Can SpRY recognize any PAM in human cells?

The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)|-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.

A patient with severe congenital neutropenia harbors a missense ELANE mutation due to paternal germline mosaicism.

BACKGROUND: Clinical and genetic characteristics of ELANE mutation of a 3-year-old male who had a severe congenital neutropenia (SCN) were examined. We then investigated whether CRISPR/Cas9-mediated gene editing could correct the mutation. PROCEDURE: The proband underwent extensive clinical assessments, such as exome sequencing and bioinformatics analysis, so that pathogenic genes could be identified. Sanger sequencing was also utilized for confirmation. The cell line, 293-ELANE, harboring ELANE mutation was generated, and the mutation was then corrected by CRISPR/Cas9-mediated homology-directed repair (HDR). RESULTS: The ELANE gene test in the proband unveiled a heterozygous de novo missense mutation: c. 248T > A (p.V83D), which was not detected in his asymptomatic parents who had provided peripheral blood samples. We found that 46.01% of his father's sperm cells had the same mutation. These results demonstrate that the proband inherited the ELANE mutation from his father, who had an average neutrophil count but had a germline mosaicism. The highest repair efficiency of CRISPR/Cas9-mediated HDR for 293-ELANE is 4.43%. CONCLUSIONS: We identified a missense mutation (p.V83D) in ELANE that causes SCN. This is the first report on paternal semen mosaicism of an ELANE mutation. Our study paves the way for preimplantation genetic diagnosis (PGD) based on ELANE mutation prevention and clinical treatment of congenital disabilities.

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.