CD34+ HSPCs give rise to all the types of blood cells, and the CD34+ marker differentiates these immature cells from fully-formed blood cells. Hematopoietic stem cell transplantation (HSCT) of CD34+ HSPCs is currently clinically used in treatment of many disorders, but presents many challenges such as limited numbers of appropriate human donors, rejection of the transplanted cells, graft-versus-host disease (GVHD), and side effects of immunosuppression. Therefore, gene editing in this primary cell type to improve clinical applications is an area of active research. CRISPR-Cas9 gene editing in particular holds much promise for such work, so Shapiro et al. undertook an optimization study using the IDT Alt-R CRISPR-Cas9 system for genome editing in these cells. The dual goals of the study were to optimize a CRISPR genome editing method for CD34+ HSPCs and to build a workflow by which off-target effects could be identified. The researchers studied the human recombination-activating genes RAG1 and RAG2, because mutations of these genes in HSPCs are known to cause severe combined immunodeficiency (SCID), a disease often treated with HSCT. Optimizing CRISPR genome editing for these specific genes might improve this treatment.
As a first step, Shapiro et al. wanted to determine the best guide RNA (gRNA) sequences to study. Using a plasmid expression system, they tested 5 target gRNA sequences each for the RAG1 and RAG2 genes in immortalized K562 cells and selected the sequence with the highest efficiency (on-target editing activity) for each of these genes for further experiments in primary CD34+ HSPCs. After selecting the sequences, the researchers wanted to determine the optimal format of these sequences to use. The selected gRNA sequences were therefore chemically synthesized in 3 formats: Alt-R 2-part gRNA, Alt-R 2-part XT gRNA, and Alt-R sgRNA. All of these formats consist of RNA with chemical modifications that improve genome editing. The sgRNA format is a single fusion of crRNA and tracrRNA. Both of the 2-part gRNA formats utilize separate crRNA and tracrRNA which must be allowed to anneal for a brief time before use. The XT 2-part format has more chemical modifications than the standard 2-part format. After complexing to Alt-R Cas9, these gRNAs were transfected into the HSPCs by electroporation with a Lonza 4D-Nucleofector™ at different concentrations. Shapiro et al. examined percentage of indel formation by next generation sequencing (NGS) with and without Alt-R Cas9 Electroporation Enhancer (an ssODN with no homology to human DNA) or excess gRNA. They also investigated genomic integration frequencies of the Alt-R Electroporation Enhancer itself. They went on to study the robustness of GUIDE-seq off-target identification in immortalized cells vs. HSPCs. They also tested how to quantify CRISPR off-target editing events in CD34+ HSPCs by rhAmpSeq targeted sequencing.
Shapiro et al. observed that equal levels of genome editing were obtained with all 3 gRNA formats when RAG2 was targeted. When RAG1 was targeted, standard Alt-R 2-part gRNA did not work as well as Alt-R 2-part XT gRNA and Alt-R sgRNA. Therefore, they concluded that potency of gRNA is target sequence dependent.
The researchers also determined that a low but detectable level of genomic integration of the Alt-R Electroporation Enhancer occurred in primary CD34+ HSPCs. This kind of integration would present a dangerous situation in clinical trials; unwanted DNA integration into a patient’s cells potentially could cause cancer or unknown diseases. Therefore, the researchers next tested the effect of using excess gRNA in place of Alt-R Electroporation Enhancer. They determined that excess sgRNA in a 1:2.5 Cas9:gRNA molar ratio compensated for the lack of Alt-R Electroporation Enhancer, thus eliminating the danger of integration of Alt-R Electroporation Enhancer in the cells’ genome. In their study of GUIDE-seq off-target identification, Shapiro et al. determined that in HEK293 cells stably expressing Cas9, a much wider range of off-target sites were nominated than in CD34+ HSPCs with transient RNP transfection. Finally, the rhAmpSeq system was used to quantify edits in the HEK293 cells and CD34+ HSPCs. Several off-target sites that were nominated only in the HEK293 cells by GUIDE-seq were determined by the rhAmpSeq method to have also undergone editing in the CD34+ HSPCs. This demonstrated that HEK293 cells stably expressing Cas9 had significant value as a source of data for nominating potential off-target sites in primary CD34+ HSPCs.
To decrease the level of off-target effects, Shapiro et al. then switched from wild-type Cas9 to Alt-R HiFi Cas9. For RAG2, the off-target effects dramatically decreased while the on-target editing was unaffected. For RAG1, the off-target effects similarly decreased, but on-target editing also decreased from 83% to 60%. The authors concluded that optimization should always be performed for each target site. Importantly, however, they also noted that the protein-coding regions of the RAG1 and RAG2 genes showed no off-target effects above 0.1%.
Shapiro et al. concluded that CRISPR-Cas9 genome editing in CD34+ HSPCs is very promising, and may in the future be used to cure a wide range of diseases. However, they pointed out that it will be necessary first to discover optimal methods for identifying and quantifying off-target effects as well as specific optimal methods for targeting precise genomic sites. They also observed that the combination of the Alt-R CRISPR-Cas9 system with HEK293 cells, GUIDE‑seq methodology, and the rhAmpSeq system made for a streamlined workflow in studying off-target effects in preclinical research.