CRISPR/Cas9 increases mitotic gene conversion in human cells.

Gene conversion is a process of transferring genetic material from one homologous sequence to another. Most reported gene conversions are meiotic although mitotic gene conversion is also described. When using CRISPR/Cas9 to target the human hemoglobin subunit beta (HBB) gene, hemoglobin subunit delta (HBD) gene footprints were observed in HBB gene. However, it is unclear whether these were the results of gene conversion or PCR-mediated sequence shuffling between highly homologous sequences. Here we provide evidence that the HBD footprints in HBB were indeed results of gene conversion. We demonstrated that the CRISPR/Cas9 facilitated unidirectional sequence transfer from the homologous gene without double-strand breaks (DSB) to the one with DSBs, and showed that the rates of HBD footprint in HBB were positively correlated to the HBB insertion and deletion rates. We further showed that when targeting HBD gene, HBB footprints could also be observed in HBD gene. The mitotic gene conversion was observed not only in immortalized HEK293T cells, but also in human primary cells. Our work reveals mitotic gene conversion as an often overlooked effect of CRISPR/Cas9-mediated genome editing.

Delivering Cas9/sgRNA ribonucleoprotein (RNP) by lentiviral capsid-based bionanoparticles for efficient 'hit-and-run' genome editing.

Transient expression of the CRISPR/Cas9 machinery will not only reduce risks of mutagenesis from off-target activities, but also decrease possible immune response to Cas9 protein. Building on our recent developing of a system able to package up to 100 copies of Cas9 mRNA in each lentivirus-like particle (LVLP) via the specific interaction between aptamer and aptamer-binding proteins (ABP), here we develop a lentiviral capsid-based bionanoparticle system, which allows efficient packaging of Cas9/sgRNA ribonucleoprotein (RNP). We show that replacing the Tetraloop of sgRNA scaffold with a com aptamer preserves the functions of the guide RNA, and the com-modified sgRNA can package Cas9/sgRNA RNP into lentivirus-like particles via the specific interactions between ABP and aptamer, and sgRNA and Cas9 protein. These RNP bionanoparticles generated Indels on different targets in different cells with efficiencies similar to or better than our recently described Cas9 mRNA LVLPs. The new system showed fast action and reduced off-target rates, and makes it more convenient and efficient in delivering Cas9 RNPs for transient Cas9 expression and efficient genome editing.

Delivering SaCas9 mRNA by lentivirus-like bionanoparticles for transient expression and efficient genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system discovered using bacteria has been repurposed for genome editing in human cells. Transient expression of the editor proteins (e.g. Cas9 protein) is desirable to reduce the risk of mutagenesis from off-target activity. Using the specific interaction between bacteriophage RNA-binding proteins and their RNA aptamers, we developed a system able to package up to 100 copies of Staphylococcus aureus Cas9 (SaCas9) mRNA in each lentivirus-like bionanoparticle (LVLP). The SaCas9 LVLPs mediated transient SaCas9 expression and achieved highly efficient genome editing in the presence of guide RNA. Lower off-target rates occurred in cells transduced with LVLPs containing SaCas9 mRNA, compared with cells transduced with adeno-associated virus or lentivirus expressing SaCas9. Our LVLP system may be useful for efficiently delivering Cas9 mRNA to cell lines and primary cells for in vitro and in vivo gene editing applications.

No evidence of genome editing activity from Natronobacterium gregoryi Argonaute (NgAgo) in human cells.

The argonaute protein from the thermophilic bacterium Thermus thermophilus shows DNA-guided DNA interfering activity at high temperatures, complicating its application in mammalian cells. A recent work reported that the argonaute protein from Natronobacterium gregoryi (NgAgo) had DNA-guided genome editing activity in mammalian cells. We compared the genome editing activities of NgAgo and Staphylococcus aureus Cas9 (SaCas9) in human HEK293T cells side by side. EGFP reporter assays and DNA sequencing consistently revealed high genome editing activity from SaCas9. However, these assays did not demonstrate genome editing activity by NgAgo. We confirmed that the conditions allowed simultaneous transfection of the NgAgo expressing plasmid DNA and DNA guides, as well as heterologous expression of NgAgo in the HEK293T cells. Our data show that NgAgo is not a robust genome editing tool, although it may have such activity under other conditions.

Accuracy of Surgeon's Intraoperation Diagnosis of Acute Appendicitis, Compared with the Histopathology Results.

OBJECTIVE: To evaluate the accuracy of surgeons' intraoperative diagnosis in open appendectomy and compare it with the histopathology examination results afterwards. METHODS: This was a cross-sectional retrospective study accomplished in Namazee hospital affiliated with Shiraz University of Medical Sciences, in a one-year period from 2007 to 2008. Medical charts of all the patients who were admitted with impression of acute appendicitis and underwent open appendectomy in our center were included. Demographic information, intraoperative findings as in the operation note based on a method used by our  surgeons, and histopathology examination  of the removed appendix were recorded and reported. RESULTS: A total of 342 patients were studied including 229 (67%) males and 113 (33%) females, with the mean age of 16.02 ± 9.89 (range 3 to 76) years, with a large proportion from 10 to 15 years. Surgeons reported 97.4% of the patients to have acute appendicitis, 29.5%, 10.2% and  5.6% with severe, moderate  and  mild inflammation  respectively, whereas 26.6%  and 9.4% with suppurated  and gangrenous  appendicitis  separately, 14.6% to  have perforated appendicitis and only 1.5%hadperforated appendicitis with peritonitis. However, 79.5% of cases showed appendicitis in the histopathology review. The accuracy of surgeons' intraoperative diagnosis is 81.6%, 85.2% for men and 72.6% for women. CONCLUSION: The method used by our surgeon is not completely indicative in mild to severe inflamed appendix but it is almost always compatible with the pathology results in suppurated, gangrened, and perforated appendix. Therefore surgeons' gross observation of the  inflamed appendix  may not  always be in  concordance  with the  histopathology examination of the resected appendix.

Short-term Outcome of Open Appendectomy in Southern Iran: A Single Center Experience.

OBJECTIVES: To evaluate the short-term outcome of open appendectomy, the rate of negative appendectomy as well as pathology reports after surgery in patients with suspected acute appendicitis. METHODS: This was a retrospective cross-sectional study being performed in Nemazee hospital affiliated with Shiraz University of Medical Science during a 2-year period between 2008 and 2010. The medical records of all consecutive patients who underwent open appendectomy in our center due to acute appendicitis were included in the study. The elective and laparoscopic appendectomies were excluded. The demographic information, clinical findings, laboratory investigations and the histopathological examination of the appendix were recorded and reported. RESULTS: A total of 337 patient including 137 (36.4%) females, and 240 (63.6%) males with the mean age of 16.26 ± 9.81 (range 3 to 76) years were stduied. Anorexia (64.7%) and fever (20.7%) were more prevalent symptoms. The mean duration between pain initiation and operation ranged from 0 to 14 days with mean 1.88 ± 1.63 days. Right lower quadrant (RLQ), periumbilical, epigastria, left lower quadrant (LLQ), and Right upper quadrant (RUQ), pain were manifest in 78.8%, 41.6%, 12.2%, 3.2%, and    1.3% of patients, respectively. Pathological evaluation of the appendix showed appendicitis in 70.4% of patients. CONCLUSION: The higher rate of negative appendectomy accounts for wasteful tapping of medical resources and causing further complication in patients. Therefore it is essential to conduct more accurate studies to detect the root cause of the disease. This would help improve the management of appendicitis which is an emergency condition with high incidence.

Accuracy of Ultrasonography in Diagnosing Acute Appendicitis.

OBJECTIVES: To evaluate the accuracy of sonography in diagnosing acute appendicitis in patients with Alvarado score 4-7. METHODS: This is a retrospective cross-sectional study being performed in Namazee hospital affiliated with Shiraz University of Medical sciences during a one year period from 9/2007 to 9/2008. We evaluated all patients with Alvarado score 4-7 and divided them in two groups: those with Ultrasound study prior to surgery and those without any imaging modalities for diagnosis of AA. The demographic information, histopathology, physical examination, laboratory data, sonography report and histopathological reports of patients were gathered. RESULTS: A total of 238 patients had Alvarado scores 4-7 including 160 males and 78 females. 110 patients did not have any imaging and 128 had undergone sonography before operation. Ultrasound had overall sensitivity of 75 %, specificity 69.2 %, PPV 88 %, NPV 46.1% and accuracy of 73.6 %. Negative appendectomy rate was 20.9 % and 23.4 % in those without sonography and inpatients with sonography respectively, with a higher rate in females. CONCLUSION: Ultrasound is more useful when the patient is female and the result of sonography is positive; however, it is not reliable when the result is negative and maybe other diagnostic modalities such as CT scan can help us in better diagnosis of Acute Appendicitis.