Panda's In Class Group Things

CRISPR PPT (1-16) + Isothermal Amp Methods (17-end)

How does a real-time hydrolysis probe (like TaqMan) generate a signal during amplification?

• Probes have a 5' fluorescent reporter dye and a 3' quencher dye

• Binds to specific target sequences

• When Taq polymerase runs into it, the 5' reporter dye is cleaved

• Now separated from the quencher, it releases a measureable signal

Contrast TaqMan with CRISPR platforms that use isothermal pre-amplification followed by Cas detection

•TaqMan requires thermal cycling (PCR)

•TaqMan relies on probe cleavage during target amplification. CRISPR uses a 2-step process where isothermal amplified product (RPA/LAMP)  activates Cas enzyme – Trans-cleavage breaking probes to release signal

Discuss trade-offs: What are the contamination risks of putting a post-amplification product on a lateral flow strip compared to a closed-tube real-time PCR?

On a lateral flow strip, there is potential for open environment exposure (eg. Airborne particles); on the other hand, with a closed-tube system, owing to the requirement that the system must the entire time, cross-contamination between different samples before the specimens are sealed will dramatically lower the accuracy of the system owing to its increased sensitivity.

CRISPR is better for qualitative results and when speed is important and there are low levels of target, whereas TaqMan qPCR methods are preferred when quantitative results are desired.

What is "collateral" or trans-cleavage activity?

refers to the phenomena that occurs whenever the enzyme binds to the nucleic acid, becomes activated, and cuts DNA/RNA at targeted locations.

Which Cas enzyme performs this indiscriminate cutting on single-stranded DNA, and which performs it on RNA?

Cas12 -> cleaves ssDNA

Cas13 -> cleaves RNA

How do these systems use reporter molecules (fluorophore attached to a quencher) to create a visible signal when cleaved by the activated Cas enzyme?

Concept:

• Add a reporter molecule:
  ssDNA or RNA tagged with:

  • Fluorophore (F)

  • Quencher (Q)

Before cleavage:

• F and Q are close → no fluorescence

After target recognition:

1. Cas12/Cas13 binds its specific target

2. Enzyme becomes activated

3. Collateral cleavage cuts reporter

4. F separates from Q → fluorescence signal ON

Which specific enzyme does DETECTR rely on, and what kind of target does it naturally bind and cut?

DETECTR relies on the Cas12a ssDNAase enzyme, naturally binding to and cutting the target of trace DNA samples. DETECTER makes use of a guide RNA (gRNA) to find its target sequence. The results in outcomes similar to PCR and is applicable to both bacteria and viruses.

Explain the sequence of events: how does combining an isothermal pre-amplification step (like RPA) with Cas12a collateral activation allow for rapid virus detection (e.g., HPV in under 60 mins)

• Isothermal pre-amplification increases the concentrations of DNA, to increase signal

• CAS12a protein detects target region of DNA with self-designed crRNA

• DNA probes are made similar to TaqMan. One end of the probe is bound to a fluorophore.

• Cas12a recognition of target degrades the DNA probes due to the collateral cuts, therefore releasing fluorophores

Which specific enzyme does SHERLOCK use, and what is its primary target?

uses Cas13a enzyme to target RNA (specifically ssRNA) for coronavirus and zika virus detection

Why does this platform require an extra in vitro transcription step to convert the isothermally pre-amplified DNA into RNA before the Cas enzyme can detect it?

• Requires extra in vitro transcription step because of the isothermal pre-amplification and how it identifies DNA

• Amplified DNA must be transcribed into RNA before Cas 13

• Cas13  is a programmable single-effect RNA-guided ribonuclease that works for  type VI CRISPR-Cas

Which type of nucleic acid (DNA vs. RNA) does Cas12 target compared to Cas13?

Cas12: dsDNA

Cas13: ssRNA

Based on these targets, give an example of a specific virus each enzyme has been used to detect (e.g., HPV, SARS-CoV-2, Dengue)

Cas12: HBV

Cas13: SARS-CoV-2

Furthermore, describe how their "collateral" (trans-cleavage) activity differs regarding the specific type of reporter molecule (ssDNA vs. ssRNA) they must cut to generate a fluorescent signal

Cas 12

• DETECTR

• trans-cutting activity

• early diagnosis tool

• rapid, high sensitivity

• can’t cut RNA

Cas13

• SHERLOCK

• broad, used for emerging infectious diseases

• antiviral drug development

• can’t cut DNA

Why does the indiscriminate nature of collateral trans-cleavage make multiplexing (detecting multiple pathogens in one tube) so inherently difficult, and what is the current target limit for platforms like SHERLOCK v2

A1：Off-target effect. Activated Cas enzymes generated non-specific trans-cleavage, cutting off other reporters which does not belong to it. Hard to tell which target is positive. Four target is limited.*

Explain how the strict requirement of a PAM (Protospacer Adjacent Motif) sequence restricts where you can design a diagnostic probe.

This PAM requirement limits the target sequences by only allowing sequences with nearby PAMs to be selected which could lead to the selection of less optimal sequences (Also likely fail if there is new mutations). This in turn reduces the probe versatility by  forcing researchers to choose sequences situated near a PAM instead of choosing the most optimal diagnostic sequence based on GC content and hybridization efficiency alone.

Finally, why do these assays require an isothermal pre-amplification step to achieve a clinically relevant Limit of Detection (LOD)?

Isothermal amplification nucleic acid sequence-based amplification and CRISPR-Cas system is combined as diagnostic tools for rapid detection of infectious diseases. This step is necessary to enhance analytical sensitivity by increasing the amount of target DNA/RNA especially in the case of low abundance pathogens. This replaces complex equipment by keeping constant temperature and enables faster turnaround times for critical diagnostic applications and field testing.

Which isothermal amplification method specifically targets RNA

NASBA

Which isothermal amplification method contains a simple primer design of 1 primer?

Rolling Circle Amplification (RCA)

Which isothermal amplification method has simple primer design that has DNA-RNA-DNA chimeric style with nicking site?

Isothermal Chain Amplification (ICA)

Which isothermal amplification methods have nicking sites on their primers

iSDA and ICA

Which isothermal amplification method uses a T7 promoter

NASBA

Which isothermal amplification outputs RNA transcripts?

NASBA

Which isothermal amplification method has extremely high amplification power?

LAMP

Which isothermal amplification method has much lower to moderate amplifcation power?

RCA

Which isothermal amplification method use thermal breathing and the Bst polymerase?

CPA and LAMP

Which isothermal amplification method uses spontaneous DNA breathing (thru nicking)

iSDA

Which isothermal amplification method uses an enzymatic initiation strategy with reverse transcription and transcription (enzyme: reverse transcriptase)?

NASBA

Which isothermal amplification method uses helicase and DNA polymerase?

helicase-dependent amplification (HDA)

Which isothermal amplification method uses recombinase?

recombinase polymerase amplification (RPA)

Which isothermal amplification methods use nicking endonuclease or nicking enzyme with DNA polymerase?

iSDA and Ica

Which isothermal amplification method uses Phi29 DNA polymerase?

Rolling Circle Amplification (RCA)