Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 phox -deficient chronic granulomatous disease.

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets.

Streptococcus pyogenes Cas9 (SpCas9) has been employed as a genome engineering tool with a promising potential within therapeutics. However, its off-target effects present major safety concerns for applications requiring high specificity. Approaches developed to date to mitigate this effect, including any of the increased-fidelity (i.e., high-fidelity) SpCas9 variants, only provide efficient editing on a relatively small fraction of targets without detectable off-targets. Upon addressing this problem, we reveal a rather unexpected cleavability ranking of target sequences, and a cleavage rule that governs the on-target and off-target cleavage of increased-fidelity SpCas9 variants but not that of SpCas9-NG or xCas9. According to this rule, for each target, an optimal variant with matching fidelity must be identified for efficient cleavage without detectable off-target effects. Based on this insight, we develop here an extended set of variants, the CRISPRecise set, with increased fidelity spanning across a wide range, with differences in fidelity small enough to comprise an optimal variant for each target, regardless of its cleavability ranking. We demonstrate efficient editing with maximum specificity even on those targets that have not been possible in previous studies.

Position-dependent sequence motif preferences of SpCas9 are largely determined by scaffold-complementary spacer motifs.

Streptococcus pyogenes Cas9 (SpCas9) nuclease exhibits considerable position-dependent sequence preferences. The reason behind these preferences is not well understood and is difficult to rationalise, since the protein establishes interactions with the target-spacer duplex in a sequence-independent manner. We revealed here that intramolecular interactions within the single guide RNA (sgRNA), between the spacer and the scaffold, cause most of these preferences. By using in cellulo and in vitro SpCas9 activity assays with systematically designed spacer and scaffold sequences and by analysing activity data from a large SpCas9 sequence library, we show that some long (>8 nucleotides) spacer motifs, that are complementary to the RAR unit of the scaffold, interfere with sgRNA loading, and that some motifs of more than 4 nucleotides, that are complementary to the SL1 unit, inhibit DNA binding and cleavage. Furthermore, we show that intramolecular interactions are present in the majority of the inactive sgRNA sequences of the library, suggesting that they are the most important intrinsic determinants of the activity of the SpCas9 ribonucleoprotein complex. We also found that in pegRNAs, sequences at the 3' extension of the sgRNA that are complementary to the SL2 unit are also inhibitory to prime editing, but not to the nuclease activity of SpCas9.

SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e.

Several advancements have been made to SpCas9, the most widely used CRISPR/Cas genome editing tool, to reduce its unwanted off-target effects. The most promising approach is the development of increased-fidelity nuclease (IFN) variants of SpCas9, however, their fidelity has increased at the cost of reduced activity. SuperFi-Cas9 has been developed recently, and it has been described as a next-generation high-fidelity SpCas9 variant, free from the drawbacks of first-generation IFNs. In this study, we characterize the on-target activity and the off-target propensity of SuperFi-Cas9 in mammalian cells, comparing it to first-generation IFNs. SuperFi-Cas9 demonstrates strongly reduced activity but high fidelity features that are in many aspects similar to those of some first-generation variants, such as evo- and HeFSpCas9. SuperFi-cytosine (CBE3) and -adenine (ABE7.10) base editors, as well as SuperFi-prime editor show no meaningful activity. When combined with ABE8e, SuperFi-Cas9, similarly to HeFSpCas9, executes DNA editing with high activity as well as high specificity reducing both bystander and SpCas9-dependent off-target base editing.

PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells.

Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency of prime editing, which typically achieves editing rates of around 10%-30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.

Identification of New Interactions between Endolysosomal Tethering Factors.

Proper functioning of the precisely controlled endolysosomal system is essential for maintaining the homeostasis of the entire cell. Tethering factors play pivotal roles in mediating the fusion of different transport vesicles, such as endosomes or autophagosomes with each other or with lysosomes. In this work, we uncover several new interactions between the endolysosomal tethering factors Rabenosyn-5 (Rbsn) and the HOPS and CORVET complexes. We find that Rbsn binds to the HOPS/CORVET complexes mainly via their shared subunit Vps18 and we mapped this interaction to the 773-854 region of Vps18. Based on genetic rescue experiments, the binding between Rbsn and Vps18 is required for endosomal transport and is dispensable for autophagy. Moreover, Vps18 seems to be important for ß1 integrin recycling by binding to Rbsn and its known partner Vps45.

A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs.

Detailed target-selectivity information and experiment-based efficacy prediction tools are primarily available for Streptococcus pyogenes Cas9 (SpCas9). One obstacle to develop such tools is the rarity of accurate data. Here, we report a method termed 'Self-targeting sgRNA Library Screen' (SLS) for assaying the activity of Cas9 nucleases in bacteria using random target/sgRNA libraries of self-targeting sgRNAs. Exploiting more than a million different sequences, we demonstrate the use of the method with the SpCas9-HF1 variant to analyse its activity and reveal motifs that influence its target-selectivity. We have also developed an algorithm for predicting the activity of SpCas9-HF1 with an accuracy matching those of existing tools. SLS is a facile alternative to the much more expensive and laborious approaches used currently and has the capability of delivering sufficient amount of data for most of the orthologs and variants of SpCas9.

Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5'G-extended sgRNAs.

Increased fidelity mutants of the SpCas9 nuclease constitute the most promising approach to mitigating its off-target effects. However, these variants are effective only in a restricted target space, and many of them are reported to work less efficiently when applied in clinically relevant, pre-assembled, ribonucleoprotein forms. The low tolerance to 5'-extended, 21G-sgRNAs contributes, to a great extent, to their decreased performance. Here, we report the generation of Blackjack SpCas9 variant that shows increased fidelity yet remain effective with 21G-sgRNAs. Introducing Blackjack mutations into previously reported increased fidelity variants make them effective with 21G-sgRNAs and increases their fidelity. Two "Blackjack" nucleases, eSpCas9-plus and SpCas9-HF1-plus are superior variants of eSpCas9 and SpCas9-HF1, respectively, possessing matching on-target activity and fidelity but retaining activity with 21G-sgRNAs. They facilitate the use of existing pooled sgRNA libraries with higher specificity and show similar activities whether delivered as plasmids or as pre-assembled ribonucleoproteins.

Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases.

The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have generated new PAM mutant variants of the four Cas12a orthologs that are active in mammalian and plant cells, by combining the mutations of their corresponding RR and RVR variants with altered PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a) was found to further increase the activity of that variant in a PAM-dependent manner. Due to the overlapping but still different PAM preferences of impLbCas12a and the recently reported enAsCas12a variant, they complement each other to provide increased efficiency for genome editing and transcriptome modulating applications.

Developmentally regulated autophagy is required for eye formation in Drosophila.

The compound eye of the fruit fly Drosophila melanogaster is one of the most intensively studied and best understood model organs in the field of developmental genetics. Herein we demonstrate that autophagy, an evolutionarily conserved selfdegradation process of eukaryotic cells, is essential for eye development in this organism. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (Atg) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. We also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog Lab (Labial), a master regulator of early development. Atg8a transcription is repressed in front of, while activated along, the MF by Lab. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought. ABBREVIATIONS: αTub84B, α-Tubulin at 84B; Act5C, Actin5C; AO, acridine orange; Atg, autophagy-related; Ato, Atonal; CASP3, caspase 3; Dcr-2; Dicer-2; Dfd, Deformed; DZ, differentiation zone; eGFP, enhanced green fluorescent protein; EM, electron microscopy; exd, extradenticle; ey, eyeless; FLP, flippase recombinase; FRT, FLP recognition target; Gal4, gene encoding the yeast transcription activator protein GAL4; GFP, green fluorescent protein; GMR, Glass multimer reporter; Hox, homeobox; hth, homothorax; lab, labial; L3F, L3 feeding larval stage; L3W, L3 wandering larval stage; lf, loss-of-function; MAP1LC3, microtubule-associated protein 1 light chain 3; MF, morphogenetic furrow; PE, phosphatidylethanolamine; PBS, phosphate-buffered saline; PI3K/PtdIns3K, class III phosphatidylinositol 3-kinase; PZ, proliferation zone; Ref(2)P, refractory to sigma P, RFP, red fluorescent protein; RNAi, RNA interference; RpL32, Ribosomal protein L32; RT-PCR, reverse transcription-coupled polymerase chain reaction; S.D., standard deviation; SQSTM1, Sequestosome-1, Tor, Target of rapamycin; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay; UAS, upstream activation sequence; qPCR, quantitative real-time polymerase chain reaction; w, white.

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage.

BACKGROUND: The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects. RESULTS: These three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a matching 5' G extension being more detrimental to their activities than a mismatching one. HeFSpCas9 exhibit substantially improved specificity for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. The targets can also be ranked by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s. CONCLUSIONS: No single nuclease variant shows generally superior fidelity; instead, for highest specificity cleavage, each target needs to be matched with an appropriate high-fidelity nuclease. We provide here a framework for generating new nuclease variants for targets that currently have no matching optimal nuclease, and offer a simple means for identifying the optimal nuclease for targets in the absence of accurate target-ranking prediction tools.

A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid.

The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell. The use of 'self-cleaving' GFP-plasmids containing universal gRNAs and corresponding targets alleviates cloning burdens when this method is applied. These universal gRNAs mediate efficient plasmid cleavage and are designed to avoid genomic targets in several model species. The method combines the advantages of the straightforward FACS detection provided by applying fluorescent reporter systems and of the PCR-based approaches being capable of testing targets in their genomic context, without necessitating any extra cloning steps. Additionally, we show that NHEJ-cloning can also be used in mammalian cells for targeted integration of donor plasmids up to 10 kb in size, with up to 30% efficiency, without any selection or enrichment.

Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.

The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease--a Body Double of the Type IIP enzyme--is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.