CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10797759PMC
http://dx.doi.org/10.1186/s13059-023-03132-3DOI Listing

Publication Analysis

Top Keywords

library cloning
8
cloning protocol
8
compact crispr
4
crispr genetic
4
genetic screens
4
screens enabled
4
enabled improved
4
improved guide
4
guide rna
4
rna library
4

Similar Publications

The determinate inflorescence trait of L. is associated with various desirable agricultural characteristics. ( and ), which encode the transcription factor have previously been identified as candidate genes controlling this trait through map-based cloning.

View Article and Find Full Text PDF

Construction of Immune Single Domain Antibodies Library for Development of Specific Nanobodies Using Phage Display Strategy.

Recent Pat Biotechnol

January 2025

Center of Excellence in Recombinant Biopharmaceutical Proteins, Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, Egypt.

Background: poses a considerable global public health challenge. In Egypt, approximately 60% of the inhabitants in the Northern and Eastern areas of the Nile Delta are affected by this parasite, whereas the Southern region experiences a significantly lower infection rate of 6%.

Aim: Construction of an immune phage display Nbs library based on the VHH framework for selecting -specific Nbs for seeking cost-effective, sensitive, and specific diagnostic tools for rapidly detecting mansoni.

View Article and Find Full Text PDF

BK virus is implicated in polyomavirus-associated nephropathy (PVAN) and hemorrhagic cystitis, particularly in kidney transplant recipients, affecting the functionality of the transplanted kidney and posing a risk of graft loss. Despite these challenges, specific antiviral drugs targeting BK virus remain elusive. Agnoprotein, a small, positively charged protein encoded by the BK virus late gene, functions in the assembly, maturation, and release of the virus.

View Article and Find Full Text PDF

The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!