A comparative analysis of different reduced-port laparoscopic surgical procedures after non-curative endoscopic resection for early colorectal cancer.

Introduction: Surgery serves as a salvage procedure for non-curative resection of early-stage colorectal cancer under endoscopy. A standard method for performing additional surgery after endoscopic submucosal dissection (ESD) for early colorectal cancer has yet to be established. Aim: To enhance the understanding of different surgical outcomes by discussing additional treatment strategies following non-complete curative endoscopic resection of early colorectal cancer. Material and methods: This retrospective study included 88 patients who were divided into three groups based on the surgical approach: conventional laparoscopic surgery (CLS), single-incision plus one-port laparoscopic surgery (SILS+1), and three-port laparoscopic surgery combined with natural orifice specimen extraction surgery (three-port NOSES). The study aimed to compare the surgical outcomes, safety, and postoperative recovery among these groups. Results: The SILS+1 and three-port NOSES groups demonstrated comparable safety and efficacy to the CLS group in terms of blood loss, complications, number of lymph node dissections, and length of bowel resection. However, the SILS+1 and three-port NOSES groups had advantages in terms of incision length (7.11 ±0.38, 4.24 ±0.33, 3.16 ±0.22, p < 0.001), postoperative pain (4.000 [3.0,5.0], 3.500 [3.0,4.0], 3.000 [3.0,4.0]; p = 0.003), cosmetic result (4.000 [3.8,5.0], 7.000 [7.0,8.0], 7.000 [7.0,8.0]; p < 0.001), and hospital stay (8.000 [7.0,9.0], 7.000 [6.3,8.0.], 7.000 [6.3,8.0]; p = 0.035). Conclusions: Different strategies of reduced-port laparoscopic surgery have been demonstrated to be effective and safe in additional surgery after non-curative ESD. These techniques have shown reduced pain and increased satisfaction among patients. Reduced-port laparoscopic surgery is expected to become the preferred treatment option for these patients.

Correction: Pu et al. Exosome miRNA Expression in Umbilical Cord Blood of High-Parity Sows Regulates Their Reproductive Potential. Animals 2022, 12, 2456.

In the original publication [...].

Comparative Transcriptomic Profiling in Ovarian Tissues of Lohmann Hens and Chengkou Mountain Chicken.

BACKGROUND: As a crucial economic characteristic and a major indicator of reproductive performance in layers, egg production is controlled by a series of complex regulatory heredity basis. In particular, the interacting regulatory function between noncoding RNAs (ncRNAs) and coding RNA plays important roles in regulating laying performance. METHODS: In this study, the RNA sequencing (RNA-seq) of ovarian tissues from Lohmann hens (n = 3) and Chengkou Mountain chicken (n = 3) under the laying peak period was performed to identify RNA transcriptional differences among different laying-performance populations. RESULTS: Results showed that the expression level of 303 mRNAs, 68 long ncRNAs (lncRNAs), 533 circular RNAs (circRNAs), and 79 microRNAs (miRNAs) was significantly different among the groups. Functional enrichment analysis of these differentially expressed (DE) mRNAs revealed that the laying process was implicated in numerous significantly enriched pathways (p < 0.05), such as the neuroactive ligand-receptor interaction, steroid hormone biosynthesis, and calcium-signaling pathway. Furthermore, the lncRNA/circRNA-miRNA-mRNA regulatory networks related to the regulation of laying performance were constructed. Some randomly selective DE RNAs were verified by Real Time Quantitative (RT-qRCR), indicating that the bioinformatics analysis results of RNA-seq data were credible. CONCLUSIONS: This study could increase our understanding of the heredity basis of transcriptome in the laying performance of chicken.

Association of leisure sedentary behavior and physical activity with the risk of nonalcoholic fatty liver disease: a two-sample Mendelian randomization study.

Introduction: Previous observational studies have demonstrated the relationship between leisure sedentary behavior, physical activity, and nonalcoholic liver disease (NAFLD). However, whether these associations are causal or confounding factors remains unclear. Methods: Pooled genetic data from the UK Biobank and other large genome-wide association studies (GWAS) were used to extract instrumental variables representing sedentary television watching, computer use, driving, vigorous physical activity (VPA), and moderate-to-vigorous physical activity (MVPA). The two-sample Mendelian randomization (MR) method was used to explain the causal relationship between them and NAFLD. The inverse variance of the weighted method was used as the main analysis method, and MR-Egger, weighted median, MR-PRESSO, and other supplementary methods were also used. A sensitivity analysis was also performed. Simultaneously, the common risk factors for NAFLD were further analyzed for potential mediating associations. Results: We observed that sedentary television viewing (odds ratio (OR): 1.84; 95% confidence interval (CI): 1.09-3.10; p = 0.021) and genetically predicted VPA duration (OR: 0.0033; 95% CI: 0.000015-0.70; p = 0.036) were suggestively associated with the risk of NAFLD. Using a computer (OR: 1.51; 95% CI: 0.47-4.81; p = 0.484), driving (OR: 0.78; 95% CI: 0.05-11.94; p = 0.858), and MVPA time (OR: 0.168; 95% CI: 0.01-2.81; p = 0.214) were not significantly associated with NAFLD. The role of heterogeneity versus pleiotropy was limited in all the analyses. Discussion: This study supports the association between sedentary television watching and an increased risk of NAFLD, along with vigorous physical activity as a possible protective factor for NAFLD.

Identification of differentially expressed long non-coding RNAs and messenger RNAs involved with muscle development in Dazu black goats through RNA sequencing.

This study aimed to explore the genetic basis of muscle development in goats. The transcriptome dataset for differentially expressed lncRNAs (DELs) and differentially expressed genes (DEGs) of goat muscle at different developmental stages were obtained using RNA-Seq. A total of 447,806,481 and 587,559,465 clean reads in the longissimus dorsi muscle of Dazu black goats between 75d embryonic stage and 1d after birth were generated through Illumina paired-end sequencing, and their mapping rates were 89.82 and 90.99%, respectively. Moreover, 4517 DEGs and 648 DELs were identified, and 4784 lncRNA-mRNA targeting relationships were predicted. Gene function annotation results showed that 4101 DEGs were significantly enriched to 1098 GO terms, and 2014 DEGs were significantly enriched to 40 KEGG pathways, including many GO terms and pathways related to muscle development, such as cell differentiation and Wnt signaling pathway. Then, 10 DELs and 20 DEGs were randomly selected for RT-qPCR verification, and the agreement rate between the verification and RNA-Seq results was 90%, indicating the high reliability of the RNA-Seq data analysis. In conclusion, this study obtained several mRNAs and lncRNAs related to the muscle development of Dazu black goats and identified several targeted regulatory pairs of lncRNA-mRNA. This study may serve as a reference to understand the genetic basis and molecular mechanism of muscle development in goats.

Carbon nanoparticle-assisted natural orifice specimen extraction surgery with left colic artery preservation: a retrospective study.

Introduction: Natural orifice specimen extraction surgery (NOSES) has been widely regarded as a new technology in minimally invasive surgery. Meanwhile, carbon nanoparticles have been increasingly used for lymph node tracing in colorectal cancer surgery. Aim: To evaluate the effectiveness and feasibility of carbon nanoparticle-assisted natural orifice specimen extraction surgery with left colic artery preservation for total laparoscopic colorectal resection. Material and methods: We retrospectively reviewed the medical records of 83 patients diagnosed with sigmoid colon cancer or mid- and upper-rectal cancer from October 2017 to June 2020. These patients were divided into the NOSES group who underwent left colic artery preservation NOSES, being injected with a carbon nanoparticle suspension under colonoscopy the day before surgery, and the LA group, who underwent left colic artery preservation laparoscopic surgery. Surgical outcomes were retrospectively analyzed. Results: The mean number of harvested lymph nodes (p < 0.001) in the NOSES group was higher than in the LA group. Conversely, as regards pain score (p < 0.001) and postoperative hospital stay (p = 0.035), the LA group has higher mean values. The incidence of perioperative complications (p = 0.385) was 5.3% for the NOSES group compared to 13.3% for the LA group. Conclusions: Preoperative colonoscopic injection of a carbon nanoparticle suspension is a feasible and practical solution to dissect lymph nodes surrounding the inferior mesenteric artery without affecting the left colic artery in patients with colorectal cancer and about to receive NOSES. Moreover, NOSES combined with this approach leads to less postoperative pain and shorter hospital stays.

Exosome miRNA Expression in Umbilical Cord Blood of High-Parity Sows Regulates Their Reproductive Potential.

The objective of modern pig breeding is to improve the genetic reproduction performance potential of sows, including the litter size and weight of piglets. During the gestation period, the umbilical cord facilitates placenta−fetal communication; thus, it plays an indispensable role in intrauterine embryonic development and fitness. Herein, we analyzed the molecular mechanism in declining reproductive potential in high-parity sows by assessing the changes in the umbilical cord blood. Firstly, we analyzed the reproductive characteristics data of sows, followed by histological analysis of the umbilical cord phenotype. Next, we evaluated the effect of umbilical cord blood exosomes (UCB-EXO) on angiogenesis. Finally, the miRNA expression in UCB-EXO from high-parity sows with poor reproductive performance (OS) and multiparous sows with excellent reproductive performance (MS) was assessed. Overall, the best reproductive performance was at parity 3−7, gradually decreasing after parity 8 and angiogenesis was repressed in OS. However, exosomes derived from MS (Exo-MS) exhibited pro-angiogenesis properties but were diminished in exosomes derived from OS (Exo-OS). Additionally, the angiogenesis of sows was significantly decreased, increasing the risk of disease with the increase in parity, greatly limiting the reproductive potential of the sows. At the same time, miR-188-5p expression in Exo-OS was significantly higher than in Exo-MS (p < 0.01), implying that it may play an important role in regulating the lifespan and reproductive potential of sows. These findings demonstrated that miRNAs in UCB-EXO play a central role in intrauterine development. Further, the findings suggest novel insights on reproductive potential, which provide a reference for increasing the sow reproductive efficiency.

Synergistic antitumor effect of Andrographolide and cisplatin through ROS-mediated ER stress and STAT3 inhibition in colon cancer.

Colon cancer is one of the most leading death-causing cancers in the world. Cisplatin has been widely used as the first-line treatment of cancer. However, its clinical application is limited by the side effects or acquired drug resistance. Hence, it is of vital clinical significance to develop novel agents that synergize with cisplatin and decrease its side effects. The aim of this study was to investigate whether Andrographolide (AP) synergistically potentiates the anti-tumor effect of cisplatin on colon cancer cells. Here, we found that AP synergizes with cisplatin in exerting anticancer activity in colon cancer cells. Further studies showed that AP potentiates cisplatin-induced endoplasmic reticulum stress and STAT3 inhibition through increasing intracellular ROS. Notably, pre-treatment of NAC, a ROS scavenger, reversed apoptosis induced by combined treatment of AP and cisplatin, while relieving the activation of endoplasmic reticulum stress as well as STAT3 inhibition. These findings indicated that ROS plays a pivotal role in mediating synergistic anticancer effects of AP and cisplatin on colon cancer cells. Overall, this study presents a potential new therapeutic strategy for the treatment of colon cancer.

Small-molecule compounds boost genome-editing efficiency of cytosine base editor.

Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.

Lb2Cas12a and its engineered variants mediate genome editing in human cells.

Cas12a-mediated targeted genome engineering strategies have enabled a broad range of research and clinical applications. However, the limited target-selection spectrum and low activity/fidelity remain a bottleneck for its widespread application in precision site-specific human genome editing. Therefore, there exists an acute need to identify novel Cas12a nucleases with improved features for genome editing. By screening a range of candidate Cas12a nucleases, here we demonstrate that Lb2Cas12a possesses genome editing activity in human cells and it has greater flexibility in PAM (5'-BYYV-3') selection. Furthermore, we engineered Lb2Cas12a to generate variants (Lb2Cas12a-RVR and Lb2Cas12a-RR), which greatly expands the target-selection spectrum. Our study illustrated that Lb2Cas12a could be harnessed as additional genome editing tool for the manipulation of human genome.

Engineered FnCas12a with enhanced activity through directional evolution in human cells.

Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.

Modified procedure for prolapse and hemorrhoids: Lower recurrence, higher satisfaction.

BACKGROUND: Hemorrhoidal prolapse is a common benign disease with a high incidence. The treatment procedure for prolapse and hemorrhoids (PPH) remains an operative method used for internal hemorrhoid prolapse. Although it is related to less pos-operative pain, faster recovery and shorter hospital stays, the postoperative recurrence rate is higher than that of the Milligan-Morgan hemorrhoidectomy (MMH). We have considered that recurrence could be due to shortage of the pulling-up effect. This issue may be overcome by using lower purse-string sutures [modified-PPH (M-PPH)]. AIM: To compare the therapeutic effects and the patients' satisfaction after M-PPH, PPH and MMH. METHODS: This retrospective cohort study included 1163 patients (M-PPH, 461; original PPH, 321; MMH, 381) with severe hemorrhoids (stage III/IV) who were admitted to The 2nd Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University from 2012 to 2014. Early postoperative complications, efficacy, postoperative anal dysfunction and patient satisfaction were compared among the three groups. Established criteria were used to assess short- and long-term postoperative complications. A visual analog scale was used to evaluate postoperative pain. Follow-up was conducted 5 years postoperatively. RESULT: Length of hospital stay and operating time were significantly longer in the MMH group (8.05 ± 2.50 d, 19.98 ± 4.21 min; P < 0.0001) than in other groups. The incidence of postoperative anastomotic bleeding was significantly lower after M-PPH than after PPH or MMH (1.9%, 5.1% and 3.7%; n = 9, 16 and 14; respectively). There was a significantly higher rate of sensation of rectal tenesmus after M-PPH than after MMH or PPH (15%, 8% and 10%; n = 69, 30 and 32; respectively). There was a significantly lower rate of recurrence after M-PPH than after PPH (8.7% and 18.8%, n = 40 and 61; P < 0.0001). The incidence of postoperative anal incontinence differed significantly only between the MMH and M-PPH groups (1.3% and 4.3%, n = 5 and 20; P = 0.04). Patient satisfaction was significantly greater after M-PPH than after other surgeries. CONCLUSION: M-PPH has many advantages for severe hemorrhoids (Goligher stage III/IV), with a low rate of anastomotic bleeding and recurrence and a very high rate of patient satisfaction.

Rational Selection of CRISPR-Cas Triggering Homology-Directed Repair in Human Cells.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) nucleases have been widely applied for genome engineering. Cas9 (Streptococcus pyogenes Cas9 [SpCas9] and Staphylococcus aureus Cas9 [SaCas9]) and Cpf1 (i.e., Francisella novicida U112 Cpf1 [FnCpf1], also named FnCas12a) were harnessed to perform gene editing in human cells. Precise genetic modification by homology-directed repair (HDR) is an attractive approach for in situ gene correction. However, so far, the comparative efficiencies of HDR mediated by different CRISPR orthologs remain unknown. To address this question, in this study, we developed a reporter system to investigate HDR efficiencies triggered by various CRISPR orthologs. We found that SpCas9 and SaCas9, the two most commonly used Cas9 enzymes, possessed a similar ability to induce HDR. Interestingly, with the increasing amount of coding plasmids or additional nuclear localization sequences, FnCpf1 could improve the HDR efficacy. Collectively, our study provides insights for the rational selection of appropriate tools for human genome manipulation.

Highly multiplexed quantifications of 299 somatic mutations in colorectal cancer patients by automated MALDI-TOF mass spectrometry.

BACKGROUND: Detection of somatic mutations in tumor tissues helps to understand tumor biology and guide treatment selection. Methods such as quantitative PCR can analyze a few mutations with high efficiency, while next generation sequencing (NGS) based methods can analyze hundreds to thousands of mutations. However, there is a lack of cost-effective method for quantitatively analyzing tens to a few hundred mutations of potential biological and clinical significance. METHODS: Through a comprehensive database and literature review we selected 299 mutations associated with colorectal cancer. We then designed a highly multiplexed assay panel (8-wells covering 299 mutations in 109 genes) based on an automated MADLI-TOF mass spectrometry (MS) platform. The multiplex panel was tested with a total of 319 freshly frozen tissues and 92 FFPE samples from 229 colorectal cancer patients, with 13 samples also analyzed by a targeted NGS method covering 532 genes. RESULTS: Multiplex somatic mutation panel based on MALDI-TOF MS detected and quantified at least one somatic mutation in 142 patients, with KRAS, TP53 and APC being the most frequently mutated genes. Extensive validation by both capillary sequencing and targeted NGS demonstrated high accuracy of the multiplex MS assay. Out of 35 mutations tested with plasmid constructs, sensitivities of 5 and 10% mutant allele frequency were achieved for 19 and 16 mutations, respectively. CONCLUSIONS: Automated MALDI-TOF MS offers an efficient and cost-effective platform for highly multiplexed quantitation of 299 somatic mutations, which may be useful in studying the biological and clinical significance of somatic mutations with large numbers of cancer tissues.

High-fidelity SaCas9 identified by directional screening in human cells.

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2- to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSaCas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.

Development of a Simple and Quick Method to Assess Base Editing in Human Cells.

Base editing is a form of genome editing that can directly convert a single base (C or A) to another base (T or G), which is of great potential in biomedical applications. The broad application of base editing is limited by its low activity and specificity, which still needs to be resolved. To address this, a simple and quick method for the determination of its activity/specificity is highly desired. Here, we developed a novel system, which could be harnessed for quick detection of editing activity and specificity of base editors (BEs) in human cells. Specifically, multiple cloning sites (MCS) were inserted into the human genome via lentivirus, and base editing targeting the MCS was performed with BEs. The base editing activities were assessed by specific restriction enzymes. The whole process only includes nucleotide-based targeting the MCS, editing, PCR, and digestion, thus, we named it NOTEPAD. This straightforward approach could be easily accessed by molecular biology laboratories. With this method, we could easily determine the BEs editing efficiency and pattern. The results revealed that BEs triggered more off-target effects in the genome than on plasmids including genomic indels (insertions and deletions). We found that ABEs (adenine base editors) had better fidelity than CBEs (cytosine base editors). Our system could be harnessed as a base editing assessment platform, which would pave the way for the development of next-generation BEs.

For colorectal cancer patients with type II diabetes, could metformin improve the survival rate? A meta-analysis.

OBJECTIVE: Current research is controversial about whether metformin can improve the survival rate of patients with colon cancer. Therefore, we conducted a meta-analysis to identify the association between metformin and the survival rate of colorectal cancer (CRC) patients with type II diabetes. METHODS: We conducted a search in databases including Pubmed, EMBASE and Cochrane Library. All articles were published in the last decade, and the quality of each study was evaluated by the Newcastle-Ottawa Scale. Odds ratios (ORs) and its corresponding 95% confidence intervals (CIs) for each study were calculated and summary relative risk estimates with corresponding 95% CIs were generated using the random-effects model. Heterogeneity and publication bias were assessed. RESULTS: Ten articles were included in this meta-analysis. The included articles were all cohort studies. In a pooled analysis of all studies, metformin using was associated with increased overall survival (OS) rate (OR, 0.54; 95% CI, 0.47 to 0.63) and cancer-specific survival (CS) rate (OR 0.59; 95% CI 0.43 to 0.82) of CRC patients with diabetes. We found that the effect of metformin is associated with geographical region through subgroup meta-analysis. CONCLUSIONS: Metformin using was associated with an increased OS rate and CS rate of colorectal cancer.

Metabolic Syndrome, as Defined Based on Parameters Including Visceral Fat Area, Predicts Complications After Surgery for Rectal Cancer.

BACKGROUND/OBJECTIVES: Metabolic syndrome (MetS) has become a major public health problem. However, few studies have examined the impact of MetS on the postoperative complications of colorectal cancer and the conclusions remain controversial. The present study aimed to investigate whether MetS, as defined based on visceral fat area (VFA) instead of BMI or waist circumference, would predict complications after surgery for rectal cancer. SUBJECTS/METHODS: We conducted a retrospective study of patients who underwent surgery for rectal cancer at our department between January 2013 and August 2018. Univariate and multivariate analyses evaluating the risk factors for postoperative complications were performed. A receiver operating characteristic curve analysis was used to determine the gender-specific cut-off values for VFA. RESULTS: A total of 381 patients were included in the study. The optimal cut-off values for VFA were 117.9 cm2 for men and 76.9 cm2 for women, and 153 patients were diagnosed as having MetS. The rate of postoperative complication was significantly higher in the MetS group than that in the non-MetS group (34.6% versus 15.8%, P < 0.001). The multivariate logistic regression analysis demonstrated that MetS (OR 3.712, P < 0.001), NRS 2002 scores ≥ 3 (OR 2.563, P = 0.001), and tumor located at the lower 1/3 (OR 3.290, P = 0.001) were independent risk factors for complications after surgery for rectal cancer. CONCLUSION: Metabolic syndrome, as defined based on parameters including visceral fat area, was an independent risk factor for complications after surgery for rectal cancer.

Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells.

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) of bacterial adaptive immunity have been adopted as a powerful and versatile tool for manipulation of the genome. This paradigm has been widely applied in biological research and treatments of animal or cellular disease models. A critical feature of CRISPR-Cas is the protospacer adjacent motif (PAM), which dictates the DNA target recognition mechanism of Cas proteins. While, direct identifying functional PAM sequences in human cells remains a challenge. Here, we developed a positive screen system termed PAM-DOSE (PAM Definition by Observable Sequence Excision) to delineate the functional PAMs in human cells. Specifically, the PAM libraries for CRISPR-Cas (SpCas9, SpCas9-NG, FnCas12a, AsCas12a, LbCas12a and MbCas12a) were generated and the corresponding CRISPR-Cas mediated cleaved fragments with functional PAM in human cells were harvested for DNA sequencing, which could be tracked and visualized with either florescence microscopy or flow cytometry analysis. With this system, we identified the functional PAMs of CRISPR-Cas members. We also found that spacer sequence affects the PAM preference of Cas proteins. This method will facilitate identification of functional PAMs for Cas-mediated human genome editing applications.

Engineering the Direct Repeat Sequence of crRNA for Optimization of FnCpf1-Mediated Genome Editing in Human Cells.

FnCpf1-mediated genome-editing technologies have enabled a broad range of research and medical applications. Recently, we reported that FnCpf1 possesses activity in human cells and recognizes a more compatible PAM (protospacer adjacent motif, 5'-KYTV-3'), compared with the other two commonly used Cpf1 enzymes (AsCpf1 and LbCpf1), which requires a 5'-TTTN-3' PAM. However, due to the efficiency and fidelity, FnCpf1-based clinical and basic applications remain a challenge. The direct repeat (DR) sequence is one of the key elements for FnCpf1-mediated genome editing. In principle, its engineering should influence the corresponding genome-editing activity and fidelity. Here we showed that the DR mutants [G(-9)A and U(-7)A] could modulate FnCpf1 performance in human cells, enabling enhancement of both genome-editing efficiency and fidelity. These newly identified features will facilitate the design and optimization of CRISPR-Cpf1-based genome-editing strategies.