Single guide RNA

The CRISPR/Cas9 system, composed of a single-guide RNA for target recognition and a Cas9 protein for DNA cleavage, has the potential to revolutionize agriculture as well as medicine. Even though extensive work has been done to improve the gene editing activity of CRISPR/Cas9, little is known about the regulation of this bacterial system in eukaryotic host cells, especially at the post-transcriptional level.

15p vigalileogalilei 27-02-2022 14 1   Download

Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing.

11p vigalileogalilei 27-02-2022 8 1   Download

CRISPR-Cas12a/Cpf1, a single RNA-guided endonuclease system, provides a promising tool for genome engineering. However, only three Cas12a orthologs have been employed for mammalian genome editing, and the editing efficiency as well as targeting coverage still requires improvements.

6p vigalileogalilei 27-02-2022 15 1   Download

RNA sequencing using the latest single-molecule sequencing instruments produces reads that are thousands of nucleotides long. The ability to assemble these long reads can greatly improve the sensitivity of long-read analyses. Here we present StringTie2, a reference-guided transcriptome assembler that works with both short and long reads.

13p vielonmusk 30-01-2022 9 0   Download

Single-cell RNA-sequencing (scRNA-seq) allows studying heterogeneity in gene expression in large cell populations. Such heterogeneity can arise due to technical or biological factors, making decomposing sources of variation difficult. We here describe f-scLVM (factorial single-cell latent variable model), a method based on factor analysis that uses pathway annotations to guide the inference of interpretable factors underpinning the heterogeneity.

13p vialfrednobel 29-01-2022 14 0   Download

We report that engineered Cas9 variants with improved specificity—eCas9-1.1 and Cas9-HF1—are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5’ terminus, relative to target DNA sequences. Because the nucleotide at the 5’ end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites.

6p vialfrednobel 29-01-2022 6 0   Download

CRISPR is widely used to disrupt gene function by inducing small insertions and deletions. Here, we show that some single-guide RNAs (sgRNAs) can induce exon skipping or large genomic deletions that delete exons. For example, CRISPR-mediated editing of β-catenin exon 3, which encodes an autoinhibitory domain, induces partial skipping of the in-frame exon and nuclear accumulation of β-catenin.

8p vialfrednobel 29-01-2022 9 0   Download

CRISPR-based approaches have quickly become a favored method to perturb genes to uncover their functions. Here, we review the key considerations in the design of genome editing experiments, and survey the tools and resources currently available to assist users of this technology.

21p viaristotle 29-01-2022 23 0   Download

Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)–transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency.

10p viaristotle 29-01-2022 12 0   Download

Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes. Results: Here we describe CRISPR library designer (CLD), an integrated bioinformatics application for the design of custom single guide RNA (sgRNA) libraries for all organisms with annotated genomes.

10p viaristotle 29-01-2022 10 0   Download

Prime editing is a revolutionary genome-editing technology that can make a wide range of precise edits in DNA. However, designing highly efficient prime editors (PEs) remains challenging. We develop Easy-Prime, a machine learning–based program trained with multiple published data sources.

11p viarchimedes 26-01-2022 9 0   Download

Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) is an acquired immune system found in bacteria and archaea that can specifically silence or degrade a foreign single or double strand nucleic acid to protect it from infection. In recent years, the CRISPR/Cas9 system has rapidly been evolved into a genome editing technology, in which the Cas9 endonuclease can be targeted to specific DNA sequences by guide RNAs (gRNAs) that are easily programmable.

16p tudichquannguyet 29-11-2021 21 1   Download

The beauty and power of the genome editing mechanism, CRISPR Cas9 endonuclease system, lies in the fact that it is RNA-programmable such that Cas9 can be guided to any genomic loci complementary to a 20-nt RNA, single guide RNA (sgRNA), to cleave double stranded DNA, allowing the introduction of wanted mutations. Unfortunately, it has been reported repeatedly that the sgRNA can also guide Cas9 to off-target sites where the DNA sequence is homologous to sgRNA.

8p vilarryellison 29-10-2021 8 1   Download

Single-cell RNA sequencing (scRNA-seq) has quickly become one of the most dominant techniques in modern transcriptome assessment. In particular, 10X Genomics’ Chromium system, with its high throughput approach, turn key and thorough user guide made this cutting-edge technique accessible to many laboratories using diverse animal models.

13p vitzuyu2711 29-09-2021 10 1   Download

Standard RNAseq methods using bulk RNA and recent single-cell RNAseq methods use DNA barcodes to identify samples and cells, and the barcoded cDNAs are pooled into a library pool before high throughput sequencing. In cases of single-cell and low-input RNAseq methods, the library is further amplified by PCR after the pooling.

9p vicolorado2711 23-10-2020 11 0   Download

One of the main challenges for the CRISPR-Cas9 system is selecting optimal single-guide RNAs (sgRNAs). Recently, deep learning has enhanced sgRNA prediction in eukaryotes. However, the prokaryotic chromatin structure is different from eukaryotes, so models trained on eukaryotes may not apply to prokaryotes.

14p vicolorado2711 23-10-2020 7 1   Download

microRNAs (miRNAs) are endogenous small (~21 nucleotide) single-stranded non-coding RNAs that typically function by guiding cleavage of target genes. To find the miRNAs that may be involved in dark-induced leaf senescence, we identified miRNAs by microarray platform using Arabidopsis thaliana leaves from both whole darkened plants (DPs) and individually darkened leaves (IDLs).

12p vihashirama2711 21-05-2020 11 1   Download