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: 2

assistant: {'Thoughts': 'The user has selected Auto-Mode, indicating a preference for customized guidance based on their specific request.', 'Choice': '2'}

assistant: Please directly type in your need.

user: I'd like to activate human LTBR gene in primary T cells.

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: Please directly type in your need.

assistant: {'Thoughts': 'The user wants to activate the LTBR gene in primary T cells, which falls under CRISPR activation (CRISPRa). Therefore, the necessary steps involve designing guideRNAs specifically for CRISPRa and completing the experimental protocols.', 'Tasks': ['act_rep.StateStep3', 'act_rep.StateStep4']}

assistant: 
Step 3. Designing guideRNA for gene activation or repression

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: 
In order to complete the current task, we would need you to answer the following questions. 

If you are not sure how to select, we recommend you use the Meta-mode where we could generate case-specific guidance based on your needs, please open a new chat and enter meta-mode (select option 1 for the initial prompt).

1. Which Cas system are you planning to use? (We currently support SpCas9, enCas12a, SaCas9, SpRYCas9)
2. Are you hoping to activate or repress the target gene expression?


assistant: Here is the GPT-suggested answer: 

1. SpCas9
2. Activate


 Reason: 
The user wants to activate the human LTBR gene in primary T cells. For this task, we'll need to choose the appropriate Cas system and specify that we are aiming to activate the gene expression.

assistant: Press Enter to continue with the generated answer. Enter other text to correct answer

user: 

assistant: {'Thoughts': 'The user has specified that they are planning to use SpCas9 and they aim to activate the target gene expression. SpCas9 is a widely used Cas protein known for its efficiency in gene editing. To activate gene expression, we typically use a CRISPR activation (CRISPRa) system, which involves fusing transcriptional activators to a deactivated Cas9 (dCas9) that binds to the target DNA sequence without cutting it.', 'Answer': 'SpCas9', 'Choice': 'activate'}

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; I hope to design a guide RNA for CRISPRa that locates within 108146000 to 108146262.)

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


assistant: Here is the GPT-suggested answer: 

I hope to design 2 sgRNAs targeting the promoter region of the human LTBR gene for CRISPRa.


 Reason: 
The user intends to activate the human LTBR gene in primary T cells using the SpCas9 protein for a CRISPR activation (CRISPRa) system. To accomplish this, we need to design guide RNAs (sgRNAs) that can target the promoter region of the LTBR gene without cutting the DNA, enabling the recruitment of transcriptional activators to enhance gene expression.

assistant: Press Enter to continue with the generated answer. Enter other text to correct answer

user: 

assistant: https://crispr-gpt.s3.amazonaws.com/d818c332-298e-4d6a-9e62-54492acc3f25?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=mEO2Sxy%2B6D02rguA4m5pBovB2%2FM%3D&Expires=1725806427

| Target Gene Symbol   | CRISPR Mechanism   | sgRNA Sequence       | PAM Sequence   | Target Domain   |   TSS Position |   sgRNA 'Cut' Position |   On-Target Efficacy Score |   Off-Target Rank |   Combined Rank |
|:---------------------|:-------------------|:---------------------|:---------------|:----------------|---------------:|-----------------------:|---------------------------:|------------------:|----------------:|
| LTBR                 | CRISPRa            | GGGAGTTCCAGAGAGCGCAA | GGG            | [TSS-300,TSS+0] |        6384169 |                6383870 |                     1.08   |                11 |               1 |
| LTBR                 | CRISPRa            | CCTGTCGCAATCGCAGCCCA | GGG            | [TSS-300,TSS+0] |        6384169 |                6384094 |                     0.9069 |                 8 |               2 |

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


assistant: Here is the GPT-suggested answer: 

I don't know


 Reason: 
Input/Output may contain sensitive sequences. This is not supported in Automation mode! Please manually enter your response!

assistant: Press Enter to continue with the generated answer. Enter other text to correct answer

user: 

assistant: Pick Delivery Method

assistant: 
In order to complete the current task, we would need you to select one delivery method you are planning to use from the following options. 

If you are not sure how to select, we recommend you use the Meta-mode where we could generate case-specific guidance based on your needs, please open a new chat and enter meta-mode (select option 1 for the initial prompt).
 
a. Plasmid transfection
b. Lentivirus/Retrovirus delivery
c. RNP/mRNA Electroporation
d. RNP/mRNA Microinjection
e. mRNA LNP
f. AAV (Adeno-Associated Virus)


assistant: Here is the GPT-suggested answer: 

c. RNP/mRNA Electroporation


 Reason: 
To activate the human LTBR gene in primary T cells, choosing a suitable delivery method is crucial. Each option has its own advantages and limitations depending on efficiency, safety, and ease of use. Given the primary T cells context, RNP/mRNA Electroporation is often preferred due to its high efficiency and lower risk of integration-related issues.

assistant: Press Enter to continue with the generated answer. Enter other text to correct answer

user: b

assistant: {'Thoughts': 'The user has chosen option b for delivery method. Since the user has already made a selection, I will confirm this choice and provide the relevant information.', 'Choice': 'select', 'Answer': 'b'}

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.


assistant: Here is the GPT-suggested answer: 

start


 Reason: 
The user has chosen option b for the delivery method and is ready to proceed with the detailed protocols of CRISPR experiments as per Giuliano et al., Curr Protoc Mol Biol., 2019.

assistant: Press Enter to continue with the generated answer. Enter other text to correct answer

user: 

assistant: {'ChatResponse': {'Thoughts': "The user wants to initiate the process of testing and validating the sgRNA using the steps by Giuliano et al. They have entered 'start', which corresponds to the first step in the process.", 'Choice': '1'}}

assistant: Please find the protocols below:
[BP1.docx](https://crispr-gpt.s3.amazonaws.com/BP1.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=kjK4LMo6UQwQCdRT7VEEmQdCZN8%3D&Expires=1725806464)



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.


assistant: Here is the GPT-suggested answer: 

SP3


 Reason: 
The user is at the first step in the process to test and validate the sgRNA for the activation of the human LTBR gene in primary T cells. They have the option to access support protocols associated with this step.

assistant: Press Enter to continue with the generated answer. Enter other text to correct answer

user: 

assistant: Please find the protocols below:
[SP3.docx](https://crispr-gpt.s3.amazonaws.com/SP3.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=sTT3Ij3P473dqYhfebIPEeyx7Pk%3D&Expires=1725806471)



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.


assistant: Here is the GPT-suggested answer: 

SP3


 Reason: 
The user is asked if they want to access any of the support protocols related to obtaining guide RNA oligonucleotides. The user should be directed to the specific protocol number if they want to proceed.

assistant: Press Enter to continue with the generated answer. Enter other text to correct answer

user: SP3

assistant: Please find the protocols below:
[SP3.docx](https://crispr-gpt.s3.amazonaws.com/SP3.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=9CbciG4uOy8doXhWVeCjy8hpUJQ%3D&Expires=1725806478)



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.


assistant: Here is the GPT-suggested answer: 

SP3


 Reason: 
The user inquiry is about whether they want to access any of the listed support protocols. The list includes SP1, SP2, and SP3. Given the user's meta request, it seems relevant to access the protocol for obtaining guide RNA oligonucleotides (SP3).

assistant: Press Enter to continue with the generated answer. Enter other text to correct answer

user: 

assistant: Please find the protocols below:
[SP3.docx](https://crispr-gpt.s3.amazonaws.com/SP3.docx?AWSAccessKeyId=AKIA6ODU3LTXQWKWTRED&Signature=TyiVPH8zJpm3tB5Oy%2BtUSP6aPCU%3D&Expires=1725806484)



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.


assistant: Here is the GPT-suggested answer: 

SP3


 Reason: 
The user is inquiring about accessing specific protocols related to the CRISPR design tool for activating the LTBR gene in primary T cells. The user has provided a link to SP3 and has been asked if they want to access one of the three protocols.

assistant: Press Enter to continue with the generated answer. Enter other text to correct answer