Cas13 can lead to random RNA degradation if there's too much of it in the system. CRISPR/Cas9 is a KNOCK-OUT since it targets the original DNA itself and removes it from genome.