CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors

Abstract

Summary The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of ~4.5 kilobases limits the donor size. Here we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing. We use the methodology in primary human T cells and CD34+ hematopoietic stem and progenitor cells to site-specifically integrate an expression cassette that as a single donor vector would otherwise amount to a total of 6.5 kilobases. This approach now provides an efficient way to integrate large transgene cassettes into the genomes of primary human cells using HR-mediated genome editing with AAV vectors.

CRISPR/Cas9 genome editing in human hematopoietic stem cells

“…Although current literature may suggest that rAAV6 is a more efficient donor than IDLV 21 , a thorough comparison has yet to be performed. In comparison with ssODNs, which are usually ~50–200 bp in total size and can therefore mediate only small genomic changes, rAAV6 donor templates can mediate precise SNPs, as well as insert transgene cassettes up to ~4 kb in size if a single donor vector is used, or can insert even larger transgene cassettes if a sequential HR strategy with two donor vectors is used 32 . If single-point mutations are the desired genomic change, ssODNs may be a useful donor template for use in HSPCs, as they are easily produced and have been shown to work well in vitro 10 .…”

The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

“…Studies have found that the site‐specific DNA cleavage could significantly increase the efficiency of HDR‐based knock‐in at the nearby region by up to ~1000‐folds, which widely prompts the application of CRISPR‐HDR‐based methods to introduce various genomic modifications through sequence replacement. The homologous templates have been provided in various forms, ranging from a small single‐stranded oligonucleotide (ssODN) with a size of around 90‐120 nt, large circular (plasmids) or linear dsDNA, to ssDNA delivered via AAV vectors . Through CRISPR‐induced HDR repair, gene function analysis and disease modelling/correction have become feasible via introduction and correction of specific point mutations, or targeted insertions of desired sequences ranging from a few nucleotides to large DNA fragments up to 7.5 kb .…”

Therapeutic gene editing in haematological disorders with CRISPR/Cas9

“…AAV vectors are generally considered the safer choice because the parental wild type virus is not known to cause disease, in direct contrast to human immunodeficiency virus‐derived lentiviral vectors. On the other hand, AAV suffers from the 4700 nt packaging capacity, which is limiting for integration of large gene segments, although this has been circumvented by the use of a split donor system, in which a large gene cassettes is split onto two separate AAV6 donors designed to undergo sequential HR to fuse the two gene parts seamlessly (Bak & Porteus, ). This does however come with the cost of decreased efficiencies.…”