Abstract
CRISPR/Cas9 systems have been used in a wide variety of biological studies; however, the large size of CRISPR/Cas9 presents challenges in packaging it within adeno-associated viruses (AAVs) for clinical applications. We identified a two-cassette system expressing pieces of the S. pyogenes Cas9 (SpCas9) protein which splice together in cellula to form a functional protein capable of site-specific DNA cleavage. With specific CRISPR guide strands, we demonstrated the efficacy of this system in cleaving the HBB and CCR5 genes in human HEK-293T cells as a single Cas9 and as a pair of Cas9 nickases. The trans-spliced SpCas9 (tsSpCas9) displayed ~35% of the nuclease activity compared with the wild-type SpCas9 (wtSpCas9) at standard transfection doses, but had substantially decreased activity at lower dosing levels. The greatly reduced open reading frame length of the tsSpCas9 relative to wtSpCas9 potentially allows for more complex and longer genetic elements to be packaged into an AAV vector including tissue-specific promoters, multiplexed guide RNA expression, and effector domain fusions to SpCas9. For unknown reasons, the tsSpCas9 system did not work in all cell types tested. The use of protein trans-splicing may help facilitate exciting new avenues of research and therapeutic applications through AAV-based delivery of CRISPR/Cas9 systems.
Highlights
Polyadenylation sequences has produced functional SpCas[9] constructs that can be effectively packaged in AAVs14,15, but such constraints on promoter selection will limit their application
After testing different inteins and split points within SpCas[9], we identified a system using the gyrase A (GyrA) intein from Mycobacterium xenopi[17] which robustly recapitulates the nuclease activity of the wild-type clustered regularly interspaced palindromic repeats (CRISPR)/Cas[9] system[2]
Our trans-splicing system consists of two cassettes, each less than 4 kb, which express two single guide RNAs and the two halves of SpCas[9] fused to the split portions of the Mxe GyrA intein (Fig. 1c)
Summary
Polyadenylation sequences has produced functional SpCas[9] constructs that can be effectively packaged in AAVs14,15, but such constraints on promoter selection will limit their application. Fusing effector domains to Cas[9] further increases the size of the protein thereby further increasing the degree of difficulty of packaging it in an AAV vector. Our trans-splicing system consists of two cassettes (created by modifying the pX330 plasmid2), each less than 4 kb, which express two single guide RNAs (sgRNAs) and the two halves of SpCas[9] fused to the split portions of the Mxe GyrA intein (Fig. 1c). Since the trans-splicing approach dramatically reduces the length of protein coding sequence each cassette is required to contain, there is ample room remaining to add sequences in future studies that allow expression of additional sgRNAs (which have been shown to promote highly synergistic effects5,6) and fusion of effector domains with dCas[9] (Supplemental Figure S1). Effector domains that are small enough to be used in our system include FokI (nuclease)[3,4], VP64 (gene upregulation)[5,6], KRAB (gene repression)[13], and SID4X (histone deacetylation)[7], all of which have been previously fused successfully to engineered DNA binding domains
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