Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

The overall goal of this procedure is to generate lineage specific human induced pluripotent stem cell or H-I-P-S-C knockin reporters using CRISPR Cas nine mediated homologous recombination. This is accomplished by first designing and constructing targeting vectors. The second step is to design and construct single guide RNAs for the CRISPR Cas nine system or the CAS nine N double Nick case system.

2 93 FT.Cells are then cot transfected with the single guide RNAs and CAS nine expression vector and the DNA is extracted and amplified for evaluation by a T seven ENDONUCLEASE one assay. The final step is to perform in silico prediction of potential off-target sites.Sites. Ultimately HI PSCs will be transfected with the targeting vector and CRISPR Cas nine system components to make neural lineage knockin reporter cell lines.

The main advantage of this technique over existing methods like the conventional homologous recombination. Instead, CRISPR cast nine mediated gene targeting has a much higher targeting efficiency. Generally individuals new to this method will struggle because it might take some practice to design single GA rna.

Visual demonstration of this method is critical as the steps for single guide RA design and in sical prediction of off target sites are difficult to learn because the CRISPR Cas nine concept was just recently introduced for gene editing and the new online resource tools are employed Upon determination of the lineage specific marker. Locate the genomic sequence of the gene and design the homology arms accordingly. The A LDH one L one genomic locus is used in this example.

The targeting construct is made by sub cloning GFP downstream of the genomic sequence right before the stop code on connected by self cleaving two A peptide sequences add a positive selection cassette such as a F fluxed neomycin cassette downstream of the reporter include a negative selection cassette, for example thymidine kinase outside of the three prime homology arm. Begin this procedure by determining the lineage specific marker that will be targeted to make the reporter A LDH one L one is used in this demonstration. Copy the CDNA sequence from the NCBI database.

Go to the human blatt search to obtain a genomic DNA alignment. Click the browser tab to view the assembly of the gene under the view tab. Click DNA to obtain the full genomic DNA sequence in fast a format.

Locate the stop code on within the DNA sequence where the two-way sequence and the reporter cassette will be tagged. Search for the proto spacer adjacent motif or Pam for example NGG within the vicinity of the stop code on scan both strands to locate the appropriate NGG sequence. Copy the sequence of the 20 basis immediately upstream of NGG and paste it into the web Designing tool Z fit design oligos with overhangs compatible with MLM 3 6 3 6.

The human GRNA expression vector with a U six promoter. After both forward and reverse oligos have been synthesized, proceed to make double stranded single guide RNA for a 50 microliter reaction. Add 22.5 microliters of forward and reverse oligos respectively and five microliters of Anne kneeling buffer denature both oligos at 70 degrees Celsius for 15 minutes and gradually cool down to room temperature.

Next mix and ligate the oligos with BSMB one digested MLM 3 6 3 6. For a 20 microliter reaction add one microliter of oligos 100 nanograms of MLM 3 6 3 6 and one microliter of T four ligase incubate at room temperature overnight on the following day, transform DH five alpha component cells with a ligation product by heat shocking at 42 degrees Celsius for 30 seconds. Grow the bacteria on an LB auger ampicillin plate at 37 degrees Celsius overnight.

On the following day, pick three to five colonies for plasmid preparation. Subsequently, the plasmid DNA is confirmed by commercial sequencing and the correct plasmid is prepared using a standard maxi prep protocol to begin this procedure, obtain a double nick case CAS nine ND 10 a vector Design a pair of forward and reverse oligos for each targeting locus following the same procedure demonstrated for the CRISPR Cas nine system. A LDH one L one will again be the example here.

Copy the sequence of 20 bases immediately upstream of NGG and paste it into the web. Designing tool Z fit to obtain oligos with over hank's compatible with the vector. Remove the first base and the last base of both forward and reverse oligos that are shown by the Z fit designing tool.

Synthesize the forward and reverse oligos and name them as follows. Lowercase FN uppercase F and lowercase FN uppercase R For PAMs on one side of the target site and lowercase R and uppercase F and lowercase R and uppercase R for PAMs on the other side of the target site of these N is the number to designate different PAMs lowercase f and r represent the location of the PAMs either at the sense or antisense strand and uppercase f and r represent forward and reverse oligos. After identifying the optimized pair of single guide RNAs to be used for CAS nine n by using the T seven E one assay, proceed to make double stranded single guide RNA for a 50 microliter reaction add 22.5 microliters of forward and reverse oligos respectively and five microliters of a kneeling buffer.

Denature both oligos at 70 degrees Celsius for 15 minutes and gradually cool down to room temperature. Mix and ligate the oligos with bbbs. One digested double nase CAS nine ND 10 a vector.

For a 20 microliter reaction add one microliter of single guide RNA 100 nanograms of bbbs, one digested vector and one microliter of T four ligase incubate at room temperature overnight transform DH five alpha component cells with a ligation product by heat shock at 42 degrees Celsius for 30 seconds. Grow the bacteria in lb agar ampicillin at 37 degrees Celsius overnight. On the following day, pick three to five colonies for plasmid preparation.

Subsequently, the plasmid DNA is confirmed by a commercial sequencing vendor and the correct plasmid is prepared using a standard maxi prep protocol to begin this assay. Co transfect 2 93 ft cells with the single guide RNA plasmid and CAS nine expression vector at 0.5 micrograms of single guide RNA vector 0.5 micrograms of CAS nine expression vector and 1.5 microliters of lipectomy 2000 to the cells. Incubate the cells at 37 degrees Celsius for 72 hours.

After 72 hours, harvest the cells by removing the culture medium from each well. Adding 100 microliters of DNA quick extraction buffer and trier rating vigorously transfer the cells to tubes. Incubate the mixture 68 degrees Celsius for 15 minutes, followed by 95 degrees Celsius for eight minutes.

This is the template for the subsequent PCR experiments. Set up the PCR to amplify the sequence flanking the proto spacer adjacent motif. For a 50 microliter reaction, combine one microliter of the template, 0.5 microliters of Hercules, two 0.5 microliters of DMSO 10 microliters of five x buffer, 0.5 microliters of A-D-N-T-P mix and one microliter of a forward and reverse primer mix.

Perform the PCR following the parameters described in the protocol when the PCR is complete and the DNA concentrations of the PCR products have been measured at 400 nanograms of PCR products to two microliters of digestion buffer two, ADD DD H2O to a final reaction. Volume of 19 microliters incubate in boiling water for five minutes. Let the reaction sit on the bench for 45 to 60 minutes to naturally cool it down to room temperature.

Next, add one microliter of T seven E one and incubate at 37 degrees Celsius for 15 minutes. Stop the reaction by adding two microliters of 0.25 molar EDTA mixed with six XDNA loading dye. Load the entire 20 microliter reaction to a 2.5%AROS gel.

Run the gel at 100 to 150 volts for 30 minutes. Inspect the gel under UV light to evaluate single guide RNA mediated cleavage. Subsequently create neuro lineage reporters by electro human induced pluripotent stem cells or HI PSCs with a linearized targeting vector CAS 9 0 0 3 expression vector and appropriate single guide RNA vector or vectors Potential off-target sites can be predicted using an online open source tool called CS ot, which is a pearl based program.

For a window system, download the CS OT program and the human genome database files to a local hard drive. Prepare the target file of the designed single guide RNA in the FASTA format. Open the file named cas ot.

A command window will appear. Leave it as is for now. Next, open the file named opt generator.

To generate an option string paste the path and name of the target file into the dialogue of the minus T or minus target option. Paste the path of the genome sequence file into the dialogue of the minus G or minus genome option. Copy the option string and paste it into the CAS OT command window.

Shown earlier at the completion of the cas OT program, run a folder will be generated. Open the CSV file within the folder with a spreadsheet program. This file contains the following information for every off-target site, chromosomal and genomic location.

Sequence of the site mismatched type, total number of mismatched bases and exon information relating to the off-target site before transecting. HI PSCs. The single guide RNAs are evaluated in 2 93 ft cells by the T seven E one assay.

In this representative result, the single guide RNAs are designed for targeting the human oli. Two gene lane's one through three are transfected with a CAS nine expression vector and lanes four through 11 are transfected with a CAS nine N nase expression vector. GFP is used as a control.

Lane two contains single guide RNA for oli. Two targeting using CAS nine lane five contains binding sense strand only. Lane six contains single guide RNA binding antisense strand only and lanes nine and 10 contain a pair of single guide RNAs for both DNA strands.

The undigested PCR product is about 614 base pairs In lane two. A portion of PCR products are digested into two bands of about 470 base pairs and 140 base pairs with an insertion and deletion percentage of 33%in lane's nine and 10. The two digested bands are about 410 base pairs and 200 base pairs with insertion and deletion percentages between three and 8%Once master the vector and the single guide only design and the evaluation can be done in about a week.

It is performed properly After its development. This technique paved the way for researchers in the field of stem cell biology with broad gene functions in development and disease in human systems. After watching this video, you should have a good understanding of how to design and construct single guide arrays for the CRISPR Cas nine and CAS nine N double N system.