Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration

Abstract

Inserting custom designed DNA sequences into the mammalian genome plays an essential role in synthetic biology. In particular, the ability to introduce foreign DNA in a site-specific manner offers numerous advantages over random DNA integration. In this review, we focus on two mechanistically distinct systems that have been widely adopted for targeted DNA insertion in mammalian cells, the CRISPR/Cas9 system and site-specific recombinases. The CRISPR/Cas9 system has revolutionized the genome engineering field thanks to its high programmability and ease of use. However, due to its dependence on linearized DNA donor and endogenous cellular pathways to repair the induced double-strand break, CRISPR/Cas9-mediated DNA insertion still faces limitations such as small insert size, and undesired editing outcomes via error-prone repair pathways. In contrast, site-specific recombinases, in particular the Serine integrases, demonstrate large-cargo capability and no dependence on cellular repair pathways for DNA integration. Here we first describe recent advances in improving the overall efficacy of CRISPR/Cas9-based methods for DNA insertion. Moreover, we highlight the advantages of site-specific recombinases over CRISPR/Cas9 in the context of targeted DNA integration, with a special focus on the recent development of programmable recombinases. We conclude by discussing the importance of protein engineering to further expand the current toolkit for targeted DNA insertion in mammalian cells.

Structural basis for topological regulation of Tn3 resolvase

“…While serine recombinases lack the flexibility in target sequences of CRISPR-Cas systems, they do not create R-loops or leave double-strand breaks for the host to repair. Furthermore, the modular nature of small serine recombinases facilitates their engineering to alter target specificity: for example, constitutively active mutants of their catalytic domains can be fused to other DNA binding domains such as zinc fingers and even Cas9 (Mercer et al, 2012; Proudfoot et al, 2011; Zhang et al, 2021). However, because those chimeras are constitutively active, they will synapse any pair of sites regardless of topological context, and will catalyze (usually undesirable) further rounds of recombination between the product sites.…”

Hematopoietic Stem Cell Gene-Addition/Editing Therapy in Sickle Cell Disease

“…CRISPR editing consists of an endonuclease (often Cas9) and a single guide RNA (sgRNA). The sgRNA includes a ∼20 nucleotide target sequence and an ∼80 nucleotide RNA scaffold [ 79 ]. To edit with CRISPR, there must be a protospacer-adjacent motif (PAM) downstream of the target sequence.…”

“…To edit with CRISPR, there must be a protospacer-adjacent motif (PAM) downstream of the target sequence. For Cas9, this is usually a 2–4 nucleotide guanine-rich region [ 79 ]. Other Cas endonucleases can be used as well, such as Cas12, which has a thymine-rich PAM region, and Cas13, which is an RNase as opposed to a DNase.…”

STRAIGHT-IN: A platform for high-throughput targeting of large DNA payloads into human pluripotent stem cells

“…For various research and clinical applications, there is an increasing need to precisely integrate large DNA fragments (Zhang et al, 2021). STRAIGHT-IN could potentially simplify the generation of cell lines or animal models containing these large and complex genetic circuits.…”

“…CRISPR-Cas9) have made it significantly easier to perform small-scale genomic modifications in hPSCs. However, strategies to insert multi-kilobase (kb) payloads that consist of several transgenes or a genomic fragment, for example, are limited since the efficiency of homology directed repair-mediated targeting decreases significantly with increasing insert size (Byrne et al, 2015;Zhang et al, 2021).…”