assistant: Welcome to CRISPR-GPT. I can help with the following tasks. Please select 
one to continue.

1. Meta-Mode (Step-by-Step Guidance on Pre-defined Meta-Task)
2. Auto-Mode (Customized Guidance on Free-style User Request)

Note: Chat with ChatGPT augmented with our up-to-date knowledge base 
directly by entering Questions followed by "Q:". For example, "Q: What is 
CRISPR?"


user: 1

assistant: {'Thoughts': 'The user has chosen Meta-Mode, which involves step-by-step guidance on a pre-defined meta-task. This indicates that the user is looking for a structured approach to a specific CRISPR-related task.', 'Choice': '1'}

assistant: Please select the general gene editing scenarios to continue.
1. Generating a Knockout Using CRISPR.
2. CRISPR Base Editing Without Double-Strand Breaks.
3. Generating Small Insertion/deletion/base editing through Prime Editing.
4. Activation or Repression of Target Genes Using CRISPR.


user: 1

assistant: {'Thoughts': 'The user has selected the option to generate a knockout using CRISPR, which typically involves introducing a double-strand break at a specific location in the genome to disrupt the function of a target gene.', 'Choice': '1'}

assistant: Now, let's start designing and planning your knockout experiment using CRISPR system, we will go through a step by step process as listed below:
1. Selecting a CRISPR/Cas system.
2. Selecting a delivery approach for CRISPR/Cas system.
3. Designing guideRNA for gene knockout.
4. Collecting appropriate experimantal protocols and validating the editing outcomes.


assistant: 
Step 1. Selecting a CRISPR/Cas system 

We support four types of CRISPR/Cas system for CRISPR knockout, SpCas9, enCas12a, SaCas9, and SpRYCas9, representing distinct variants with unique properties and applications.

1. SpCas9  (Jinek et al.,Science,2012;Cong et al.,Science,2013;Mali et al.,Science,2013;Jinek et al.,eLife,2013)
- Pros: Highly effective, well-studied, proven applications in many cell types/scenarios.  
- Cons: Large, potential off-target effects.  
- Uses: Genetic research, gene therapy, genetically modified models/organisms (GMMs/GMOs).

2. enCas12a  (Kleinstiver et al.,Nat Biotechnol.,2019;DeWeirdt et al.,Nat Biotechnol.,2020)
- Pros: Smaller, precise, low off-target rate, multi-target editing capability using a single guideRNA array.  
- Cons: May be less efficient than SpCas9 when using a single guideRNA (mitigated if using multi-guide array), limited target sites vs. SpCas9 due to more restrictive PAM.  
- Uses: High-precision and multiplexed genome editing (for genetic interaction, target redundant genes/pathways).

3. SaCas9  (Ran et al.,Nature,2015)
- Pros: Compact, suitable for AAV vectors (knockout genes in primary cells or in vivo).  
- Cons: limited target sites vs. SpCas9 due to more restrictive PAM.  
- Uses: In vivo gene therapy and some primary cells when using AAV delivery.

4. SpRYCas9  (Walton et al.,Science,2020)
- Pros: Wide PAM range, targets more genomic regions vs. typical SpCas9.  
- Cons: Higher off-target risks.  
- Uses: editing a specific target site where canonical PAMs for SpCas9/SaCas9/enCas12a are not available, and where specificity/off-target concerns are less critical.

*Please cite the corresponding papers if you decide to use the system.


assistant: 
Please select the CRISPR-Cas system you would like to use. Or please briefly describe your need (exp: I want to edit mouse liver/I want low off-target rate/I want to do multiplexed editing) and we could recommend the CRSIPR-Cas system to you.


user: I hope to do mutiplexed editing to knockout a few genes in A549 cell line.

assistant: {'Thoughts': 'The user needs to perform multiplexed editing to knockout a few genes in the A549 cell line. Multiplexed editing requires the ability to target multiple genomic locations simultaneously. Among the available Cas systems, enCas12a has a unique ability to cut multiple sites simultaneously, allowing for efficient targeting and editing of several genomic locations in a single experiment. Additionally, enCas12a offers increased genome editing fidelity due to its lower off-target rate. While it sometimes exhibits lower editing efficiency compared to SpCas9, the requirement for multiplexed editing makes enCas12a the most relevant choice.', 'Answer': 'enCas12a'} Final Result enCas12a

assistant: 
Step 2. Selecting a delivery approach for CRISPR/Cas system

Different biological systems are amenable to different delivery methods for genome-editing tools. We currently support the following delivery method:

a. Plasmid transfection
b. Lentivirus/Retrovirus delivery
c. RNP/mRNA Electroporation
d. RNP/mRNA Microinjection
e. mRNA LNP
f. AAV (Adeno-Associated Virus)


Please select one from above or please briefly describe your application cases below (exp. I hope to edit A549 cell lines/ I hope to edit mouse liver).

*Please note we currently only support mammalian cells/tissues.


user: I hope to edit A549 cell line.

assistant: Please find the recommended delivery approach below:

Top choice: 
### b. Lentivirus/Retrovirus

**Introduction**: Lentivirus and retrovirus vectors are tools for delivering genetic material into cells, allowing stable integration of the transferred genes into the host genome, leading to long-term expression.

- **Pros**:
  - **Stable Expression**: Allows for permanent gene expression modifications in the host cell.
  - **Broad Host Range**: Capable of infecting both dividing and non-dividing cells.
  - **High Efficiency**: Especially effective in hard-to-transfect cell types.

- **Cons**:
  - **Safety Concerns**: Though lentiviral vectors are designed to be safe, there remains a theoretical risk of insertional mutagenesis.
  - **Complex Production**: Virus production and purification can be technically challenging and time-consuming.
  - **Regulatory and Biosafety Considerations**: Use of viral vectors requires strict adherence to biosafety guidelines.

- **Applications**: Ideal for studies requiring stable gene expression, gene therapy research, and cell line generation.


The relevant papers we found on Google Scholar: 
- Targeting H19 by lentivirus-mediated RNA interference increases A549 cell migration and invasion: https://www.tandfonline.com/doi/abs/10.1080/01902148.2016.1223229
- Downregulation of Glutathione S‑transferase A1 suppressed tumor growth and induced cell apoptosis in A549 cell line: https://www.spandidos-publications.com/ol/16/1/467
- TMEM16A protein attenuates lipopolysaccharide-mediated inflammatory response of human lung epithelial cell line A549: https://www.tandfonline.com/doi/abs/10.3109/01902148.2014.905655
- Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line.: https://search.ebscohost.com/login.aspx?direct=true&profile=ehost&scope=site&authtype=crawler&jrnl=10093419&asa=Y&AN=84503399&h=g3mCCJQ1he%2F0qRQgcnoIGRulDb8PiLFfQjwTuxv1kHGMRz60pr2w8slCAoIrD6O%2BDocMEF0fhyS755ZN%2FxEafA%3D%3D&crl=c
- Knockdown of autophagy-related gene BECLIN1 promotes cell growth and inhibits apoptosis in the A549 human lung cancer cell line: https://www.spandidos-publications.com/mmr/7/5/1501
- Down-regulation of JAK1 by RNA interference inhibits growth of the lung cancer cell line A549 and interferes with the PI3K/mTOR pathway: https://link.springer.com/article/10.1007/s00432-011-1037-6
- Construction of the lentivirus vector over-expressing PCGF1 and establishment of A549 cell line stably over-expressing PCGF1: https://search.proquest.com/openview/a62d750911b7b52ef03625a8c8973854/1?pq-origsite=gscholar&cbl=2047934
- A newly engineered A549 cell line expressing ACE2 and TMPRSS2 is highly permissive to SARS-CoV-2, including the delta and omicron variants: https://www.mdpi.com/1999-4915/14/7/1369
- Lentivirus-mediated shRNA interference targeting SLUG inhibits lung cancer growth and metastasis: https://koreascience.kr/article/JAKO201205061575916.page
- Antitumor activity of interleukin-18 on A549 human lung cancer cell line: https://journals.lww.com/cancerjournal/fulltext/2019/15070/antitumor_activity_of_interleukin_18_on_a549_human.31.aspx

Second choice: 
### c. RNP/mRNA Electroporation

**Introduction**: This method involves introducing ribonucleoprotein (RNP) complexes or mRNA directly into cells using an electrical field to make the cell membrane more permeable.

- **Pros**:
  - **Transient and Safe**: Provides a temporary expression without integrating into the genome, reducing long-term safety concerns.
  - **High Efficiency**: Can achieve high transfection efficiencies in a broad range of cell types, including difficult-to-transfect cells.
  - **Versatile**: Suitable for various applications from gene editing (using CRISPR-Cas9 RNPs) to protein expression.

- **Cons**:
  - **Cell Viability**: The electroporation process can be harsh, resulting in reduced cell viability.
  - **Optimization Required**: Conditions must be meticulously optimized for different cell types to achieve the desired balance of efficiency and viability.

- **Applications**: Gene editing, transient protein expression, immunotherapy research, and stem cell research.


The relevant papers we found on Google Scholar: 
- Cytotoxicity studies of quantum dots with the electroporation method: https://www.sciencedirect.com/science/article/pii/S156753941830389X
- Electroporation of suspension cell lines–A proposed assay set for optimizations: https://www.sciencedirect.com/science/article/pii/S1567539421001547
- Role of Irreversible Nano Electroporation on the A549 Epithelial Cancer Cells: https://www.ingentaconnect.com/contentone/asp/jobn/2018/00000012/00000006/art00018
- Effect of irreversible electroporation on cancer cells: https://ieeexplore.ieee.org/abstract/document/6232622/
- Optimization of Non-viral Gene Delivery An Experimental Study on the Optimization of Electroporation on Adhered Epithelial Cells: https://www.researchgate.net/profile/Arun-Nambiar/publication/299716824_Optimization_of_Non_Viral_Gene_Transfer_via_Electroporation/links/57046cdc08ae74a08e24642b/Optimization-of-Non-Viral-Gene-Transfer-via-Electroporation.pdf
- Specific antitumor effect of the combined action of algal heteropolysaccharide and electroporation: https://biomed.bas.bg/bioautomation/2016/vol_20.3/files/20.3_08.pdf
- Type I interferon response against viral and non‐viral gene transfer in human tumor and primary cell lines: https://onlinelibrary.wiley.com/doi/abs/10.1002/jgm.997
- Irreversible electroporation: a drug-free cancer treatment: https://books.google.com/books?hl=en&lr=&id=onB7AwAAQBAJ&oi=fnd&pg=PA219&dq=%22A549,+cell+line%22%22electroporation%22&ots=V2KURUIGvJ&sig=prJrc0WzK54pzLuD-MvYZi6BiHM
- THE EFFECT OF ELECTROPORATION ON THE ADHESIVENESS OF DIFFERENT EPITHELIAL CELL TYPES: https://www.researchgate.net/profile/Aneliya-Kostadinova/publication/270565362_THE_EFFECT_OF_ELECTROPORATION_ON_THE_ADHESIVENESS_OF_DIFFERENT_EPITHELIAL_CELL_TYPES/links/54ad1c4f0cf2828b29f95374/THE-EFFECT-OF-ELECTROPORATION-ON-THE-ADHESIVENESS-OF-DIFFERENT-EPITHELIAL-CELL-TYPES.pdf
- EXPLORATION OF THE VARIABLES OF ELECTROPORATION FOR THE TRANSFECTION OF A MACROPHAGE CELL LINE: https://libstore.ugent.be/fulltxt/RUG01/002/482/335/RUG01-002482335_2018_0001_AC.pdf



assistant: Which delivery method would you like to use?


user: b. I would like to use lentivirus.

assistant: {'Thoughts': 'The user has selected lentivirus as the delivery method. Lentivirus vectors are beneficial for stable and long-term gene expression, which is ideal for applications such as gene therapy research and generating stable cell lines. Despite the safety and biosafety considerations, the advantages such as high efficiency in hard-to-transfect cells and broad host range make it a suitable choice for their needs.', 'Choice': 'b'}

assistant: 
Step 3. Designing guideRNA for gene knockout

Next, we would like to guide you through the design of guideRNA for your target gene. Designing an effective and specific single-guide RNA (sgRNA) is crucial for successful CRISPR/Cas9-mediated genome editing.There are several factors to consider in this process, including the selection of target sites that are unique to your gene of interest and minimizing potential off-target effects. 



assistant: 
We could help you design the guideRNA sequence based on CRISPick (https://portals.broadinstitute.org/gppx/crispick/public) (Kim et al.,Nat Biotechnol.,2018; DeWeirdt et al.,Nat Biotechnol.,2021; Doench et al.,Nat Biotechnol.,2016; Sanson et al.,Nat Commun, 2018).

Could you please describe what sgRNAs do you need? (For example, you can type in: I hope to design 2 sgRNAs targeting human EGFR; Please give me 1 sgRNA target Exon 1 of mouse Tgfbr1.)

*Please include the target and species information.
*Please cite the corresponding papers if you decide to use the designed sgRNA.


user: Could you help me design 1 sgRNA targeting human TGFBR1 with highest on-target editing efficiency?

assistant: Warning: Your input contains possible human heritable editing. Please confirm you understand the ethical risk and have read the international guideline https://www.nature.com/articles/d41586-019-00726-5. Type 'yes' to continue.

user: yes

assistant: 
We could help you design the guideRNA sequence based on CRISPick (https://portals.broadinstitute.org/gppx/crispick/public) (Kim et al.,Nat Biotechnol.,2018; DeWeirdt et al.,Nat Biotechnol.,2021; Doench et al.,Nat Biotechnol.,2016; Sanson et al.,Nat Commun, 2018).

Could you please describe what sgRNAs do you need? (For example, you can type in: I hope to design 2 sgRNAs targeting human EGFR; Please give me 1 sgRNA target Exon 1 of mouse Tgfbr1.)

*Please include the target and species information.
*Please cite the corresponding papers if you decide to use the designed sgRNA.


assistant: Sugessted exon/exons: {'Exon/exons': 'Exon 2 or Exon 3', 'Reason': {"Exon 2 and Exon 3 of the TGFBR1 gene are commonly targeted exons for CRISPR/Cas9 editing due to their essential role in the gene's function and their relatively high accessibility for sgRNA binding. Targeting these exons can potentially result in a functional knockout of the gene, which is often desired in gene editing experiments."}}

assistant: 
Based on the information you provided, it may be beneficial to design sgRNAs targeting specific exons within the genes.

Would you like to continue sgRNA design with the exon/exons we suggested?


user: no

assistant: https://crispr-gpt.s3.amazonaws.com/b935b403-5e6f-4030-b6e2-f0238ae470e2?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=JQo2BkewpNUd6kBKxSj3XCJOw8M%3D&Expires=1725759883

| Target Gene Symbol   | CRISPR Mechanism   | sgRNA Sequence          | PAM Sequence   |   Exon Number |   sgRNA Cut Position (1-based) |   On-Target Efficacy Score |   Off-Target Rank |   Combined Rank |
|:---------------------|:-------------------|:------------------------|:---------------|--------------:|-------------------------------:|---------------------------:|------------------:|----------------:|
| TGFBR1               | CRISPRko           | AGAGGGTACTACGTTGAAAGACT | TTTC           |             3 |                       99132702 |                     0.9922 |                52 |               2 |

assistant: 
Would you like to use CRISPRitz to predict sgRNA off-target effects?


user: yes

assistant: To identify potential off-target sites for your genome editing guides, we recommend you using CRISPRitz (Cancellieri et al.,Bioinformatics,2020), a rapid, high-throughput and variant-aware in silico off-target site identification package.

Please find the github page below:
https://github.com/pinellolab/CRISPRitz

*Please cite the corresponding papers if you decide to use the tool.

Our CRISPR-GPT can run CRISPRitz on our server for you, if you'd like CRISPR-GPT to help you run this step please type “Yes”, otherwise, type “No” if you prefer to run CRISPRitz locally, and we will give you a set of instructions. Would you like CRISPR-GPT to run off-target analysis for you? (Yes/No)


user: yes

assistant: Are you hoping to perform (1). Mismatches-Only search or (2). Mismatches + Bulges search?

user: 1

assistant: 
Please provide the species (human or mouse)
1. human
2. mouse


user: 1

assistant: 
Please provide the CAS system (SpCas9 or AsCas12a)
1. SpCas9
2. AsCas12a


user: 2

assistant: 
Please provide the sequence entering guide+PAM for SpCas9 (e.g. TCACCCAGGCTGGAATACAGNNN) or PAM+guide for AsCas12a (e.g. NNNNTCACCCAGGCTGGAATACAG).


user: NNNNAGAGGGTACTACGTTGAAAGACT

assistant: Running CRISPRItz mismatch_only with species: human, CAS: AsCas12a, sequence: NNNNAGAGGGTACTACGTTGAAAGACT. We will send you an email once the result files are ready to be downloaded.

assistant: Step 4. Collecting appropriate experimantal protocols and validating the editing outcomes.

Based on information from the previous step 2, we are showing you related protocols using Lentivirus/Retrovirus delivery


assistant: Now, we will help you through the detailed protocols of CRISPR experiments (Giuliano et al.,Curr Protoc Mol Biol., 2019).

1. Synthesize or clone the Cas and sgRNA system.
2. Deliver the product (synthesized or cloned the Cas and sgRNA system) into the desired cells.
3. Isolation of single cell clones.

*Please cite the corresponding papers if you decide to use the protocols.

Please type start so we can get you to the first step or enter the step numbers you are interested in. Type 'End' to finish.


user: 1

assistant: {'ChatResponse': {'Thoughts': "The user has entered '1', which corresponds to the first step in the process.", 'Choice': '1'}}

assistant: [BP1.docx](https://crispr-gpt.s3.amazonaws.com/BP1.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=qSqTc%2Fw3q4%2F%2BHfLEblR5NgRGEpA%3D&Expires=1725760627)



assistant: There are 3 support protocols associated with this step: 

- SP1: ISOLATION OF PLASMID DNA
- SP2: PREPARATION OF COMPETENT STBL3 E. COLI FOR gRNA CLONING
- SP3: OBTAINING GUIDE RNA OLIGONUCLEOTIDES
 
Do you want to access them? If yes, type in the protocal number; otherwise, type 'no' to return.


user: SP1

assistant: {'ChatResponse': {'Thoughts': "The user has chosen to access one of the support protocols. They have typed 'SP1', which indicates they want to access the first support protocol.", 'Choice': 'SP1'}}[SP1.docx](https://crispr-gpt.s3.amazonaws.com/SP1.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=PBtI%2FxgdiTqXwUnmge%2BR6wFDA8A%3D&Expires=1725760631)



assistant: There are 3 support protocols associated with this step: 

- SP1: ISOLATION OF PLASMID DNA
- SP2: PREPARATION OF COMPETENT STBL3 E. COLI FOR gRNA CLONING
- SP3: OBTAINING GUIDE RNA OLIGONUCLEOTIDES
 
Do you want to access them? If yes, type in the protocal number; otherwise, type 'no' to return.


user: SP2

assistant: {'ChatResponse': {'Thoughts': 'The user has chosen to access protocol SP2.', 'Choice': 'SP2'}}[SP2.docx](https://crispr-gpt.s3.amazonaws.com/SP2.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=3MJU8GkGquCVM%2BhRZPp2ALWpmAg%3D&Expires=1725760633)



assistant: There are 3 support protocols associated with this step: 

- SP1: ISOLATION OF PLASMID DNA
- SP2: PREPARATION OF COMPETENT STBL3 E. COLI FOR gRNA CLONING
- SP3: OBTAINING GUIDE RNA OLIGONUCLEOTIDES
 
Do you want to access them? If yes, type in the protocal number; otherwise, type 'no' to return.


user: SP3

assistant: {'ChatResponse': {'Thoughts': 'The user has indicated they want to access protocol SP3. It is important to confirm that this is a valid choice among the available support protocols.', 'Choice': 'SP3'}}[SP3.docx](https://crispr-gpt.s3.amazonaws.com/SP3.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=0BKsC5A0m%2Fna8M4gnxhlybObu0Y%3D&Expires=1725760635)



assistant: There are 3 support protocols associated with this step: 

- SP1: ISOLATION OF PLASMID DNA
- SP2: PREPARATION OF COMPETENT STBL3 E. COLI FOR gRNA CLONING
- SP3: OBTAINING GUIDE RNA OLIGONUCLEOTIDES
 
Do you want to access them? If yes, type in the protocal number; otherwise, type 'no' to return.


user: no

assistant: {'ChatResponse': {'Thoughts': 'The user has chosen not to access any of the support protocols (SP1, SP2, SP3).', 'Choice': 'no'}}

assistant: Now, we will help you through the detailed protocols of CRISPR experiments (Giuliano et al.,Curr Protoc Mol Biol., 2019).

1. Synthesize or clone the Cas and sgRNA system.
2. Deliver the product (synthesized or cloned the Cas and sgRNA system) into the desired cells.
3. Isolation of single cell clones.

*Please cite the corresponding papers if you decide to use the protocols.

Please type start so we can get you to the first step or enter the step numbers you are interested in. Type 'End' to finish.


user: 2

assistant: {'ChatResponse': {'Thoughts': 'The user has selected step 2, which involves delivering the synthesized or cloned Cas and sgRNA system into the desired cells.', 'Choice': '2'}}

assistant: [BP2.docx](https://crispr-gpt.s3.amazonaws.com/BP2.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=s00MkzpnCkwA%2FH5rMwCv42s5YgQ%3D&Expires=1725760665)



[BP4.docx](https://crispr-gpt.s3.amazonaws.com/BP4.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=yybAYjBLTLgJJ3yewPqh%2BG3l9F4%3D&Expires=1725760665)



assistant: Now, we will help you through the detailed protocols of CRISPR experiments (Giuliano et al.,Curr Protoc Mol Biol., 2019).

1. Synthesize or clone the Cas and sgRNA system.
2. Deliver the product (synthesized or cloned the Cas and sgRNA system) into the desired cells.
3. Isolation of single cell clones.

*Please cite the corresponding papers if you decide to use the protocols.

Please type start so we can get you to the first step or enter the step numbers you are interested in. Type 'End' to finish.


user: end

assistant: {'ChatResponse': {'Thoughts': 'The user has chosen to end the process, indicating they do not wish to proceed with any steps.', 'Choice': 'end'}}

assistant: In this protocol, you can validate the knockout through the following ways, including: 

1. Staining of target protein by antibody, and measure level of knockout using flow cytometry or western blot.
2. Mutation sequencing by Sanger Sequencing
3. Mutation sequencing by Next-generation Sequencing (NGS)

Which one would you like to use?


user: 3

assistant: [SP4.docx](https://crispr-gpt.s3.amazonaws.com/SP4.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=Hr0fMIgC%2BrH6A2Yh6tWPJClAdZI%3D&Expires=1725760675)

[BP8_NGS.docx](https://crispr-gpt.s3.amazonaws.com/BP8_NGS.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=dZWIhbbQwrr9%2BA63ANbe0m4KhtA%3D&Expires=1725760675)

 
 
 (add amplicon for NGS and add Crispresso website)

assistant: We could help you design the related PCR primers using Primer3 (Untergasser et al.,Nucleic Acids Res.,2012) through following options:

1. Primer3 - command-line script to design primers - we run the script for you.
2. Primer-blast (Web tool, Manual design one by one)
3. Primer3 - command-line script to design primers - you run the code yourself

*Please cite the corresponding papers if you decide to use the tool.

Which one do you prefer?



user: 1

assistant: {'ChatResponse': {'Thoughts': 'The user prefers to use Primer3 through a command-line script and wants us to run the script for them.', 'Choice': '1'}}

assistant: To help you design the PCR primers for mutation sequencing by NGS, we would need the following information:

- Species: For example: human, mouse.
- Sequence of your target site: Please include 200bp upstream and 200bp downstream of the gRNA.

Please type in the two answers in two lines sequentially. (Press Shift+Enter to type in a new line)


user: human
GTTTATTTCACTCGAGGCCCTTTTTCAGTAAAGTCATCACCTGGCCTTGGTCCTGTGGAACTGGCAGCTGTCATTGCTGGACCAGTGTGCTTCGTCTGCATCTCACTCATGTTGATGGTCTATATCTGCCACAACCGCACTGTCATTCACCATCGAGTGCCAAATGAAGAGGACCCTTCATTAGATCGCCCTTTTATTTCAGAGGGTACTACGTTGAAAGACTTAATTTATGATATGACAACGTCAGGTTCTGGCTCAGGTAACATAATTGTTTCTCCTTTTTCCTAAGACATCTTTTTAAAATTAAGCGATACTTATTTTATTAGTTTTCTAATAAGCCAACTTGCTAGAATGTGAGATAGTATAAATAATATTGCAGGTTTGAACCTAATAAAAATCT

assistant: status: success
result: [{'primer set 1': ['ACCGCACTGTCATTCACCAT', 'CTGAGCCAGAACCTGACGTT'], 'primer set 2': ['ATTGCTGGACCAGTGTGCTT', 'CTGAGCCAGAACCTGACGTT']}]
Please don’t forget to add corresponding illumina adaptors for the downstream library construction.

assistant: 
Next, we could help you analyze the NGS data using
CRISPResso2 (Clement et al., Nat Biotechnol.,2019).

1. CRISPResso2 - command-line script to analyze data - we run the script for you.
2. CRISPResso2 (Web tool)

*Please cite the corresponding papers if you decide to use the tool.
Which one do you prefer?


user: 1

assistant: {'ChatResponse': {'Thoughts': 'The user has chosen the command-line script option for analyzing the NGS data using CRISPResso2.', 'Choice': '1'}}

assistant: 
To help you analyze the NGS data, we would need the following information:

- Amplicon:


user: ACCGCACTGTCATTCACCATCGAGTGCCAAATGAAGAGGACCCTTCATTAGATCGCCCTTTTATTTCAGAGGGTACTACGTTGAAAGACTTAATTTATGATATGACAACGTCAGGTTCTGGCTCAG

assistant: 
To help you analyze the NGS data, we would needthe following information:

- sgRNA (only include spacer, do not include PAM):


user: AGAGGGTACTACGTTGAAAGACT

assistant: 
To help you analyze the NGS data, we would need the following information:

- **Fastq file R1**: Required (upload)
- Fastq file R2: Optional (upload)

Make sure to name the files appropriately (e.g. *.r1.fastq.gz and *.r2.fastq.gz)


user: 

assistant: Running CRISPResso with amplicon: ACCGCACTGTCATTCACCATCGAGTGCCAAATGAAGAGGACCCTTCATTAGATCGCCCTTTTATTTCAGAGGGTACTACGTTGAAAGACTTAATTTATGATATGACAACGTCAGGTTCTGGCTCAG, sgRNA: AGAGGGTACTACGTTGAAAGACT, CAS: enCas12a. We will send you an email once the result files are ready to be downloaded.

assistant: Finished. Clear the current chat or start a new one.