Induced pluripotent stem cells (iPSCs), have the capacity to give rise to differentiated progeny arising from of all germ layers of the body including: ectoderm, endoderm, and mesoderm. The ability to expand patient derived human iPSCs in vitro and subject them to cell-type specific differentiation protocols is the basis for generating “disease-in-a-dish” cellular models for basic stem cell research and drug-discovery applications. Recently, gene editing technologies such as CRISPR/CAS9 have allowed the generation of isogenic control human iPS cell lines to study the genetic mechanism behind disease and cellular functionality. Relying on our extensive experience in both gene editing and stem cell culture, we have developed a step-by-step protocol guide that can be followed to perform gene editing in human iPSCs using CRISPR/CAS9 technology.
Figure 1. Overview of CRISPR gene editing of human iPSCs. Gene editing of human iPSCs using CRISPR/CAS9 allows for the generation of isogenic disease controls for stem cell research applications.
It is important to start with high quality iPS cells prior to initiating any CRISPR/Cas9 gene editing experiment. Ensure that no less than 90% of the overall stem cell culture remains pluripotent and have not spontaneously differentiated. Follow our complete step-by-step human iPS cell culture protocol guide including ECM coating, thawing, culturing and freezing of iPSCs prior to starting a CRISPR experiment.
This protocol is based on using 1 x 106 cells per nucleofection. This protocol is for two samples. For more samples, adjust the following protocol as necessary. Where targeted integration is desired, donor molecules must be designed and included in the transfections as well, and will require specific detection assays to determine integration efficiency. These methods will be governed by overall cleavage efficiency of the nuclease, distance from the cut site to the desired mutation site, and local sequence composition. For best results, it is highly recommended that the end user optimizes donor designs based on these criteria.
For difficult to edit cell lines, we recommend the use of a 3-part synthetic CRISPR system with purified recombinant Cas9 protein and synthetic gRNA which is divided into a tracrRNA and a crRNA. The crRNA is variable and complementary to the target of interest, while the tracrRNA sequence is static.
Following transfection of the nuclease reagents, cells should be incubated for 24-72 hours before assessment of nuclease activity. The SURVEYOR nuclease assay is a widely accepted method to determine nuclease efficiency. Protocols for this method do not vary between ZFN and CRISPR nuclease formats, and a detailed method for this assay can be found in pages 5-8 of the CompoZr Custom Zinc Finger Nuclease Technical Bulletin.
It is highly recommended that clonal isolates be generated for genotypic screening in order to obtain pure populations of either knock-out cells or SNP converted cells. Limiting dilution and FACS are among the most common methods for cloning, and we recommend optimization of the cloning conditions for your desired cell line prior to attempting gene editing.
Figure 2. Alzheimer’s Human iPS Cell Lines. ApoE polymorphic alleles are the principal genetic determinants of Alzheimer disease (AD) risk. The EBiSC stem cell bank contains a complete set of isogenic lines, CRISPR engineered by Bioneer A/S, with the main ApoE genotypes: ApoE 2/2 (BIONi010-C-6), ApoE 3/3 (BIONi010-C-2) and ApoE 4/4 (BIONi010-C-1) as well as an ApoE knockout line (BIONi010-C-3) and TREM2 gene knockouts with homozygous R47H SNPs (BIONi010-C-7) or a homozygous T66M SNPs (BIONi010-C-8). A) iPS cells display normal undifferentiated phenotypes with colonies having clear defined borders and B) express the pluripotency marker Oct-4 (B).
Figure 3. Neural Differentiation of Human iPS Cell Lines. CRISPR engineered human iPS cells can be differentiated into neural phenotypes with Nestin (A, red) and b-Tubulin (A, green) expression or glial phenotypes with GFAP (B, green) expression.