Credit: Salk Institute
The gene-editing technology CRISPR can precisely and efficiently alter human stem cells, according to research published in Molecular Therapy.
Using JAK2 and other genes as models, researchers showed that CRISPR offers advantages over TALEN, another gene-editing technique, for manipulating induced pluripotent stem cells (iPSCs).
And, unlike in a previous study, CRISPR did not produce any off-target effects.
The team believes their findings could streamline and speed up efforts to modify human iPSCs for use as treatments or in the development of model systems to study diseases and test drugs.
“Stem cell technology is quickly advancing, and we think that the days when we can use iPSCs for human therapy aren’t that far away,” said study author Zhaohui Ye, PhD, of the Johns Hopkins University School of Medicine in Baltimore, Maryland.
“This is one of the first studies to detail the use of CRISPR in human iPSCs, showcasing its potential in these cells.”
CRISPR originated from a microbial immune system that contains DNA segments known as “clustered regularly interspaced short palindromic repeats.” The system makes use of an enzyme that nicks together DNA with a piece of small RNA that guides the tool to where researchers want to introduce cuts or other changes in the genome.
Previous research has shown that CRISPR can generate genomic changes or mutations through these interventions more efficiently than other gene-editing techniques, such as TALEN, which is short for “transcription activator-like effector nuclease.”
Despite CRISPR’s advantages, a recent study suggested it might also produce a large number of off-target effects in human cancer cell lines; specifically, modification of genes that researchers didn’t mean to change.
To see if this unwanted effect occurred in other human cell types, Dr Ye and his colleagues pitted CRISPR against TALEN in human iPSCs. The researchers compared the ability of both techniques to either cut out pieces of known genes in iPSCs or cut out a piece of these genes and replace it with another.
As model genes, the researchers used JAK2, a gene that, when mutated, causes myeloproliferative neoplasms; SERPINA1, a gene that, when mutated, causes alpha1-antitrypsin deficiency, an inherited disorder that may cause lung and liver disease; and AAVS1, a gene that’s been recently discovered to be a “safe harbor” in the human genome for inserting foreign genes.
The comparison showed that, when simply cutting out portions of genes, the CRISPR system was significantly more efficient than TALEN in all 3 gene systems, inducing up to 100 times more cuts.
However, when using these genome-editing tools for replacing portions of the genes, such as the disease-causing mutations in JAK2 and SERPINA1 genes, CRISPR and TALEN showed about the same efficiency in patient-derived iPSCs.
Contrary to results of the human cancer cell line study, both CRISPR and TALEN had the same targeting specificity in human iPSCs, hitting only the genes they were designed to affect.
The researchers also found that the CRISPR system has a second advantage over TALEN. It can be designed to target only the mutation-containing gene without affecting the healthy gene in patients where only one copy of a gene is affected.
These findings, according to the researchers, offer reassurance that CRISPR will be a useful tool for editing the genes of human iPSCs with little risk of off-target effects.
