G. Grant Welstead

Delivery and Specificity of CRISPR-Cas9 Genome Editing Technologies for Human Gene Therapy.

Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) technology is revolutionizing the study of gene function and likely will give rise to an entire new class of therapeutics for a wide range of diseases. Achieving this goal requires not only characterization of the technology for efficacy and specificity but also optimization of its delivery to the target cells for each disease indication. In this review we survey the various methods by which the CRISPR-Cas9 components have been delivered to cells and highlight some of the more clinically relevant approaches. Additionally, we discuss the methods available for assessing the specificity of Cas9 editing; an important safety consideration for development of the technology.

572. Successful Generation of CAR+PD-1-Primary T Cells Using Cas9-Mediated Genome Editing

Cancer immunotherapy is an exciting area of cancer treatment bolstered by recent success of anti-immune checkpoint antibodies in the clinic. Engineered T cells that are programmed to attack tumors via chimeric antigen receptors (CARs) have also shown promise in early clinical trials. It is speculated, however, that the PD-1/PD-L1 axis dampens the effectiveness of CAR T therapy in certain cancer types. In order to improve the function of transplanted CAR T cells in these contexts, we have deleted PDCD1 using the genome editing system, CRISPR/Cas9. Delivery of the Cas9 protein and a PDCD1 specific gRNA to primary T cells resulted in the deletion of PD1 expression in over 90% of cells. Additionally, we were able to achieve similar editing efficiencies in the context of a lentivirus CAR infection and successfully generated CAR+/PD-1 knockout primary T cells with a very high level (>60%) of duel CAR expression and PD-1 editing. Utilizing a variety of assays, we have determined that the CAR+/PD-1− primary T cells are viable, proliferate normally and can kill target expressing cells both in the presence and absence of PD-L1 on the target cells. In conclusion, we have successfully generated CAR+/PD-1− primary T cells using Cas9-mediated editing that can be directed to kill PD-L1 expressing target cells.

124. Cas9-Mediated Genome Editing in Hematopoietic Stem/Progenitor Cells

Transplantation of genetically modified autologous hematopoietic stem/progenitor cells (HSPCs) has proven to be an effective clinical treatment for patients with hematologic disease. Genome editing with the CRISPR/Cas9 platform has been shown to precisely alter endogenous gene targets in multiple human cell lines and animal models. Here, we compared Cas9-induced gene modification in primary human HSPCs, including mobilized peripheral blood and steady state bone marrow CD34+ cells. Co-delivery of Streptococcus pyogenes or Staphylococcus aureus Cas9 paired with locus-specific guide RNAs induced targeted gene editing in CD34+ cells and hematopoietic progeny, as determined by T7E1 assay and DNA sequence analysis. Multiple gene targets and delivery strategies were evaluated in these primary human CD34+ cells. Importantly, hematopoietic progeny differentiated from Cas9 gene-edited HSPCs maintained ex vivo hematopoietic colony forming potential. This study provides further evidence of Cas9-mediated genome editing in clinically relevant primary cell populations.

561. Staphyloccocus aureus Cas9: An Alternative Cas9 for Genome Editing Applications

The CRISPR/Cas9 system that is broadly used for genome editing in myriad model organisms and cell lines is derived from the bacterial species Streptoccocus pyogenes. Here we report the use and characterization of another Type II CRISPR endonuclease Cas9, from the bacterium Staphylococcus aureus. This Cas9, encoded by ≈3.2kb of DNA, is substantially smaller than the S. pyogenes Cas9 yet delivers comparable DNA cleavage rates as measured by NHEJ-induced inserts and deletions in HEK293T cells. The S. aureus Cas9 has a Protospacer Adjacent Motif (PAM) that is well defined and different from the S. pyogenes Cas9 PAM, thus extending the range of specific target sites for any locus of interest. We characterize single guide RNAs specific to the S. aureus Cas9 with a range in the length of targeting sequences, investigating differences in on-target DNA cleavage efficiency as well as off-target activity. Off target activity assays include detection of cleavage events at chromosomally-integrated target sequences and at endogenous loci genome-wide. Taken together, our results show that the S. aureus Cas9 is an effective tool for genome editing with properties similar to but distinct from the more widely used S. pyogenes Cas9.

166. Biophysical Characterization and Direct Delivery of S. Pyogenes Cas9 Ribonucleoprotein Complexes

Several groups have demonstrated efficient genome editing in various mammalian cells by cationic lipid mediated delivery of purified Cas9 protein complexed with in-vitro translated or chemically synthesized guide-RNA (gRNA). Such “direct delivery” of the Cas9 ribonucleoprotein (RNP) complex allows for efficient gene-editing while minimizing off-target activity owing to the rapid turnover of the Cas9 protein in cells. Efficiency of gene-editing mediated by RNP delivery varies by locus, depends on the length of guide-RNA and on the amount and ratio of Cas9 protein and gRNA delivered.The Cas9 complex with gRNA has been well characterized structurally and biophysically revealing a large contact area and a high affinity. Thermal melt curves are a useful property to detect the binding and stability of complexes. We have used the large increase in the melting temperature from apo-Cas9 to the Cas9 complexed with gRNA to characterize the affinity of Cas9 for gRNA. Multiple gRNAs with differing lengths were complexed with Cas9 at differing stoichiometries and the tightness of the interaction was measured using thermal shift. These biophysically characterized complexes were then transfected into 293T cells and the efficiency of indel generated was measured. We have found that subtle differences in the gRNA length and base composition affect the binding and formation of RNP complex. Correlating binding affinity with efficiency of genome editing informs the design of an optimal composition of RNPs for cationic lipid mediated direct delivery.

348. Characterization of Cas9-Mediated Genome Editing in Human T Cells

Genome editing via CRISPR/Cas9 promises to provide a novel class of therapies for a variety of human diseases. To unlock the potential of the CRISPR/Cas9 technology, a deeper understanding of its efficacy in different primary cell types is required. A cell type of particular interest for gene editing is the human T cell due to its central role in the evolving cancer immunotherapy field. To better understand the utility of Cas9-mediated gene editing for engineering human T cells, we surveyed a variety of delivery modalities including electroporation of RNA and administration of ribonucleic acid-protein complexes. Additionally, we assessed the functionality of different Cas9 variants in human T cells. Here we report our findings including genome editing in human T cells using CRISPR/Cas technology.