Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Abstract

The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9). We define functional protospacer adjacent motif (PAM) sequences and variables required for robust and efficient editing in vitro. Expression of holoSt1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver rescued lethality and metabolic defects in a mouse model of hereditary tyrosinemia type I demonstrating effective cleavage activity in vivo. Furthermore, we identified potent anti-CRISPR proteins to regulate the activity of both St1Cas9 and the related type II-A Staphylococcus aureus Cas9 (SaCas9). This work expands the targeting range and versatility of CRISPR-associated enzymes and should encourage studies to determine its structure, genome-wide specificity profile and sgRNA design rules. INTRODUCTIONClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins form a prokaryotic adaptive immune system and some of its components have been harnessed for robust genome editing 1 . Type II-based editing tools rely on a large multidomain endonuclease, Cas9, guided to its DNA target by an engineered single-guide RNA (sgRNA) chimera 2 (See 3, 4 for a classification of CRISPR-Cas systems). The Cas9-sgRNA binary complex finds its target through recognition of a short sequence called the protospacer adjacent motif (PAM) and subsequent base pairing of the guide RNA with the DNA to generate a specific doublestrand break (DSB) 1,5 . While Streptococcus pyogenes (SpCas9) remains the most widely used Cas9 variant for genome engineering, the diversity of naturally occurring RNA-guided nucleases is astonishing 4 . Hence, Cas9 enzymes from different microbial species can contribute to the expansion of the CRISPR toolset by increasing targeting density, improving activity and specificity as well as easing delivery 1,6 .In principle, engineering complementary CRISPRCas systems from distinct bacterial species should be relatively straightforward, as they have been minimized to only two components. However, many such enzymes were found inactive in human cells despite being accurately reprogrammed for DNA binding and cleavage in vitro [7][8][9][10] . Nevertheless, the full potential of selected enzymes can be unleashed using machine learning to establish sgRNA design rules 11,12 .Perhaps the most striking example of the value of alternative Cas9 enzymes is the implementation of the type II-A Cas9 from Staphylococcus aureus (SaCas9) for in vivo editing using recombinant adeno-associated virus (rAAV) vectors 7,13, 14 . More recently, Campylobacter jejuni and Neisseria meningitidis Cas9s from the type II-C 15 CRISPRCas systems have been added to this repertoire 16,17 .In vivo genome edi...

Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6

“…To further explore the functional impact of such decreased DNA-binding affinity in vivo, we turned to a base editing system that provides a sensitive readout of DNA binding. We adapted the architecture of a previously described DNA base editor (Koblan et al, 2018) and created a fusion between catalytically impaired St1Cas9 (St1Cas9-D9A) and a cytidine deaminase in order to convert C$G base pairs to T$A in human cells, referred to as St1BE4max (Agudelo et al, 2019). While St1BE4max efficiently converted target cytosines into thymines at two distinct loci, co-expression of AcrIIA6 resulted in complete inhibition of base editing (Figures 4C and S5A).…”

State‐of‐the‐art 2023 on gene therapy for phenylketonuria

“…For instance, genetically modified fumarylacetoacetate hydrolase (FAH) positive hepatocytes enjoy a tremendous selective advantage in animal models of human tyrosinemia type 1 and will almost completely repopulate the liver of Fah À/À animals. [96][97][98][99][100] A similar selective advantage exists for hepatocytes expressing methylmalonyl-CoA mutase in murine methylmalonic acidemia. 101 No such selective mechanism exists for PAH-expressing hepatocytes in PAH-deficient mice, 102 so methods that might provide a selective advantage to genetically modified hepatocytes regardless of target disease or therapeutic transgene have been avidly sought.…”

Dead Cas Systems: Types, Principles, and Applications

“…To reduce or even avoid potential off-target activity, a plethora of technical refinements has been made, including (a) advanced design of gRNAs using in situ CRISPR-Cas design tools (CRISPR design, E-Crisp, CROP-IT, Cas-OFFinder) [178], (b) modifications of gRNAs (truncations [179], introduction of secondary structures [180] etc. ), (c) rationally engineered SpCas9 variants (eSpCas9 [181], Sp-HF1 [182], evoCas9 [183], Hypa-Cas9 [184]) with limited non-specific cleavage and off-target activity, (d) Cas proteins with altered PAM-specificity [185], (e) orthologous CRISPR-Cas systems [77,186,187], (f) engineered dCas proteins fused with FokI nuclease [188], and (g) delivery of CRISPR-Cas components as short-lived ribonucleoprotein complexes [189]. These technical achievements have minimized, but still not completely erased, potential off-target activity.…”