Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing.

Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.2-fold that of wild-type Bxb1) in human cell lines with pre-installed recombinase landing sites. In single-transfection experiments at safe-harbour and therapeutically relevant sites, PASSIGE with eeBxb1 led to an average targeted-gene-integration efficiencies of 23% (4.2-fold that of wild-type Bxb1). Notably, integration efficiencies exceeded 30% at multiple sites in primary human fibroblasts. PASSIGE with evoBxb1 or eeBxb1 outperformed PASTE (for 'programmable addition via site-specific targeting elements', a method that uses prime editors fused to recombinases) on average by 9.1-fold and 16-fold, respectively. PASSIGE with continuously evolved recombinases is an unusually efficient method for the targeted integration of genes in mammalian cells.

Twin prime editing mediated exon skipping/reinsertion for restored collagen VII expression in recessive dystrophic epidermolysis bullosa.

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

Prime editing: current advances and therapeutic opportunities in human diseases.

Gene editing ushers in a new era of disease treatment since many genetic diseases are caused by base-pair mutations in genomic DNA. With the rapid development of genome editing technology, novel editing tools such as base editing and prime editing (PE) have attracted public attention, heralding a great leap forward in this field. PE, in particular, is characterized by no need for double-strand breaks (DSBs) or homology sequence templates with variable application scenarios, including point mutations as well as insertions or deletions. With higher editing efficiency and fewer byproducts than traditional editing tools, PE holds great promise as a therapeutic strategy for human diseases. Subsequently, a growing demand for the standard construction of PE system has spawned numerous easy-to-access internet resources and tools for personalized prime editing guide RNA (pegRNA) design and off-target site prediction. In this review, we mainly introduce the innovation and evolutionary strategy of PE systems and the auxiliary tools for PE design and analysis. Additionally, its application and future potential in the clinical field have been summarized and envisaged.

Phage-assisted evolution and protein engineering yield compact, efficient prime editors.

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.

[Epidemiological characteristics and serum antibody detection of a COVID-19 aggregated outbreak in vaccinated population].

An epidemiological investigation was conducted on a cluster epidemic of COVID-19 in the vaccinated population in Beijing in 2022, and serum samples were collected from 21 infected cases and 61 close contacts (including 20 cases with positive nucleic acid in the isolation observation period). The results of antibody detection showed that the IgM antibody of two infected persons was positive, and the IgG antibody positive rates of patients who were converted, not converted to positive and infected persons were 36.84% (7/19), 63.41% (26/41) and 71.43% (15/21), respectively. About 98.78% of patients had been vaccinated with the SARS-CoV-2 inactivated vaccine. The positive rate of IgG antibody in patients immunized with three doses of vaccine was 86.00% (43/50), which was higher than that in patients with one or two doses [16.12% (5/31)]. The antibody level of M (Q1, Q3) in patients immunized with three doses was 4.255 (2.303, 7.0375), which was higher than that in patients with one or two doses [0.500 (0.500, 0.500)] (all P values<0.001). The antibody level of patients who were vaccinated less than three months [7.335 (1.909, 7.858)] was higher than that of patients vaccinated more than three months after the last vaccination [2.125 (0.500, 4.418)] (P=0.007). The positive rate and level of IgG antibody in patients who were converted to positive after three doses were 77.78% (7/9) and 4.207 (2.216, 7.099), respectively, which were higher than those in patients who were converted after one or two doses [0 and 0.500 (0.500, 0.500)] (all P values<0.05).

Adenine Base Editing In Vivo with a Single Adeno-Associated Virus Vector.

Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (∼5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor (ABE) using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared with the well-characterized Streptococcus pyogenes Cas9-containing ABEs, ABEs using Nme2Cas9 (Nme2-ABE) possess a distinct protospacer adjacent motif (N4CC) and editing window, exhibit fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we show that in vivo delivery of Nme2-ABE and its guide RNA by a single AAV vector can efficiently edit mouse genomic loci and revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration.

Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.

Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing.

The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.

[Establishment and validation of prognostic risk score model for esophageal squamous cell carcinoma based on immune related genes].

Objective: To find the biomarkers that accurately predict the survival of patients with esophageal squamous cell carcinoma (ESCC). Methods: The immune related genes that were significantly related to the overall survival (OS) of patients with ESCC were screened from The Cancer Genome Atlas (TCGA) database to construct a prognostic risk score model. The prognoses of the high-risk and low-risk groups were compared by Kaplan-Meier method. The accuracy of the model was evaluated by the receiver operating characteristic (ROC) curve. Tumor tissue samples of 83 patients with pathological diagnosis of ESCC were collected from Anyang Cancer Hospital for external verification. Cox regression analysis was used to comprehensively evaluate the effects of prognostic risk score and various clinical characteristics on OS of patients with ESCC. Results: Seven immune-related genes that were significantly related to survival prognosis were selected from the TCGA database and included in the prognostic risk score model, which were S100A12, SLC40A1, FABP9, TNFSF10, IGHA2, IL1F10, and STC2. The 1- and 2-year survival rates of the low-risk group (40 cases) were 94.3% and 82.5%, respectively, while those of the high-risk group (40 cases) were 75.9% and 32.9%, respectively.The prognosis of the high-risk group was worse than that of the low-risk group (P<0.001). The 83 external validation samples obtained consistent results by using the prognostic risk score model. The prognostic risk score was positively correlated with the content of CD4(+) T lymphocytes in ESCC (r(s)=0.259, P=0.020), but not correlated with the content of B lymphocytes, CD8(+) T lymphocytes, neutrophils, macrophages or dendritic cells (P>0.05). Conclusions: S100A12, SLC40A1, FABP9, TNFSF10, IGHA2, IL1F10, and STC2 were risk genes significantly associated with OS of patients with ESCC. The prognostic risk score was an independent prognostic factor for the OS of patients with ESCC, and it was correlated with the content of CD4(+) T lymphocytes in ESCC tissue.

Anatomic morphological study of thoracolumbar foramen in normal adults.

BACKGROUND: Based on computed tomography images of the thoracolumbar intervertebral foramen and its surrounding parameters, and analysing the intervertebral foramen morphology and the correlation between the measured parameters, to provide an anatomical basis for clinical minimally invasive transvertebral surgery. MATERIALS AND METHODS: Ten fresh adult cadaveric specimens (32-50 years old) with bilateral (T1-S1) spinal segments were selected for a total of 20 sides, a total of 340 intervertebral foramens and were measured with vernier callipers in the Department of Anatomy, Inner Mongolia Medical University. The intervertebral foramen height, the minimum sagittal diameter of the foramen, the width of the spinal ganglion, the sagittal diameter of the spinal ganglion and the sagittal diameter of the intervertebral foramen were measured. This study was reviewed and approved by the local Ethics Committee. RESULTS: The results of the minimum sagittal diameter of the T9-10~L5/S1 intervertebral foramen were (6.93 ± 1.99) mm, (7.33 ± 1.44) mm, (7.41 ± 0.63) mm, (6.85 ± 1.08) mm, (6.79 ± 1.86) mm, (7.82 ± 3.25) mm, (8.23 ± 2.27) mm, (9.17 ± 2.33) mm, (8.38 ± 1.63) mm; the average height of the T2/3 to L5/S1 intervertebral space was (4.82 ± 1.88) mm, (3.95 ± 0.80) mm, (4.04 ± 0.52) mm, (4.26 ± 0.78) mm, (4.39 ± 1.16) mm, (5.15 ± 1.59) mm, (5.51 ± 1.49) mm, (5.97 ± 2.60) mm, (7.13 ± 2.07) mm, (8.94 ± 1.37) mm, (9.01 ± 1.47) mm, (11.63 ± 1.63) mm, (14.20 ± 1.37) mm, (14.22 ± 2.33) mm, (14.22 ± 2.33) mm, (13.32 ± 1.37) mm intervertebral foramen height, intervertebral foramen minimum sagittal diameter, spinal ganglion width, spinal ganglion sagittal diameter. P > 0.05 for comparison of the left and right sides of the intervertebral space, with no statistically significant difference. L4/5, L5/S1 segment left and right bilateral contrast with the middle height of the vertebral space p < 0.05, the difference is statistically significant. The remaining segments left and right bilaterally contrasted p > 0.05, and the difference was not statistically significant. CONCLUSIONS: The minimum height of intervertebral foramen in the thoracolumbar segment was T6/7, and L1/2 was the minimum height in the lumbar segment. When placing a spinal endoscopic working channel safely into intervertebral foramen, it is necessary to perform an enlarging foraminoplasty to reduce the risk of injury to the exiting nerve root.

[Mechanism of "Scutellaria barbata-Hedyotis diffusa" against breast cancer based on network pharmacology].

To explore the mechanism of Scutellaria barbata-Hedyotis diffusa against breast cancer based on network pharmacological methods. The active components and corresponding target proteins of S. barbata and H. diffusa were screened by using Traditional Chinese Medicine Systems Pharmacology Database(TCMSP), and the targets of breast cancer were screened through the Genecards and OMIM databases. Venn online software was used to obtain the common targets of drugs and the disease, and then the "compound-target-disease" network diagram was constructed by using Cytoscape 3.7.2 software. The STRING database was used to draw the protein-protein interaction(PPI) network, and the David database was used to perform GO function and KEGG pathway enrichment analysis on effective targets. The results showed that 29 S. barbata compounds, 7 H. diffusa compounds, and 109 effective targets were screened. The 59 common targets for drugs and disease were obtained through Venn diagrams. The PPI network showed that MYC, CCND1, EGFR, ESR1, CASP3, VEGFA, etc. might be the key targets for "S. barbata-H. diffusa" in the treatment of breast cancer. In the GO function analysis, 88 entries were found, involving the regulation of transcription factor activity, receptor activity, cytokine activity, enzyme activity, and biosynthetic processes. In KEGG pathway analysis, 37 entries related to cancer, cell cycle, apoptosis, angioge-nesis and other pathways were found, including p53 signaling pathway, ErbB signaling pathway, nod like receptor signaling pathway, MAPK signaling pathway, VEGF signaling pathway, Wnt signaling pathway and mTOR signaling pathway and other classic signaling pathways. This study preliminarily revealed the key targets, biological processes and signal pathway of "S. barbata-H. diffusa" against breast cancer. It was found that its function was in a multi-target and multi-channel manner, which laid a foundation for future molecular biological experiments.

[Study on the precision of the robot-assisted sacroiliac screw placement].

Objective: To evaluate the precision of the robot-assisted sacroiliac screw placement for posterior pelvis injury and the impacting factors. Methods: The clinical data of twenty-four cases of posterior pelvic fractures treated by percutaneous sacroiliac screw placement in Yantai shan Hospital from August 2016 to May 2018 were studied retrospectively. There were 17 males and 7 females with a mean age of 44.1 years (ranged from 17 to 71 years). According to AO classification, 17 cases were type B fractures (9 cases of type B1 and 8 type B2), and 7 cases were type C factures (3 cases of type C1, 2 type C2 and 2 type C3). All cases were treated by robot-assisted percutaneous sacroiliac screw placement (AO cannulated screws with a diameter of 7.3 mm). In the posterior pelvic surgeries for the 24 cases, 26 S(1) sacroiliac screws fixations and 18 S(2) sacroiliac screw fixations were placed in total, with single S(1) segmental fixation in 8 cases, single S(2) segmental fixation in 3 cases, S(1) and S(2) combined fixation in 13 cases, S(1) unidirectional one-sided fixation in 18 cases, S(1) bidirectional two-sided fixation in 3 cases, S(2) unidirectional one-sided fixation in 14 cases, S(2) bidirectional two-sided fixation in 2 cases and S(1) unidirectional double screws fixation in 2 cases. X-ray and CT examinations were taken for all 24 cases after operation. The visual analogue scale (VAS) of pain were performed before and after the operation. Results: All the sacroiliac screws were successfully implanted at once as planned with the assistance of the robot. The postoperative X-ray films and CT showed that none of the sacroiliac screws broke through the sacral body and the contralateral sacral wing's frontal cortex nor did they stray into the sacral canal and the intervertebral space. In 3 cases, the sacroiliac screws went closely against and wore out the front edge of iliac cortical density line and sacral alar slope and finally re-entered the sacral body. In 3 cases, sacroiliac screws touched upon the sacral nerve canals but did not break through the nerve canals. The mean VAS of pain was improved from 7.1 points (4-10 points) before the operation to 1.9 points (0-3 points) after. Conclusions: The robot-assisted sacroiliac screw placement shows high precision, and hence is worthy of clinical promotion; however the primary role of the surgeon could not be replaced.

CircRNA_100782 promotes roliferation and metastasis of gastric cancer by downregulating tumor suppressor gene Rb by adsorbing miR-574-3p in a sponge form.

OBJECTIVE: The aim of this study is to investigate the expression levels of circRNA_100782 in gastric cancer tissues, and its function of regulating tumor suppressor gene Rb by absorbing miR-574-3p in a sponge form. PATIENTS AND METHODS: qRT-PCR was performed to detect the expressions of circRNA_100782 at different stages during gastric cancer tissues. CCK-8 assay was performed to evaluate the osteoclast proliferation and differentiation. The correlation between miR-574-3p and circRNA_100782 was detected by statistical analysis. Bioinformatics and Luciferase assay were performed to explore the interaction and binding site of circRNA_100782 and miR-574-3p. The mice Rb 3'-UTR were cloned into the Luciferase reporter vector and miR-574-3p binding mutants were constructed to validate the inhibited regulation of miR-574-3p to the expression of Rb. RESULTS: In the current study, compared with adjacent non-cancerous normal tissues, the expressions of circRNA_100782 and Rb were both downregulated in human gastric cancer cells. Through qRT-PCR and CCK-8 assay, we found that the expression of circRNA_100782 is related to the proliferation of gastric cancer cells. Besides, we also found that circRNA_100782 regulated the migration ability of gastric cancer cells through transwell assay. The bioinformatics prediction and luciferase assay demonstrated that circRNA_100782 can serve as a molecular sponge to further regulate the expression of Rb by sponging with miR-574-3p; moreover, circRNA_100782 can serve as a ceRNA for miR-574-3p to further regulate the expression of Rb. CONCLUSIONS: In this research, we discovered that circRNA_100782 was downregulated in gastric cancer cells and is associated with cell proliferation and invasion by inhibiting tumor suppressor gene Rb by interacting with miR-574-3p.

Efficacy of orally toxic sugar baits against contact-insecticide resistant culex quinquefasciatus.

In recent years, attractive toxic sugar bait has been used in the mosquito control in nature, and achieved good control effects. However, the current researches about toxic sugar bait did not focus on whether the wild mosquito population used for control is resistant or not. The purpose of this study was to evaluate the effectiveness of the toxic sugar bait against mosquito resistant populations to test the effects of bait on the control of mosquitoes with different levels of resistance. Boric acid, dinotefuran and deltamethrin were separately formulated into toxic sugar bait to test their anti-mosquito activity against Culex quinquefasciatus. Using the sugar baits formulated with boric acid and dinotefuran, the mortality of Cx. quinquefasciatus resistant populations was significantly higher than that of sensitive populations at the same concentration. Conversely, with the use of sugar baits formulated with deltamethrin, the mortality of Cx. quinquefasciatus resistant populations was significantly lower than that of sensitive populations at the same concentration. The results suggested that toxic sugar baits might have a good application prospect in high resistant mosquito management.

Higher lncRNA CASC15 expression predicts poor prognosis and associates with tumor growth in cervical cancer.

OBJECTIVE: To investigate the role of long non-coding RNA Cancer Susceptibility Candidate 15 (CASC15) in cervical cancer and its potential molecular mechanism. PATIENTS AND METHODS: The CASC15 expression was measured in cervical cancer tissues and cell lines by using quantitative Real-time polymerase chain reaction (qRT-PCR) analysis. Cell counting kit-8 (CCK8), flow cytometry analysis and transwell cell invasion assays were employed to detect the capacities of cell proliferation and cell invasion. Furthermore, Western blot analysis was applied to detected the E-cadherin and N-cadherin expression in EMT pathway. RESULTS: We demonstrated that lncRNA CASC15 expression was higher in cervical cancer tissues compared to adjacent normal tissues. Higher lncRNA CASC15 expression associated with lymph node metastasis and FIGO stage. Moreover, our results showed that higher lncRNA CASC15 expression predicted poor prognosis of cervical cancer. Functional assays showed that knockdown of lncRNA CASC15 suppressed cell proliferation and cell cycle progression in cervical cancer. Moreover, we also found that knockdown of lncRNA CASC15 inhibited cell invasion ability and Epithelial-Mesenchymal Transition (EMT) signaling pathway by upregulating E-cadherin and downregulating N-cadherin expression in cervical cancer. CONCLUSIONS: These results indicated that lncRNA CASC15 expression may be a prognostic biomarker and contributed to cell proliferation and invasion in cervical cancer.

Adapting dCas9-APEX2 for subnuclear proteomic profiling.

Genome organization and subnuclear protein localization are essential for normal cellular function and have been implicated in the control of gene expression, DNA replication, and genomic stability. The coupling of chromatin conformation capture (3C), chromatin immunoprecipitation and sequencing, and related techniques have continuously improved our understanding of genome architecture. To profile site-specifically DNA-associated proteins in a high-throughput and unbiased manner, the RNA-programmable CRISPR-Cas9 platform has recently been combined with an enzymatic labeling system to allow proteomic landscapes at repetitive and nonrepetitive loci to be defined with unprecedented ease and resolution. In this chapter, we describe the dCas9-APEX2 experimental approach for specifically targeting a DNA sequence, enzymatically labeling local proteins with biotin, and quantitatively analyzing the labeled proteome. We also discuss the optimization and extension of this pipeline to facilitate its use in understanding nuclear and chromosome biology.

CD44 splice isoform switching determines breast cancer stem cell state.

Although changes in alternative splicing have been observed in cancer, their functional contributions still remain largely unclear. Here we report that splice isoforms of the cancer stem cell (CSC) marker CD44 exhibit strikingly opposite functions in breast cancer. Bioinformatic annotation in patient breast cancer in The Cancer Genome Atlas (TCGA) database reveals that the CD44 standard splice isoform (CD44s) positively associates with the CSC gene signatures, whereas the CD44 variant splice isoforms (CD44v) exhibit an inverse association. We show that CD44s is the predominant isoform expressed in breast CSCs. Elimination of the CD44s isoform impairs CSC traits. Conversely, manipulating the splicing regulator ESRP1 to shift alternative splicing from CD44v to CD44s leads to an induction of CSC properties. We further demonstrate that CD44s activates the PDGFRß/Stat3 cascade to promote CSC traits. These results reveal CD44 isoform specificity in CSC and non-CSC states and suggest that alternative splicing provides functional gene versatility that is essential for distinct cancer cell states and thus cancer phenotypes.

NmeCas9 is an intrinsically high-fidelity genome-editing platform.

BACKGROUND: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery. RESULTS: Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5'-N4GATT-3'), for NmeCas9 genome editing in human cells. CONCLUSIONS: Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins.

In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Neisseria meningitidis Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.IMPORTANCE As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality in vitro and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.