Comparison of pregnancy outcomes among patients of different ages who underwent frozen-thawed high-quality single blastocyst transfer.

OBJECTIVE: To investigate the feasibility of performing frozen-thawed high-quality single blastocyst transfer in women of different ages. METHODS: A total of 1,279 women were divided into four groups: a 38-40-year-old group (n = 147), 35-37-year-old group (n = 164), 30-34-year-old group (n = 483), and < 30-year-old group (n = 485). Intergroup comparisons of baseline characteristics and pregnancy and neonatal outcomes were made. RESULTS: The clinical pregnancy rate (47.6%), and live birth rate (34.0%) in the 38-40-year-old group were significantly lower than those in the 30-34-year-old group (64.4%, 50.9%, respectively; all P < 0.001) and < 30-year-old group (62.9%, 50.7%, respectively; all P < 0.001). However, the 35-37-year-old group did not differ from the other three groups in these two dimensions (all P > 0.05). Moreover, there were no differences in the rates of biochemical pregnancy, miscarriage, or obstetric or neonatal complications among the four groups (all P > 0.05). According to the multivariate logistic regression analysis, the 35-37-year-old group was not associated with non-live birth outcomes, adverse pregnancy outcomes, or obstetric or neonatal complications. However, being 38-40 years of age was a risk factor for non-live birth (OR = 2.121, 95% CI: 1.233-3.647) and adverse pregnancy outcomes (OR = 1.630, 95% CI: 1.010-2.633). Post hoc power analysis showed that the study was sufficiently powered to detect meaningful differences. CONCLUSION: Frozen-thawed high-quality single blastocyst transfer produces the same satisfactory pregnancy outcomes for women aged 35-37 years as younger patients. Future prospective randomized controlled studies with larger populations are needed to verify the feasibility and safety of this method.

Comparison of pregnancy outcomes between 4th day morula and 5th day blastocyst after embryo transfer: a retrospective cohort study.

BACKGROUND: This study was designed to evaluate pregnancy outcomes between morulae transferred on day 4 (D4) and blastocysts transferred on day 5 (D5). METHODS: From September 2017 to September 2020, 1963 fresh transfer cycles underwent early follicular phase extra-long protocol for assisted conception in our fertility center were divided into D4 (324 cases) and D5 (1639 cases) groups, and the general situation and other differences of patients in both groups were compared. To compare the differences in pregnancy outcomes, the D4 and D5 groups were further divided into groups A and B based on single and double embryo transfers. Furthermore, the cohort was divided into two groups: those with live births (1116 cases) and those without (847 cases), enabling a deeper evaluation of the effects of D4 or D5 transplantation on assisted reproductive outcomes. RESULTS: In single embryo transfer, there was no significant difference between groups D4A and D5A (P > 0.05). In double embryo transfer, group D4B had a lower newborn birthweight and a larger proportion of low birthweight infants (P < 0.05). The preterm delivery rate, twin delivery rate, cesarean delivery rate, and percentage of low birthweight infants were lower in the D5A group than in the D5B group (P < 0.05). Analysis of factors influencing live birth outcomes further confirmed the absence of a significant difference between D4 and D5 transplantation in achieving live birth (P > 0.05). CONCLUSION: When factors such as working life and hospital holidays are being considered, D4 morula transfer may be a good alternative to D5 blastocyst transfer. Given the in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) success rate and risk of twin pregnancy, D4 morula transfer requires an adapted decision between single and double embryo transfer, although a single blastocyst transfer is recommended for the D5 transfer in order to decrease the twin pregnancy rate. In addition, age, endometrial thickness and other factors need to be taken into account to personalize the IVF program and optimize pregnancy outcomes.

Association between blood lead levels and unfavorable IVF outcomes: potential involvement of endoplasmic reticulum stress response in granulosa cells.

PURPOSE: To investigate the relationship between blood lead levels (BLLs) and IVF clinical outcomes in infertile females and to further explore the possible involvement of granulosa cell (GC) endoplasmic reticulum (ER) stress in the process. METHODS: One hundred twenty-three infertile women undergoing IVF cycles were included in the current study. All participants were divided into three (low, medium, and high) groups determined by BLL tertiles. Gonadotropin releasing hormone (GnRH) agonist regimen for ovarian stimulation was used for all patients, with follicular fluids being collected on the day of oocyte retrieval. Lactate dehydrogenase (LDH) levels in follicular fluid and the endoplasmic reticulum stress-signaling pathway of granulosa cells (GCs) were examined. RESULTS: The oocyte maturation rate and high-quality embryo rate on cleaved stage decreased significantly as BLL increased. For lead levels from low to high, live birth rate (68.29%, 56.10%, 39.02%; P=0.028) showed negative correlations with BLLs. Also, follicular fluid Pb level and LDH level was significantly higher in the high lead group versus the low group. Binomial regression analysis revealed significant negative correlation between BLLs and live birth rate (adjusted OR, 0.38; 95% CI, 0.15-0.95, P=0.038). Further analysis of the endoplasmic reticulum stress (ER stress) signaling pathway of GCs found that expressions of GRP78, total JNK, phosphorylated JNK, and CHOP increased and BCL-2 decreased with increasing BLLs. CONCLUSIONS: BLLs are negatively associated with final clinical outcomes in IVF patients that may be related to increased ER stress response and GC apoptosis. Thus, reducing Pb exposure before IVF procedures may improve final success rates.

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.

A precise and efficient adenine base editor.

Adenine base editors (ABEs) are novel genome-editing tools, and their activity has been greatly enhanced by eight additional mutations, thus named ABE8e. However, elevated catalytic activity was concomitant with frequent generation of bystander mutations. This bystander effect precludes its safe applications required in human gene therapy. To develop next-generation ABEs that are both catalytically efficient and positionally precise, we performed combinatorial engineering of NG-ABE8e. We identify a novel variant (NG-ABE9e), which harbors nine mutations. NG-ABE9e exhibits robust and precise base-editing activity in human cells, with more than 7-fold bystander editing reduction at some sites, compared with NG-ABE8e. To demonstrate its practical utility, we used NG-ABE9e to correct the frequent T17M mutation in Rhodopsin for autosomal dominant retinitis pigmentosa. It reduces bystander editing by ∼4-fold while maintaining comparable efficiency. NG-ABE9e possesses substantially higher activity than NG-ABEmax and significantly lower bystander editing than NG-ABE8e in rice. Therefore, this study provides a versatile and improved adenine base editor for genome editing.

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.

Pubertal lead exposure affects ovary development, folliculogenesis and steroidogenesis by activation of IRE1α-JNK signaling pathway in rat.

Epidemic studies showed that lead exposures are associated with various female reproductive dysfunctions, including infertility, miscarriage, preterm delivery, and early menopause. However, the mechanism involved is still unclear. In the current study, SD rats were exposed to lead at doses of 0, 5, 25, 50 or 250 mg/L through drinking water from postnatal day 21-56. Lead exposures did not affect the body weight or ovary weight. However, the puberty initiation (ages by which vagina opens and estrous cycle occurs) was significantly delayed by as many as 5.8 and 6.8 days respectively (P < 0.05). Also, lead exposures disrupted the estrous cycles, reduced the numbers of primordial and primary follicles and increased the number of atretic follicles by adult. Furthermore, for the highest does group, serum levels of progesterone and testosterone decreased by 80.2% (P < 0.01) and 49.9% (P < 0.05) respectively, while estradiol level increased by 69.8% (P < 0.01). Western blot analyses indicated that lead exposures specifically down-regulated the expressions of steroidogenic protein STAR, CYP17A1, and HSD3B1, while up-regulated FSHR and CYP19A1. Also, the exposure stimulated the endoplasmic reticulum stress (ERS)-related IRE1α-JNK signaling pathway members. Such activation may also result in apoptosis since the death-signaling molecules CHOP and cleaved-CASP3 were up-regulated while BCL2 was down-regulated. In conclusion, lead exposure during juvenile and puberty significantly affected ovary development and functions. The effects may relate to ERS response since the 6 members related to the pathway were all consistently activated.

Does vitamin D level associate with pregnancy outcomes in Chinese women undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer? A retrospective cohort study.

AIM: To explore the relationship between serum 25(OH)D level and pregnancy outcomes (clinical pregnancy rate [CPR] and live birth rate [LBR]) in Chinese women receiving in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) (IVF/ICSI-ET). METHODS: A total of 612 patients included in the study were divided into four cohorts according to serum 25(OH)D with the threshold of 20 ng/ml, 25 ng/ml, 30 ng/ml, and retrospectively analyzed. RESULTS: None of the baseline characteristics of participants was significantly different in the four cohorts except gravid status. The trend of 25(OH)D concentration was positively correlated with CPR and LBR. The younger (age: p < 0.001 both in CPR and LBR) women with primary infertility (infertility type: p = 0.004 in LBR) were more likely to get a better pregnancy outcome under the same 25(OH)D concentration stages. As shown on heatmap plots, CPR, and LBR were significantly increased for 25(OH)D concentrations above 30.00 ng/ml and women younger than 30 years old. The adjusted binary logistic regression and restricted cubic spline (RCS) showed that there existed a nonlinear positive correlation between 25(OH)D concentration and pregnancy outcome (CPR and LBR) (Pnonlinear < 0.001, respectively). The women with a sufficient 25(OH)D concentration (30 ng/ml) had 1.07 (clinical pregnancy) and 1.05 (live birth) times higher successful birth outcomes compared to women with an insufficient 25(OH)D concentration (25 ng/ml). (OR25 ng/ml, ref = 30 ng/ml [95% CI] = 0.935 [0.932-0.938] and 0.947 [0.945-0.950], p < 0.001, respectively). CONCLUSION: In Chinese women receiving IVF/ICSI-ET, the serum level of 25(OH)D demonstrated a nonlinear positive correlation with pregnancy outcomes (CPR and LBR), with stronger correlations above 25 ng/ml and worse yields below 30 ng/ml. However, it could not yet be considered different in distinct ages.

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.

Two high-fidelity variants: efSaCas9 and SaCas9-HF, which one is better?

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease Cas9) nucleases have been widely applied for genome engineering. Staphylococcus aureus Cas9 (SaCas9) is compact, which can be packaged in AAV (adeno-associated virus) vector for in vivo gene editing. While, wild-type SaCas9 can induce unwanted off-target mutations and substantially limits the applications. So far, there are two reported SaCas9 variants with high-fidelity, including efSaCas9 from our previous study and SaCas9-HF. However, it remains unknown which one possessing the better fidelity and higher activity. Here, we performed a parallel comparison of efSaCas9 and SaCas9-HF in human cells through fluorescent reporter system and target deep sequencing, respectively. The results demonstrated that efSaCas9 possesses higher cleavage activity and fidelity than SaCas9-HF at the most endogenous sites in human cells. Collectively, our study provides insights for the rational selection of suitable SaCas9 for human genome editing.

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.

Investigate the effect of COVID-19 inactivated vaccine on sperm parameters and embryo quality in in vitro fertilization.

Since the reproductive toxicity of COVID-19 vaccines have not been assessed in previous clinical trials, and studies have shown that SARS-CoV-2 is associated with a decrease in sperm parameters. Although it has been reported that the mRNA SARS-CoV-2 vaccines do not adversely affect semen parameters, whether this conclusion applies to inactivated vaccines remains unclear. Here, we conducted a study among patients who accepted in vitro fertilization (IVF) at the reproductive centre between June and August of 2021. In the enrolled cases, men who have completed two doses of COVID-19 inactivated vaccine were included in "vaccine group" (N = 105), and those who were not vaccinated were included in "control group" (N = 155). In this study, we compare the sperm parameters and embryo quality between these two groups. Our data showed that the sperm parameters were similar in terms of volume, sperm concentration, sperm count, progressive motility, total motility and total motile sperm count between these two groups. Similarly, no significant differences were observed in IVF outcomes. The mean number of 2PN, cleavage-stage embryos, blastocysts, and good-quality blastocysts was 8.59 ± 4.47, 5.06 ± 3.17 and 2.08 ± 1.79 in vaccine group, 7.75 ± 4.14, 4.34 ± 3.06 and 1.74 ± 1.54 in control group, respectively. The high-quality blastocyst rate was 41.05% (218 of 531) in vaccine group and 40.03% (269 of 672) in control group (p > 0.05). In addition, no differences were observed in biochemical and clinical pregnancy rates between the two groups. In summary, our results revealed that COVID-19 inactivated vaccine administration exhibited no negative effect on sperm parameters and embryo quality in IVF.

Comparison of Pregnancy Outcomes Between Single-Morula Embryo Transfer and Single-Blastocyst Transfer in Fresh IVF/ICSI Cycles.

BACKGROUND This study investigated the effectiveness and feasibility of day 4 (D4) morula embryo transfer (ET) in comparison with day 5 (D5) blastocyst ET, with regards to their clinical data, laboratory test results, and pregnancy outcomes. MATERIAL AND METHODS This retrospective cohort study enrolled 1070 patients, including 178 cases in group D4 and 892 cases in group D5. The endpoint was live birth rate after fresh embryo transfer. Furthermore, the clinical outcomes of D4 embryos with different morphology were compared and assigned to 3 groups: in group 1 (n=66) the embryos were compacted but not expanded, in group 2 (n=102) the embryos were compacted and expanded (early blastocyst), and in group 3 (n=10) the embryos were not compacted. RESULTS Groups D4 and D5 had comparable clinical pregnancy rates (53.37% vs. 59.97%) and live birth rates (43.25% vs 50.89%), and there were no significant differences between the 2 groups. In group 3, there was only 1 clinical pregnancy and no live birth. In comparison between group 1 and group 2, the clinical pregnancy rate of group 2 showed an upward trend (48.48% vs 60.78%), but there was no significant difference. There was also no statistically significant difference in the live birth rate between the 2 groups (42.42% vs 49.01%). CONCLUSIONS Transferring of compacted embryos or early blastocysts can result in high clinical pregnancy rates and live birth rates. In addition to the cleavage and blastocyst ET, morula ET may serve as an alternative option for the clinician.

Frozen versus fresh single blastocyst transfer in ovulatory women: a multicentre, randomised controlled trial.

BACKGROUND: Elective single embryo transfer (eSET) has been increasingly advocated, but concerns about the lower pregnancy rate after reducing the number of embryos transferred have encouraged transfer of multiple embryos. Extended embryo culture combined with electively freezing all embryos and undertaking a deferred frozen embryo transfer might increase pregnancy rate after eSET. We aimed to establish whether elective frozen single blastocyst transfer improved singleton livebirth rate compared with fresh single blastocyst transfer. METHODS: This multicentre, non-blinded, randomised controlled trial was undertaken in 21 academic fertility centres in China. 1650 women with regular menstrual cycles undergoing their first cycle of in-vitro fertilisation were enrolled from Aug 1, 2016, to June 3, 2017. Eligible women were randomly assigned to either fresh or frozen single blastocyst transfer. The randomisation sequence was computer generated, with block sizes of two, four, or six, stratified by study site. For those assigned to frozen blastocyst transfer, all blastocysts were cryopreserved and a delayed frozen-thawed single blastocyst transfer was done. The primary outcome was singleton livebirth rate. Analysis was by intention to treat. This trial is registered at the Chinese Clinical Trial Registry, number ChiCTR-IOR-14005405. FINDINGS: 825 women were assigned to each group and included in analyses. Frozen single blastocyst transfer resulted in higher rates of singleton livebirth than did fresh single blastocyst transfer (416 [50%] vs 329 [40%]; relative risk [RR] 1·26, 95% CI 1·14-1·41, p<0·0001). The risks of moderate or severe ovarian hyperstimulation syndrome (four of 825 [0·5%] in frozen single blastocyst transfer vs nine of 825 [1·1%] in fresh single blastocyst transfer; p=0·16), pregnancy loss (134 of 583 [23·0%] vs 124 of 481 [25·8%]; p=0·29), other obstetric complications, and neonatal morbidity were similar between the two groups. Frozen single blastocyst transfer was associated with a higher risk of pre-eclampsia (16 of 512 [3·1%] vs four of 401 [1·0%]; RR 3·13, 95% CI 1·06-9·30, p=0·029). INTERPRETATION: Frozen single blastocyst transfer resulted in a higher singleton livebirth rate than did fresh single blastocyst transfer in ovulatory women with good prognosis. The increased risk of pre-eclampsia after frozen blastocyst transfer warrants further studies. FUNDING: The National Key Research and Development Program of China.

Assisted oocyte activation with calcium ionophore 44 hours after intracytoplasmic sperm injection resulting in successful pregnancy.

Assisted Oocyte Activation (AOA) with Calcium Ionophore, is possible to manually activate the oocytes and cure globozoospermia, thus leading to successful pregnancy in 1 h after ICSI. But in this case, we report a case that 44 h after ICSI, the arrest zygotes assisted oocyte activation with calcium ionophore, obtained clinical pregnancy and live birth. Accordingly, AOA may provide us with an immediate treatment for embryonic arrest in the future.

Perfluorododecanoic Acid Blocks Rat Leydig Cell Development during Prepuberty.

Perfluorododecanoic acid (PFDoA) has been used as a surfactant and may have reproductive toxicity. However, whether PFDoA influences Leydig cell development during prepuberty remains unknown. In the present study, 21-day-old male Sprague-Dawley rats were gavaged 0, 5, or 10 mg/kg PFDoA from postnatal day 21 to 35. PFDoA decreased the serum concentrations of testosterone, luteinizing hormone, and follicle-stimulating hormone at doses of 5 and 10 mg/kg without influencing Leydig cell number and proliferation. However, PFDoA down-regulated the expression of Leydig cell genes ( Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, and Hsd11b1) or their proteins. PFDoA dose-dependently reduced SIRT1 and PGC-1α levels. PFDoA did not affect AMPK and AKT2 levels but decreased their phosphorylation. We also treated primary progenitor Leydig cells purified from prepubertal rat testes with PFDoA for 24 h. It in vitro lowered viability and decreased mitochondrial membrane potential of progenitor Leydig cells, but it stimulated the generation of the intracellular reactive oxygen species and induced Leydig cell apoptosis at 10 µM. In conclusion, PFDoA blocks rat Leydig cell development during the prepubertal period possibly via targeting AMPK/SIRT1/PGC-1α and AKT2 signaling pathways.

Delayed development influences the outcome of different grades of D5 and D6 blastocysts during freeze-thaw cycle.

To analyze the effects of blastocysts on the 5th day (D5) and 6th day (D6) of frozen-thawed blastocyst transplantation on pregnancy outcome and provide evidence for further improvement of the strategy. This study included transfers from the Reproductive Medicine Center of the Second Affiliated Hospital of Wenzhou Medical University during freeze-thaw cycles from January 2016 to December 2017. They were divided into D5 group (1616 cases) and D6 group (619 cases) according to blastocyst formation and development. Each group was further divided into 5 groups according to the quality of the blastocyst and the number of transplants, making a total of 10 groups. Following the frozen transplantation cycle, the transplanting rate was significantly higher for D5 (41.73%) than for D6 (23.98%) (P < 0.05); the ongoing pregnancy rate (47,40%) was also significantly higher than that of D6 (28.43%) (P < 0.05).In the frozen-thawed blastocyst resuscitation transplantation, compared to D6 blastocysts, D5 blastocysts were more conducive to blastocyst implantation and could be used to achieve better clinical pregnancy outcome. In blastocyst selection, a single D5 excellent blastocyst transplant is preferred. Only at the 6th day of non-excellent D6, 2 blastocysts are recommended for transplantation.

A Single Multiplex crRNA Array for FnCpf1-Mediated Human Genome Editing.

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome.

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.

A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.

Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5'-TTTN-3' protospacer adjacent motif (PAM) at the 5' end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5'-TTN-3' as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications.