Cloning sgRNA Template for Cas9 Gene Editing

To bind target DNA, Cas9 nuclease requires a CRISPR RNA (crRNA) containing ~20 bases of the target sequence and a trans-activating CRISPR RNA (trRNA ) with a non-variable scaffold sequence. Commonly, these two RNA sequences are fused into a single sequence known as single guide RNA (sgRNA).

Cells or organisms can be transformed with two separate plasmids: one encoding sgRNA and one encoding Cas protein. Alternatively, a single template can be constructed with a CRISPR/Cas expression vector and one or several sgRNA target sequences. Additional homologous repair templates are useful with either approach for precise indel formation.

Single guide RNA templates and plasmid libraries enable straightforward, scalable, and cost-effective in vivo and in vitro CRISPR experiment workflows. A variety of vectors have been validated for different cells and model organisms, as well as for final applications, ranging from cutting or nicking to activating genes and screening libraries.

Selection of the optimal cloning technique for sgRNA template construction can be based on the number of sgRNA inserts, vector characteristics and desired workflow efficiency. Conventional cloning methods require synthesis, phosphorylation, annealing, and ligation of two oligos into a digested and dephosphorylated vector. Q5^® Site-directed Mutagenesis may be used to insert target sequence into a Cas9-sgRNA construct. However, NEBuilder^® HiFi DNA Assembly is highly recommended to streamline the construction of virtually error-free sgRNA templates, sgRNA/Cas expression constructs and sgRNA plasmid libraries. 

Improve cloning efficiency of sgRNA libraries for CRISPR screening

NEBuilder HiFi DNA Assembly optimizes cloning efficiency and accuracy for sgRNA plasmid libraries for CRISPR screening studies. Cloning efficiency enhances the uniformity of sgRNA libraries, supporting accuracy. NEBuilder vastly improves the efficiency of assembly reactions compared to other assembly methods. The single-tube, isothermal reaction enables seamless cloning without the bias limitations that exist in sgRNA libraries generated with restriction enzyme-based cloning.

Learn more about constructing sgRNA libraries in the Application Note:

Application note: Bridging dsDNA with an ssDNA oligo and NEBuilder HiFi DNA Assembly  to create an sgRNA-Cas9 Expression Vector  View application note

sgRNA template construction strategies