Build sgRNA library targeting the human whole genome from oligo pools.

 

Library Construction

 

A customized CRISPR sgRNA library, comprising 65,383 unique sgRNAs designed to target the human whole genome, has been meticulously constructed. Leveraging a high-throughput DNA synthesis platform, all sgRNAs have been simultaneously synthesized with unparalleled accuracy and efficiency. Subsequently, the synthesized oligos are amplified and cloned into a lentiviral vector.

Figure1. Workflow of sgRNA Library Construction

 

 

Low Error Rate

 

Following the completion of library construction, a batch of 30 clones was randomly selected and subjected to Sanger sequencing. The resulting sequencing data revealed a remarkable outcome: 100% of the sgRNA sequences within the randomly chosen clones perfectly aligned with the theoretical sequence, affirming the precision and reliability of our oligo pool synthesis.

 

Figure2. Sanger Sequencing Results

 

 

High Coverage, Uniform Distribution, High Stability Between Batches

 

The uniformity of the library's coverage and distribution are important indicators for evaluating its quality. Leveraging Next-Generation Sequencing (NGS), we have meticulously assessed the coverage and consistency across batches. 

 

Coverage refers to the proportion of actually detected sequences to designed target sequences within the library. Ideally, different sgRNA sequences are evenly distributed in the library. Despite the inevitable biases introduced during the PCR and cloning processes, resulting in varying sgRNA read levels. Some sgRNA reads are relatively low, while others are relatively high. 

 

Consequently, uniformity is a measure of the sequence distribution's consistency within the library. The distribution skew ratio, a widely recognized metric, deems libraries with values <10 as qualified, with smaller values indicating superior uniformity.

 

 

In pursuit of these standards, we have developed four batches of the aforementioned library, and the NGS detection analysis results are as follows:

 

Batch number	Diversity of designed library	Actual diversity of the library	Coverage	skew ratio
01	63950	63950	100%	2.24
02	63950	63940	99.98%	2.39
03	63950	63937	99.98%	2.91
04	63950	63943	99.99%	2.29