CRISPR - Science method

There is no point. Why introduce cell type and that vector situation as new variables? This is not going to tell you anything about what happens in your final intended system. Optimize for what you want, as directly as possible.

Didier Poncet Thanks again for your kindly help, and I have learned a lot from it! So i would take TCDI50 as consideration, for it represents infectious particles and shows the viral infectivity.

As Nicolas mentioned, size matters. Larger transgenes result in lower titers. If you are not experienced at making lenti vectors then your titer may be low for smaller transgenes like GFP. With this larger construct, your titer may fall low enough that you don't detect it at all.

Did you titer the GFP virus? If so, what was the titer? Did you transduce the experimental cells with the GFP virus or did you transduce 293?

Have you tried adding your Cas9 virus to 293 to see if you can detect it? The 293 line will probably be more permissive to lenti transduction. I would titrate both virus stocks on 293 to get a handle on titer and, if you can detect virus from the Cas9 prep, the difference in titer between the two stocks.

I hope you are doing well.

Yes, flow cytometry, commonly known as Fluorescence-Activated Cell Sorting (FACS), has been widely used on CRISPR-Cas9 transfected cells, and it serves as a powerful tool for the analysis and sorting of these modified cells. The application of FACS in this context generally focuses on the identification, quantification, and isolation of cells that have been successfully edited by CRISPR-Cas9.

Here's a detailed explanation of how FACS is utilized with CRISPR-Cas9 transfected cells:

These applications underscore the versatility of FACS in conjunction with CRISPR-Cas9 technology, providing both qualitative and quantitative insights into the gene editing process. Proper optimization of FACS parameters, including antibody selection, gating strategies, and fluorescence compensation, is essential for achieving accurate results.

If you have any specific questions or require further details, please do not hesitate to reach out.l Reviewing the protocols listed here may offer further guidance in addressing this issue

l Reviewing the protocols listed here may offer further guidance in addressing this issue

I'm sorry but this is not my area of expertise. Enjoy your research!

I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a revolutionary gene-editing tool that uses a guide RNA to direct the Cas9 enzyme to a specific genome location for precise DNA modifications. Despite its promise, CRISPR faces significant limitations and challenges in agriculture, particularly regarding off-target effects. Off-target effects can introduce unwanted, potentially harmful genome alterations, complicating the development of safe, reliable crop traits [4][5]. Additionally, technical and governance challenges, such as regulatory hurdles and the intricacy of gene drive mechanisms, further hinder its agricultural application [1][3]. Advances in detection methods like digital PCR and improvements in guide RNA synthesis are being developed to mitigate these issues [2][6].

[1] Chen, K., Wang, Y., Zhang, R., Zhang, H., & Gao, C. (2019). CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture.. Annual review of plant biology, 70, 667-697 .

[2] El-Mounadi, K., Morales-Floriano, M. L., & Garcia-Ruiz, H. (2020). Principles, Applications, and Biosafety of Plant Genome Editing Using CRISPR-Cas9. Frontiers in Plant Science, 11.

[3] Son, S., & Park, S. (2022). Challenges Facing CRISPR/Cas9-Based Genome Editing in Plants. Frontiers in Plant Science, 13.

[4] Saini, H., Thakur, R., Gill, R., Tyagi, K., & Goswami, M. (2023). CRISPR/Cas9-gene editing approaches in plant breeding. GM Crops & Food, 14, 1 - 17.

[5] Zhang, Y., Chen, S., Yang, L., & Zhang, Q. (2023). Application progress of CRISPR/Cas9 genome-editing technology in edible fungi. Frontiers in Microbiology, 14.

[6] Sato, G., & Kuroda, K. (2023). Overcoming the Limitations of CRISPR-Cas9 Systems in Saccharomyces cerevisiae: Off-Target Effects, Epigenome, and Mitochondrial Editing. Microorganisms, 11.

Hort Tandem Repeat (STR) testing, often used in forensic science and genetic analysis, is a powerful tool for identifying individuals based on the number of repeating units at specific locations in their DNA. STR testing can still identify someone with a germ-line mutation by detecting changes in the repeat patterns at specific DNA loci. If a germ-line mutation alters an STR region, the individual’s STR profile may show a new allele or a different number of repeats than expected based on their relatives. This can result in mismatches in parent-child STR profiles, which might indicate a mutation. While STR testing is generally reliable, the results must be carefully interpreted.

As I said before, even with the tag, the protein could be folded in such a way that the interaction with the nickel ions is not strong enough. If the protein becomes adsorbed at 5-10 mM imidazole and desorbs in 20-50 mM imidazole with washing, wash with the binding buffer, afterward, run an imidazole gradient (very slow) to 50 mM imidazole (or less).

Recent advancements in CRISPR technology have significantly improved its precision, efficiency, and versatility compared to traditional genetic modification methods. Here’s a simplified overview:

Overall, CRISPR technology represents a significant advancement over traditional genetic modification methods, offering greater precision, efficiency, and flexibility in genetic research and applications.

Well, creating a semi-permeable transcription bubble can be challenging in the context of the structural stability of DNA as it tends to reanneal to its double-stranded form. Next is the concern of replication, which involves the fact that semi-permanent unwinding can potentially hinder the replication machinery from proceeding with DNA replication. Lastly, to maintain the transcription bubble to its semi-permanent unwound state, RNA polymerase is required to be halted in its activity at the transcription site, which could, in turn, lead to instability and interference in the replication of the plasmid. Considering these aspects, I believe three probable yet theoretical strategies can be adopted in this regard. First is genetically engineering a modified RNA polymerase, which can maintain the plasmid DNA at its single-stranded state by getting associated at a precise plasmid location without hindering the transcription process. Second is implementing genetically engineered single-strand binding (SSB) proteins, which can keep the plasmid DNA at its unwound state without interfering with RNA synthesis. Lastly, chemical molecules such as intercalating agents are introduced, which can develop proximal unwinding by being inserted at the nitrogenous base pairs of plasmid DNA; Molecules that are enhancers or activators of helicases; Hydrogen bond destabilizers like Di-Methyl Sulfoxide (DMSO), Urea or Formamide which can perform denaturation of double-stranded DNA; Cross-linking agents like Psoralens which forms covalent cross-linkages between single-stranded DNA molecules and DNA or RNA polymerases; Ligands which associate with single-stranded DNA such as Peptide Nucleic Acids (PNAs) and nucleic analogs which stabilizes the single-stranded structures of DNA; Alkylating agents such as Nitrogen and Sulfur derivatives of Mustard gas, Ethyl Methanesulfonate (EMS), Methyl Methanesulfonate (MMS), N-Nitrosoureas and Temozolomide. Nevertheless, besides being hypothetical, all these strategies have cons of their own.

Mauro Lago Docampo thank you so much for your response!

Regarding selection of KRAS mutant clones: The authors in the paper I mention above (Clevers, Nature 2015) select for KRAS G12D mutants by removing EGF and adding the antibiotic gefitinib to culture media. My plan is to select for KRAS mutant clones using the selection media described in this paper.

The more research I have done into this experiment, I plan on introducing the triple KO in a second separate step to that described above. Again, the authors of the Clevers paper describe a media specially for selection of triple KO clones (-WNT -Noggin +Nutlin-3).

My plan was to then select and expand a single clone that has successfully been transfected with the G12D mutant/triple KO. I will confirm successful transfection with western blot and Sanger sequencing.

I hope this sufficiently addresses the points you made!

Dear Colleague,

I hope this message finds you well. The stability and half-life of guide RNA (gRNA) are critical factors in the efficacy of CRISPR/Cas9 genome editing systems. Here is a detailed and logical explanation regarding the average half-life of gRNA not bound to Cas9.

Average Half-Life of Unbound gRNA

Practical Implications for CRISPR/Cas9 Experiments

Conclusion

The average half-life of unmodified gRNA not bound to Cas9 can range from 30 minutes to 2 hours, depending on the specific cellular conditions. Chemical modifications can significantly enhance the stability of gRNA, extending its half-life to several hours or more. Understanding these factors is crucial for optimizing CRISPR/Cas9 experiments and ensuring efficient genome editing outcomes.

Should you have any further questions or require additional assistance, please feel free to reach out.

In a Crispr/Cas system, what is the average half life of a guide RNA (gRNA) not bound to Cas9?

Dear Colleague,

I hope this message finds you well. Observing extra bands on gRNA template PCR results can be attributed to several factors related to the PCR process and template quality. Here is a detailed and logical approach to understanding and troubleshooting this issue:

Potential Causes of Extra Bands in gRNA Template PCR

Troubleshooting Steps

Example Protocol Adjustments

By following these steps and making the necessary adjustments, you can reduce or eliminate extra bands in your gRNA template PCR results, ensuring more specific and accurate amplification.

Should you have any further questions or require additional assistance, please feel free to reach out.

This list of protocols might help us better address the issue.

Dear Colleague,

I hope this message finds you well. When studying glucagon secretion in pancreatic alpha cells, it is crucial to use a reliable and well-characterized cell line. One of the most commonly used and reliable pancreatic alpha cell lines for this purpose is the αTC1-6 cell line.

αTC1-6 Cell Line Overview

Protocol for Studying Glucagon Secretion in αTC1-6 Cells

Data Analysis and Interpretation

Alternative Cell Lines

While αTC1-6 cells are widely used and reliable, you may also consider other cell lines such as InR1-G9 cells, which are another pancreatic alpha cell line. However, αTC1-6 cells are generally preferred due to their extensive characterization and reproducible glucagon secretion.

By using the αTC1-6 cell line and following the outlined protocol, you can effectively study glucagon secretion and gain insights into the regulation of pancreatic alpha cell function.

Should you have any further questions or require additional assistance, please feel free to reach out.

This protocol list might provide further insights to address this issue.

researchers have used the CRISPR/Cpf1 (now often referred to as CRISPR/Cas12a) system for generating knock-in lines, similar to how CRISPR/Cas9 is used. The CRISPR/Cpf1 system has some distinct advantages and differences compared to CRISPR/Cas9, which can be beneficial in certain applications.

Overview of CRISPR/Cpf1 for Knock-In

**1. System Characteristics

**1.1 Cpf1 (Cas12a):

Different Mechanism: Cpf1/Cas12a has a different cleavage mechanism compared to Cas9. It generates sticky ends with 5' overhangs, which can be advantageous for certain types of genetic modifications.

Simpler Design: Cpf1 requires a simpler guide RNA (gRNA) design, with only one RNA molecule (as opposed to the separate crRNA and tracrRNA required for Cas9).

**1.2 Knock-In Strategy:

Targeting Vector: A donor DNA vector with the desired insert and homology arms flanks the target site. The homology arms are sequences homologous to regions flanking the double-strand break (DSB) induced by Cpf1.

Repair Mechanism: The DSB created by Cpf1 is repaired by homology-directed repair (HDR) using the donor DNA vector to incorporate the knock-in sequence.

**2. Experimental Steps

**2.1 Designing Guides:

Select Target Sites: Identify suitable target sites for Cpf1, usually requiring PAM sequences (5'-TTTV-3' for Cpf1).

Design gRNA: Design the single gRNA specific to your target site. This guide will direct the Cpf1 enzyme to the correct location in the genome.

**2.2 Preparing Components:

Cpf1 Protein: Obtain or produce the Cpf1 protein.

gRNA: Synthesize the gRNA or use a vector to express it.

Donor DNA: Clone the knock-in sequence into a donor vector with homology arms flanking the insertion site.

**2.3 Transfection:

Transfect Cells: Introduce Cpf1, gRNA, and donor DNA into your target cells via transfection methods suitable for your cell type (e.g., electroporation, lipofection, or viral transduction).

**2.4 Selection and Screening:

Selection: Use selection markers if included in the donor vector to identify successfully modified cells.

Screening: Screen for successful knock-ins using methods like PCR, sequencing, or Southern blotting to verify the incorporation of the knock-in sequence.

**3. Advantages and Considerations

**3.1 Advantages:

Sticky Ends: The sticky ends generated by Cpf1 can improve the efficiency of HDR by creating more compatible ends with the donor DNA.

Fewer Off-Target Effects: Some studies suggest Cpf1 may have fewer off-target effects compared to Cas9, although this can depend on the specific context and guide RNA design.

**3.2 Considerations:

Efficiency: The efficiency of knock-in can vary depending on the cell type and the specific CRISPR/Cpf1 system used.

Optimization: Experimental conditions, such as the ratio of Cpf1, gRNA, and donor DNA, need to be optimized for each system and cell type.

Summary

Using CRISPR/Cpf1 for generating knock-in lines is a viable and potentially advantageous alternative to CRISPR/Cas9. The system's distinct properties, such as its ability to generate sticky ends, can be leveraged to improve knock-in efficiency. As with any gene editing system, careful design, optimization, and validation are key to successful outcomes.

l With this protocol list, we might find more ways to solve this problem.

you can use a PCR product as a CRISPR HDR (Homology-Directed Repair) donor, but there are several considerations to ensure its effectiveness:

**1. Quality of the PCR Product

**1.1 Purity:

Ensure Purity: The PCR product should be pure and free from contaminants, such as residual primers, enzymes, and other by-products. Use a purification step like gel extraction or commercial PCR clean-up kits.

**1.2 Length and Integrity:

Check Integrity: Verify that the PCR product is of the correct size and does not have unintended bands or degradation products. This can be assessed by running an aliquot on an agarose gel.

**2. Design Considerations for the HDR Donor

**2.1 Homology Arms:

Include Homology Arms: The PCR product should contain homology arms flanking the desired insertion or modification site. The homology arms should be sufficiently long (typically 500-1000 bp) to promote efficient HDR.

**2.2 Donor Sequence:

Incorporate the Desired Sequence: Ensure that the sequence you want to insert or modify is correctly included in the PCR product. Verify the sequence by sequencing if necessary.

**2.3 Avoiding Repeats:

Minimize Repeats: Avoid long repeats or sequences that might cause unwanted recombination or instability.

**3. Transformation and Transfection

**3.1 Transformation Efficiency:

Assess Efficiency: If using bacteria, check the efficiency of transformation. High-quality DNA is essential for efficient transformation or transfection.

**3.2 Transfection:

Optimize Conditions: When using the PCR product in mammalian cells, optimize transfection conditions to ensure efficient delivery of the donor DNA. Methods include lipofection, electroporation, or viral delivery systems.

**4. Validation

**4.1 Confirm Insertion:

Validation: After introducing the HDR donor into the cells, validate successful insertion or modification by PCR, sequencing, or other molecular assays.

**4.2 Screening:

Screen for Correct Integration: Use appropriate screening methods to confirm that the HDR process occurred correctly and that the donor DNA has been properly integrated.

**5. Considerations for Using PCR Products

**5.1 Length Limitations:

Product Length: PCR products are typically short (e.g., 200-1000 bp). For larger insertions or modifications, consider using plasmid-based donors with larger homology arms.

**5.2 End Modification:

Blunt Ends vs. Overhangs: Ensure that the ends of the PCR product are compatible with the CRISPR-generated DSB (Double-Strand Break) ends. Depending on the CRISPR system used, you might need to modify the ends (e.g., to be blunt or to have overhangs).

**5.3 PCR Artifacts:

Check for Artifacts: Ensure that the PCR product does not contain artifacts such as nonspecific bands or residual primers that could affect the HDR process.

Alternative Approaches

**1. Plasmid-Based Donors:

Consider Using Plasmids: For larger modifications or more complex constructs, using plasmid-based HDR donors with appropriate homology arms may be more effective.

**2. Commercial Donors:

Pre-made Donors: Commercially available HDR donor plasmids often come with optimized designs for efficient integration and can save time and effort.

In summary, while PCR products can be used as HDR donors, their effectiveness depends on proper design, purification, and delivery. For larger or more complex modifications, plasmid-based donors or commercially available options might be preferable.

l Reviewing the protocols listed here may offer further guidance in addressing this issue.

Estimating the probability of forming a short CRISPR with a single spacer in a random sequence involves several steps. This calculation depends on the specific sequence characteristics and the CRISPR system's requirements. Here’s a structured approach to estimate this probability:

**1. Define the Parameters

**1.1 CRISPR System Characteristics:

Spacers: Typically, spacers in CRISPR systems are around 20 nucleotides long.

Protospacer Adjacent Motif (PAM): CRISPR systems require a PAM sequence adjacent to the target site. For example, the Streptococcus pyogenes Cas9 requires the PAM sequence "NGG."

**1.2 Random Sequence Properties:

Length: Determine the length of the random sequence where you are searching for the spacer.

Nucleotide Composition: For a truly random sequence, assume equal probabilities for each nucleotide (A, T, C, G).

**2. Calculate the Probability of a Specific Spacer Sequence

**2.1 Probability of Matching a Specific Spacer:

Calculate for PAM: If the PAM sequence is required, first calculate the probability of finding this PAM sequence in the random sequence.

Probability of Spacer Sequence: For a spacer of length L nucleotides, the probability of finding a specific sequence of length L in a random sequence is:

𝑃

(

spacer

)

=

(

1

4

)

𝐿

P(spacer)=(

4

1

​

)

L

where

1

4

4

1

​

is the probability of each nucleotide occurring at a specific position, and

𝐿

L is the length of the spacer.

**2.2 Consider PAM Sequence:

Probability of PAM: For a PAM sequence of length k nucleotides, assuming equal probability for each nucleotide, the probability of finding the PAM is:

𝑃

(

PAM

)

=

(

1

4

)

𝑘

P(PAM)=(

4

1

​

)

k

**3. Calculate the Probability of Spacer and PAM Co-occurrence

**3.1 Independent Events:

Assuming Independence: If the presence of the spacer and PAM are independent, the combined probability of finding both in the random sequence is:

𝑃

(

spacer and PAM

)

=

𝑃

(

spacer

)

×

𝑃

(

PAM

)

P(spacer and PAM)=P(spacer)×P(PAM)

**3.2 Search Space:

Length of Random Sequence: If you are searching within a sequence of length N, the number of potential positions for the spacer and PAM is N - (L + k - 1).

**4. Estimate the Expected Number of Hits

**4.1 Expected Hits:

Calculate Expected Number: Multiply the probability of finding the spacer and PAM by the number of potential positions:

Expected Number of Hits

=

𝑃

(

spacer and PAM

)

×

(

𝑁

−

(

𝐿

+

𝑘

−

1

)

)

Expected Number of Hits=P(spacer and PAM)×(N−(L+k−1))

**4.2 Adjust for Overlaps:

Overlap: Adjust calculations if the spacer and PAM are not independent or if there are constraints on their positioning relative to each other.

Example Calculation

Assuming:

Spacer length (L) = 20 nucleotides

PAM length (k) = 3 nucleotides

Random sequence length (N) = 1000 nucleotides

Probability of Spacer:

𝑃

(

spacer

)

=

(

1

4

)

20

=

9.09

×

1

0

−

13

P(spacer)=(

4

1

​

)

20

=9.09×10

−13

Probability of PAM:

𝑃

(

PAM

)

=

(

1

4

)

3

=

0.0156

P(PAM)=(

4

1

​

)

3

=0.0156

Combined Probability:

𝑃

(

spacer and PAM

)

=

9.09

×

1

0

−

13

×

0.0156

=

1.42

×

1

0

−

14

P(spacer and PAM)=9.09×10

−13

×0.0156=1.42×10

−14

Expected Hits:

Expected Number of Hits

=

1.42

×

1

0

−

14

×

(

1000

−

(

20

+

3

−

1

)

)

≈

1.42

×

1

0

−

14

×

977

≈

1.39

×

1

0

−

11

Expected Number of Hits=1.42×10

−14

×(1000−(20+3−1))≈1.42×10

−14

×977≈1.39×10

−11

Conclusion

In this example, the probability of finding a specific 20-nucleotide spacer and a 3-nucleotide PAM sequence in a random 1000-nucleotide sequence is extremely low, reflecting the challenge of finding specific CRISPR target sites. Adjust parameters accordingly based on your specific requirements and sequence characteristics.

l This protocol list might provide further insights to address this issue.

If you're experiencing inefficient CRISPR knockout results, several factors could be contributing to the suboptimal outcome. Here’s a troubleshooting guide to help identify and address common issues:

1. Design Issues

1.1 Guide RNA (gRNA) Design:

Target Sequence: Ensure that the gRNA specifically targets a unique and suitable sequence in the gene of interest. Use tools like CRISPR design tools (e.g., CRISPR design tools by Broad Institute) to assess off-target effects and efficiency.

gRNA Efficiency: Verify that the gRNA has high predicted efficiency. Tools like CRISPR-Cas9 target site predictors can help evaluate this.

1.2 PAM Sequence: Check if the protospacer adjacent motif (PAM) sequence is present and optimal for the Cas9 protein you are using (e.g., NGG for SpCas9).

2. Delivery Method

2.1 Transfection Efficiency:

Transfection Method: Ensure that your transfection method (e.g., lipofection, electroporation) is efficient. Low transfection efficiency can lead to inadequate expression of the CRISPR components.

Optimization: Optimize the transfection conditions (e.g., reagent concentrations, cell density) for your specific cell type.

2.2 Expression of CRISPR Components:

Plasmid Quality: Verify the quality and quantity of your CRISPR plasmids. Ensure they are properly prepared and free from contaminants.

Protein Expression: Confirm that the Cas9 protein and gRNA are being expressed at sufficient levels. Check for expression using methods like Western blotting or PCR.

3. Cell Line Issues

3.1 Cell Type: Some cell types are more challenging to transfect or less responsive to CRISPR. Verify that your cell line is amenable to CRISPR editing.

3.2 Cell Health: Ensure that the cells are healthy and in the appropriate growth phase. Poor cell health can affect CRISPR efficiency.

4. Validation and Detection

4.1 Off-Target Effects: Perform off-target analysis to ensure that the observed effects are due to the intended CRISPR activity and not unintended modifications.

4.2 Knockout Efficiency: Validate knockout efficiency using various methods:

Genotyping: PCR and sequencing to confirm insertions or deletions (indels) at the target site.

Western Blotting: To check the absence or reduced levels of the target protein.

Flow Cytometry: If applicable, to assess changes in protein expression at the cellular level.

4.3 Functional Assays: Assess the functional impact of the knockout to confirm that it affects the biological function or pathway you are targeting.

5. Technical Issues

5.1 Cas9 Activity: Ensure that the Cas9 protein is active and properly localized within the cell.

5.2 gRNA Stability: Check the stability of the gRNA. It should be adequately expressed and stable within the cell.

5.3 Plasmid Integration: If using plasmid-based CRISPR, ensure that the plasmids are integrated into the genome or stably expressed in the cells.

6. Alternative Approaches

6.1 Use of Different gRNAs: Try designing and using different gRNAs targeting other regions of the gene.

6.2 Cas9 Variants: Consider using different Cas9 variants or other CRISPR-associated proteins (e.g., Cpf1) that might be more effective in your system.

6.3 Experimental Conditions: Test different experimental conditions, such as different cell densities or culture conditions, to improve efficiency.

By systematically addressing these factors, you can identify and correct issues that may be causing inefficient CRISPR knockout results.

l Check out this protocol list; it might provide additional insights for resolving the issue.

“Which epistemology do you associate with biology? Why?”

- epistemology absolutely directly is associated with biology, since all points/steps in the utmost fundamental result in epistemology – “Scientific method” , see https://en.wikipedia.org/wiki/Scientific_method

“…An iterative,[43] pragmatic[12] scheme of the four points above is sometimes offered as a guideline for proceeding:[47]

The iterative cycle inherent in this step-by-step method goes from point 3 to 6 and back to 3 again. ……”

- all/every living being, even bacteria, use and perform in their lives at their behavior.

Cheers

I found the solution to the issue. The problem was caused by a small formatting error in the gRNA list file. Specifically, there was a space between the gene name "septin" and the number "5" in the entry for "septin 5". It should be "septin5" without a space. This minor formatting error prevented the data from being processed correctly, leading to the plotting error. It took a long time to identify this issue, but it's resolved now. I'm sharing this in case someone else runs into the same error.

Best,

I usually select the cells before carrying out FACS.

Hi Catherine, this might be a good place to start: http://www.leishgedit.net/Home.html

CCTOP

Yeah I found the same thing. The nanopore tools are terrible and customer support completely unresponsive. You'll be better off aligning it yourself in ClustalW.

The high random error rate between reads is still a problem. We use pacbio for this now, which has very high fidelity. Costly, but highly effective.

I think it's usually sold as a 10000X stock in DMSO, so 3.5 µL. If you have trouble seeing the band then double it to 7 µL.

Hi Zeyu;

Did you solve this problem?

It looks good to me too. I use AGL1 and GV3101 and those both have a pink coloration when you get a large pellet. They don't look very pink as colonies on plates as the color is faint.

This involves anti racist genetic engineering:

Thanks for the advice. The knock out mouse is not available but I will try the lentivirus .

Tripti

Basically, if you're publishing in any Nature journal you're obligated to make cell lines available to other qualified researchers (either via public repository or by sending them yourself), either for free or for a "reasonable" handling cost. There might be some extra hoops to jump through if the PI is from a non-profit institution / university and you're trying to acquire them at a for profit company. Also, a lot of fluorescent proteins are patented by private companies, so any use of them by for-profit entities will probably require extra licensing agreements with those companies, even if the cell line itself was created by a non-profit institution.

I think your experience with acquiring said cell line is going to be more related to the specific parties and individual PI involved than the journal publisher.

Using RT-qPCR to validate CRISPR-based knockout (KO) experiments can be highly effective but may also encounter several challenges that need careful consideration and troubleshooting. Here’s a structured approach to effectively use RT-qPCR for confirming gene knockouts and addressing potential issues:

1. Design of RT-qPCR Assays

2. RNA Extraction and Quality Control

3. cDNA Synthesis

4. qPCR Reaction Setup

5. Data Analysis

6. Troubleshooting Common Issues

Conclusion

Using RT-qPCR to validate CRISPR knockouts involves careful experimental design, precise execution, and thorough data analysis. By addressing each step with meticulous detail—from RNA extraction to data analysis—you can robustly validate the success of CRISPR-mediated gene knockouts and gain insights into the genetic manipulations within your experimental system.

Perhaps this protocol list can give us more information to help solve the problem.

The process of single cell clonal expansion of C2C12 cells, a murine myoblast cell line frequently used in muscle biology research, involves isolating and expanding individual cells to form genetically homogeneous colonies. This technique is pivotal for studies requiring uniform cell populations to ensure consistency and reproducibility of experimental results. Here’s a detailed guide on how to perform single cell clonal expansion of C2C12 cells:

Step 1: Preparation

Step 2: Single Cell Isolation

Step 3: Clonal Expansion

Step 4: Subculturing Colonies

Step 5: Validation and Characterization

Conclusion

Single cell clonal expansion of C2C12 cells is a meticulous process that requires precise technique and careful handling to ensure that colonies derived from a single cell grow and proliferate successfully. This method is essential for experiments requiring uniform cellular responses, particularly in genetic studies, drug testing, and muscle physiology research.

With this protocol list, we might find more ways to solve this problem.

Using CRISPR genome editing to generate mixed colonies of Clostridioides difficile (formerly known as Clostridium difficile) could be a challenging task due to the complexity of the organism and the intricacies of CRISPR technology. However, it's theoretically possible with careful design and execution.

Here's a general outline of how you might attempt this:

It's important to note that generating mixed colonies of C. difficile using CRISPR genome editing would require careful optimization of experimental conditions and rigorous validation of the resulting colonies.

Implementing single-cell cloning by limiting dilution in CRISPR-edited lymphoblastoid cell lines (LCLs) presents challenges and considerations, including variations in CRISPR editing efficiency, cell viability, genetic heterogeneity, selection strategies, and clone validation. While specific literature on this topic might be limited, similar techniques have been successful in other cell types. Successful implementation requires optimizing CRISPR editing protocols for LCLs, thorough validation of clones, and consultation with experts in CRISPR editing and cell biology.

When conducting experiments involving the lentiCRISPR v2 system for CRISPR-Cas9-mediated gene editing, verifying the correct insertion of the guide RNA (gRNA) sequence is a critical step. This ensures the specificity and efficacy of the gene editing process. To facilitate this, PCR amplification of the gRNA cassette followed by sequencing is commonly performed. Utilizing specific primers that flank the gRNA cloning site within the lentiCRISPR v2 vector is essential for this verification process.

Primer Design for gRNA Insert Verification:

The lentiCRISPR v2 vector, a widely used tool in gene editing, incorporates a U6 promoter driving the expression of the gRNA, followed by a cloning site where the gRNA is inserted. To amplify this region and confirm the presence and correct orientation of the gRNA insert, primers that anneal to sequences flanking the cloning site are used.

Suggested Primers:

PCR Amplification and Verification:

Conclusion:

The use of well-designed primers specific to the U6 promoter and the region downstream of the gRNA cloning site is essential for verifying the correct insertion of the gRNA in the lentiCRISPR v2 vector. This verification step is crucial for the success of CRISPR-Cas9 gene editing experiments, as it directly impacts the specificity and efficacy of the editing process. By following these guidelines for primer selection, PCR amplification, and sequencing, researchers can confidently validate the presence and accuracy of their gRNA inserts within the lentiCRISPR v2 system.

Take a look at this protocol list; it could assist in understanding and solving the problem.

It is difficult to have an single optimal way for every cell lines, each cells have their own strength driving multiple signaling pathway, upper limit of the plasmid copy received and interaction between GOI and its own proteins, hence affecting gene expression facility. We had tried at least four different transfection method with more than ten different reagents just to make a cell population to express 0.1% of GOI, while in 293T the expression was almost hitting the upper limit of detection in flow cytometry. Therefore, to generate an optimized protocol for each cell lines or primary cells are considered easier then to generate a single protocol for universal use. The cloning process or to achieve a single double KI/KO clone by CRISPR is very time consuming and troublesome, if you want to go through CRISPR, try look at the newest technique such as base/prime editing by Dr. David Liu. In the other hand, lentivirus is not, in our lab, lentivirus is an useful tool for large scale cell line production, a single production process may grant us enough amount of lentiviral particle for more then 100 times of usage. However, if a gene is already being difficult to express in such cell lines, I would rather look into an optimization of DNA construct, a small twist such as codon optimization or driving promoter sometimes grant you more then 50-fold of difference, that will be far practical then transfection process.

Best

Dear [Recipient],

I hope this message finds you well. It's clear that you are encountering challenges with your CRISPR/Cas9 cloning protocol, specifically in the insertion of your guide RNA (gRNA) sequence into the vector. This step is crucial for the successful application of CRISPR/Cas9 technology, and encountering difficulties can be frustrating. Below, I outline a series of considerations and troubleshooting tips to assist you in resolving this issue.

Assessing the Cloning Strategy:

Ligation and Transformation Efficiency:

Verification of Cloning Success:

Additional Considerations:

Conclusion:

Troubleshooting cloning protocols requires patience and systematic optimization of each step. By carefully reviewing your protocol and considering each of the points mentioned above, you can identify and rectify the issue hindering your cloning success.

Should you require further assistance or have specific questions at any step of your protocol, please do not hesitate to reach out. I am here to support you in advancing your research projects.

Best regards,

With this protocol list, we might find more ways to solve this problem.

Dear [Recipient],

I hope this message finds you well. Your inquiry regarding the use of liposomes for the delivery of Cas9/gRNA ribonucleoprotein (RNP) complexes and double-stranded oligodeoxynucleotides (dsODNs) is both timely and relevant in the context of genome editing technologies. This delivery strategy is increasingly recognized for its potential to facilitate efficient, targeted genome editing by reducing off-target effects and enhancing delivery efficiency. Below, I provide detailed advice and considerations for employing liposomes in this cutting-edge application.

Understanding the Delivery System:

Liposomes are versatile, lipid-based vesicles capable of encapsulating nucleic acids and proteins, facilitating their delivery into cells. Their biocompatibility, low immunogenicity, and ability to merge with cell membranes make them an attractive delivery system for Cas9/gRNA RNP and dsODNs.

Preparing Cas9/gRNA RNP and dsODNs:

Liposome Preparation and Complex Loading:

Cell Targeting and Uptake:

Monitoring Editing Efficiency and Off-target Effects:

Conclusion:

Liposome-mediated delivery of Cas9/gRNA RNP and dsODNs represents a promising avenue for enhancing the specificity and efficiency of genome editing applications. Success in this approach requires careful consideration of liposome composition, optimization of encapsulation processes, and thorough evaluation of delivery outcomes in target cells. Continued innovation and optimization in this area are expected to further refine these techniques, broadening their applicability and effectiveness in both research and therapeutic contexts.

Should you require further assistance or wish to explore additional aspects of this delivery method, please do not hesitate to reach out. I am here to support your research endeavors.

Best regards,

Take a look at this protocol list; it could assist in understanding and solving the problem.

You have two options:

1) As you suggested, to cut out the bands, purify them and directly sequence them with the PCR primers, but the sequence will probably be rather “dirty” because of carry-over contamination with other amplification products.

2) Cut out the bands, purify and clone them, dependent on whether you are using a Taq polymerase with proofreading activity or not in a blunt end or TA vector and sequence a couple of positive clones. Either you conduct colony PCRs with your specific primers or vector primers and directly sequence the correct amplification products with the respective primers; or make plasmid minipreps and sequence them.

Ajay Keot : Thanks. Sure will try sequencing PCR product.

Dear Esteemed Colleague,

Greetings. I trust this message finds you well and making significant progress in your research endeavors, particularly those involving the CRISPR-Cas9 system, a powerful tool for genome editing. Your inquiry regarding the selection of a control single-guide RNA (sgRNA) for CRISPR experiments is both critical and insightful, as appropriate controls are essential for validating the specificity and efficacy of your gene editing efforts. Below, I provide a detailed overview of control sgRNA selection and its importance in CRISPR studies.

Understanding Control sgRNAs in CRISPR Experiments

In CRISPR-Cas9 experiments, control sgRNAs serve as benchmarks to assess the baseline activity of the system and to distinguish between specific editing effects and nonspecific cellular responses. There are primarily two types of control sgRNAs commonly employed in CRISPR studies:

Selection Criteria for Control sgRNAs

Best Practices in Using Control sgRNAs

Conclusion

The use of control sgRNAs in CRISPR-Cas9 experiments is paramount for ensuring the reliability and specificity of gene editing studies. By carefully selecting and incorporating appropriate control sgRNAs, researchers can accurately interpret the outcomes of CRISPR experiments and advance our understanding of gene function and regulation.

Should you require further assistance in designing your CRISPR experiments or have additional questions regarding control sgRNA selection, please do not hesitate to reach out. I am here to support your scientific exploration and contribute to the success of your research endeavors.

Warm regards.

This list of protocols might help us better address the issue.

Dear Esteemed Colleague,

Greetings. I hope this message finds you well and immersed in the dynamic field of genetic engineering and regulation. Your inquiry regarding the differences between the CRISPR-dCas9 system and traditional transcriptional regulatory methods highlights a pivotal area of interest in contemporary research. Below, I provide a detailed comparison that elucidates the distinct features, applications, and implications of these two approaches to gene regulation.

Overview of CRISPR-dCas9 System

The CRISPR-dCas9 (dead Cas9) system is an innovative adaptation of the CRISPR-Cas9 genome editing technology. In this variant, the Cas9 nuclease is catalytically inactivated (dCas9), rendering it incapable of inducing double-strand breaks. Instead, dCas9 is harnessed as a precise targeting mechanism to modulate gene expression when fused to transcriptional activators or repressors, or when employed to physically block transcription machinery access.

Traditional Transcriptional Regulatory Methods

Traditional methods of transcriptional regulation encompass a broad spectrum of techniques, including the use of small molecules, RNA interference (RNAi), and overexpression or knockdown of transcription factors or co-regulators. These methods typically act by modulating the cellular concentration of regulatory molecules or by directly interfering with the transcriptional machinery.

Key Differences

Conclusion

The CRISPR-dCas9 system represents a significant advancement in the field of genetic engineering, offering specificity, flexibility, and precise control over gene expression that surpasses many traditional transcriptional regulatory methods. However, the choice between CRISPR-dCas9 and traditional methods should be informed by the specific requirements of the research project, including the desired level of control, target specificity, and potential for off-target effects.

Should you have further inquiries or wish to delve deeper into the applications of CRISPR-dCas9 in transcriptional regulation, please do not hesitate to reach out. I am here to support your scientific exploration and contribute to the advancement of genetic research.

Warm regards.

Check out this protocol list; it might provide additional insights for resolving the issue.

Dear Esteemed Colleague,

Greetings. I trust this message finds you well and engrossed in the cutting-edge realm of genome editing, particularly employing CRISPR-Cas9 technology. Your inquiry about separating homozygous from heterozygous mutations post-CRISPR is both crucial and timely for ensuring the precision and efficacy of genetic modifications. Below, I provide a comprehensive guide outlining methodologies for distinguishing between these genetic variants, thereby enhancing the resolution of your CRISPR-based experiments.

Overview

The CRISPR-Cas9 system enables targeted genomic alterations with high specificity. Following the introduction of CRISPR-Cas9 components into cells, the repair of the induced double-strand break can result in modifications leading to homozygous or heterozygous alleles. Determining the zygosity of these modifications is essential for characterizing the functional consequences of the edits.

Methodologies for Zygosity Determination

Considerations and Best Practices

By employing these strategies, you can accurately determine the zygosity of CRISPR-induced genetic modifications, a critical step in validating the success of your genome editing endeavors and understanding the functional implications of your edits.

Should you require further assistance or wish to delve deeper into any of these methodologies, please do not hesitate to reach out. I am here to support your research journey and contribute to the advancement of genome editing technologies.

Warm regards.

Take a look at this protocol list; it could assist in understanding and solving the problem.

Dear Esteemed Colleague,

Greetings. I trust this message finds you well and deeply engrossed in the exciting field of genome editing, particularly in the application of CRISPR/Cas9 technology. Your inquiry regarding the significance of the T7 Endonuclease I (T7E1) assay and the role of the HindIII site in the context of CRISPR/Cas9-mediated gene editing is both insightful and timely. These elements play pivotal roles in the optimization and evaluation of genome editing outcomes. Below, I elucidate the contributions and implications of the T7E1 assay and HindIII site in this revolutionary genetic engineering approach.

T7 Endonuclease I (T7E1) Assay in CRISPR/Cas9

The T7E1 assay is a widely used method for detecting mismatches, insertions, or deletions in DNA that result from the non-homologous end joining (NHEJ) repair mechanism following a double-strand break (DSB) induced by CRISPR/Cas9. This assay is instrumental in assessing the efficiency of CRISPR/Cas9-mediated gene editing by identifying the presence of edit-induced mutations.

HindIII Site in CRISPR/Cas9-mediated Gene Editing

The HindIII site, a recognition sequence for the HindIII restriction enzyme, is often used in molecular cloning and can also play a role in CRISPR/Cas9 genome editing, particularly in the design and validation of editing strategies.

In conclusion, both the T7E1 assay and the utilization of the HindIII site are integral components of the CRISPR/Cas9 toolkit, enhancing the precision, verification, and overall success of genome editing endeavors. These methodologies underscore the sophistication and adaptability of current genetic engineering techniques, paving the way for advanced research and therapeutic applications.

Should you seek further elucidation on these topics or wish to explore additional aspects of CRISPR/Cas9 technology, please do not hesitate to reach out. Your engagement in advancing the frontiers of genetic research is highly valued.

Warm regards.

Reviewing the protocols listed here may offer further guidance in addressing this issue

Dear Esteemed Colleague,

Greetings. I hope this message finds you well and thriving in your research endeavors. Your inquiry regarding the cultivation of HEK293T cells following single-cell sorting is of paramount importance for ensuring the success of your experiments. Single-cell sorting and subsequent culture of such cells require meticulous care to ensure viability and promote growth. Below, I provide a detailed guide designed to optimize the conditions for growing HEK293T cells after single-cell sorting.

1. Preparation Before Sorting

2. Sorting Parameters

3. Post-Sorting Culture

4. Optimal Culture Conditions

5. Troubleshooting

Adhering to these detailed tips and maintaining a meticulous approach to the post-sorting care of HEK293T cells will significantly enhance the likelihood of successful culture from single-cell sorts. It is crucial to adapt these guidelines as necessary to fit the specific requirements of your experimental setup and the unique characteristics of your cell line.

Should you have any further questions or require additional insights into cell culture techniques, please do not hesitate to reach out. I am here to support your scientific exploration and contribute to the advancement of your research.

Warm regards.

Perhaps this protocol list can give us more information to help solve the problem.

It depends. If you just want to separate your pool of GFP-positive cells and if fluorescence is always being expressed, flow is sufficient.

Single clones are when you need a specific population. The result is much more accurate, but the work is greater.

Confirming a knockout generated by the CRISPR/Cas9 system involves several steps to validate the disruption or deletion of the target gene. Here are some common methods used to confirm CRISPR/Cas9-mediated knockouts:

By using one or more of these methods, researchers can confirm the knockout generated by the CRISPR/Cas9 system and validate the loss of gene function in the targeted cells or organisms.

l Check out this protocol list; it might provide additional insights for resolving the issue.

The timing for starting selection after transfection of CRISPR vectors can depend on various factors, including the specific experimental setup, the type of CRISPR system used, and the characteristics of the target cells. However, here are some general considerations to help you determine when to initiate selection after transfection:

Based on these considerations, it's common to start selection for edited cells within 24-48 hours after transfection of CRISPR vectors. However, the optimal timing may vary depending on the specific experimental setup and cell type. It's essential to empirically determine the best timing for selection in your particular experimental context through pilot experiments and optimization studies.

l This list of protocols might help us better address the issue.

there is a protocol for knocking out genes in THP1 cells using the CRISPR/Cas9 system. Here's a general outline of the protocol:

Remember to adhere to appropriate safety guidelines and institutional regulations when working with CRISPR/Cas9 technology and genetically modified cells. Additionally, it's essential to consider potential off-target effects and validate the specificity of CRISPR/Cas9 editing in your system.

l Perhaps this protocol list can give us more information to help solve the problem.

Transfecting Raji cells efficiently can be achieved by optimizing several factors, including the choice of transfection reagent, cell density, and transfection conditions. Here's a general protocol along with some suggestions to enhance the efficiency of transfection in Raji cells:

By carefully optimizing these parameters and following the protocol, you can achieve efficient transfection of Raji cells for your experiments.

l With this protocol list, we might find more ways to solve this problem.

Dear Colleague,

Thank you for your inquiry about the application of CRISPR/Cas9 technology in MDA-MB-231 and MCF-7 cell lines. The use of CRISPR/Cas9 for genome editing has indeed been widely adopted across various cell lines, including those relevant to breast cancer research, such as MDA-MB-231 and MCF-7. The success of CRISPR/Cas9 in these cell lines is well documented in scientific literature, underscoring its potential for understanding cancer biology and developing therapeutic strategies.

MDA-MB-231, a triple-negative breast cancer cell line, and MCF-7, an estrogen receptor-positive line, have been utilized in numerous studies to investigate the role of specific genes in cancer progression, metastasis, drug resistance, and other crucial aspects of cancer biology. CRISPR/Cas9 has been employed in these contexts to knock out genes of interest, introduce specific mutations, or modulate gene expression, thereby elucidating their functions in cancer development and progression.

Several key factors contribute to the success of CRISPR/Cas9 in these cell lines:

Literature reports demonstrate successful applications of CRISPR/Cas9 in these cell lines, ranging from basic research on gene function to more applied studies on drug resistance mechanisms and potential therapeutic targets. These studies underscore the adaptability and effectiveness of CRISPR/Cas9 in manipulating the genomes of MDA-MB-231 and MCF-7 cells for diverse research purposes.

In conclusion, CRISPR/Cas9 genome editing has been successfully applied in MDA-MB-231 and MCF-7 cell lines, contributing significantly to breast cancer research. The continued refinement of CRISPR/Cas9 methodologies and an increasing understanding of cell line-specific responses to genome editing will further enhance the utility of these cell models in cancer research.

Best regards,

Reviewing the protocols listed here may offer further guidance in addressing this issue

CRISPR/Cas and dCas (dead Cas) systems have revolutionized genome engineering, allowing researchers to manipulate specific DNA and RNA sequences in living cells. However, they come with certain limitations:

In summary, while CRISPR/Cas and dCas hold immense promise, addressing these limitations is crucial for their successful application in gene function studies and epigenetic modifications.