Genome editing technologies have enabled the precise manipulation of DNA sequences at targeted sites to achieve therapeutic effects. Engineered endonucleases like CRISPR-Cas9 institute site-specific DNA breaks to either knock out a gene or enhance the homology directed repair (HDR) based gene correction with an intact donor template. High-fidelity editing, multiplexing capacity, and the ease of implementing the CRISPR-Cas9 system have expanded the scope of programmable genetic manipulations to include simultaneous deletions or insertions of multiple DNA sequences in a single round of mutagenesis. Gene editing has many applications in basic and biomedical research, including disease modeling. A promising cell therapy approach for hemoglobinopathies or cancer immunotherapy involves the ex vivo gene editing of autologous hematopoietic stem cells (HSCs) or T cells before administering patients. For hemoglobinopathies, gene editing tools like CRISPR-Cas9 can be used to install a double stranded break to either knock out a gene like BCL11A or enhance the knock-in with an intact donor DNA template to correct the mutation in the beta subunit of the hemoglobin gene (HBB). Cancer immunotherapy applications use CRISPR-Cas9 to knock out the TCR locus or checkpoint modulators to improve the efficacy and clinical outcomes of the living drug. Here, we describe the use of a clinically validated, regulatory compliant, scalable electroporation platform for the high efficiency, low toxicity gene editing of hCD34+ hematopoietic stem cells (HSCs), T cells, and iPSC cells at a clinical scale for preclinical evaluation and commercial production of cell therapy products for Sickle Cell Disease, TCR therapy or in disease modeling.

Learning Objectives:

1. llustrate therapeutic level of gene correction of the SCD mutation in patient derived CD34+ HSCs cells by electroporation of CRISPR-Cas9 gene editing tools. The gene edited cells with homology directed repair (HDR) engrafted and were detectable for a significant period of time in mouse xenograft models.

2. Paraphrase the optimization of the RNP mediated genome editing using CRISPR-Cas9 and a ssODN template to engineer iPSCs for treatment of diseases like DMD and Miyoshi Myopathy.

3. Describe a clinical trial where patients were infused with an autologous CRISPR-Cas9 genetically edited CD34+ HSC commercial product aimed knocking out the BCL11A enhancer, thereby reactivating the production of fetal hemoglobin to alleviate the debilitating symptoms of SCD and beta thalassemia.

Learning Objectives:

1. Summarize delivery vehicles for the Cas9 RNP

2. Identify Gene editing after a direct tissue injection

3. Indicate new linkers for conjugating proteins to polymers

Conventional CRISPR–Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a remarkable inversion of this paradigm, in which bacterial transposons have coopted nuclease-deficient CRISPR–Cas systems to catalyze RNA-guided integration of mobile genetic elements into the genome. Programmable transposition requires CRISPR- and transposon-associated molecular machineries, including a novel co-complex between Cascade and the transposition protein TniQ. Donor DNA integration occurs at a fixed distance downstream of target DNA sequences, accommodates variable length genetic payloads, and functions robustly in diverse bacterial species. Deep sequencing experiments reveal highly specific, genome-wide DNA integration across dozens of unique target sites. The discovery of a fully programmable, RNA-guided transposase lays the foundation for kilobase-scale genome engineering that obviates the requirements for DNA double-strand breaks and homologous recombination.

Learning Objectives:

1. Recall CRISPR-Cas systems are naturally repurposes for non-canonical functions other than cleaving RNA and DNA

2. Indicate CRISPR RNA-guided transposases integrate donor DNA without generating DNA double-strand breaks

3. Explain RNA-guided DNA integration proceeds with high-fidelity and readily accommodates kilobase-size genetic payloads

Genome Engineering allows the easy manipulation of genomes down to the nucleotide level. CRISPR is a revolutionizing tool for generating custom edited cell and pre-clinical animal models for research. Targeted deep sequencing enables the detection and quantification of low-frequency editing events. However, the large amounts of data generated by targeted deep sequencing can be difficult to interpret and quickly analyze. We have developed a Python-based computer program called CRIS.py that allows the easy analysis of multiple types of editing events. We will show examples of how rapid deep sequence analysis has guided experimental design leading to high-efficiency genome editing in a broad range of applications. Additionally, we will discuss best practices for creating knockin cell and animal models including large insertions using long ssDNA.

Learning Objectives:

1. Explain how NGS analysis enables detection and quantification of CRISPR editing outcomes on a diverse range of sample inputs including cell pools, clonal populations, and animal tissues

2. Recognize CRIS.py is a user friendly NGS analysis pipeline that is open source

3. Discuss best practices for creating knock in cell lines 4. Identify that NGS can be used to genotype both small and large insertion events

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. In collaboration with Integrated DNA Technologies, we showed that this engineered variant we refer to as AsCas12a Ultra, increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We showed that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

Learning Objectives:

1. Identify the engineered AsCas12a Ultra nuclease is commercially available to researchers from Integrated DNA Technologies

2. Recall this engineered AsCas12a nuclease forms the basis for EDIT-301, an experimental treatment for sickle cell disease which is currently being tested in the clinic

3. Recall this engineered AsCas12a nuclease breaks the observed paradigm where most natural and engineered CRISPR nuclease variants that achieve very high specificity come with a cost to on-target editing activity in most clinically relevant cell types

Learning Objectives:

1. Recall engineered CRISPR-Cas variants overcome natural limitations of wild-type enzymes

2. Know the genome is now nearly completely editable

3. Recognize minimal PAM CRISPR-Cas enzymes enable previously impossible edits