T7-Endonuclease I

To construct a sgRNA expression vector, protospacer sequences should not contain repeat sequence as follows: more than 4 continuous T nucleotides (4∼6 nucleotide poly (T) tract acts as a termination signal for RNA pol III), or other homopolymer sequences (more than 5 continuous A or C or G, more than 6 dinucleotide or trinucleotide repeats). This step can be performed by check_sgRNA_seq.pl. Once candidate CRISPR target sites are determined, selected sequences can be used to design oligonucleotides. As described above, the sequence pattern of CRISPR target sites found by sgRNAcas9.pl are 5′-GGX18NGG-3′, 5′-GX19NGG-3′ or 5′-X20NGG-3′. Therefore, the sequence of GGX18, GX19 or X20 will be extracted and used directly to design 20-nt length of sgRNAs by using sgRPrimer.pl. To describe how to use this script to batch design oligonucleotides for constructing sgRNA expression vector, the pGL3-U6-gRNA-Puromycin vector (modified from Addgene 51133) was selected as an example, which is designed for expressing customizable sgRNA under control of the U6 promoter. Annealed oligos were cloned into the vector at a Bsa I restriction site. To facilitate cloning of the 20 bp target sequence, extra bases need to be added to the ends. In this study, ‘accg’ was added to the 5′ end of the sense oligo and ‘aaac’ to the 5′ end of reverse complementary sequence (anti-sense oligo). Then, equal amounts of the sense and anti-sense strands were synthesized and annealed to generate the ds-oligo. This product can be easily ligated into the digested pGL3-U6-gRNA-Puromycin vector.
To investigate on- or off-target cleavage effects, certain lengths of predicted sequence need to be extracted from the genome by nucleotide positions. Then cleavage sites can be validated by using the T7 endonucleases I (T7E1) assay or sequencing. This is another time-consuming step. To raise experiment efficiency and save time, extraction of target sequence by nucleotide position can be performed by extract_targetSeq.pl. The length of sequences extracted from genome was set as an optional argument in this program. A default parameter value was provided to extract DNA fragments up to 1,000 bp in length. Then the sequence was used as a template to design PCR primer pairs for validation of the Cas9 cleavage effect.

We performed T7 endonuclease I digestion at 37° for 30 min using 5 U of T7 endonuclease I (T7E1; New England BioLabs) on 20 µL of unpurified PCR products (~250 ng) in a reaction volume of 50 µL. We stopped the reaction by adding 4 µL of 0.5 M ethylenediaminetetraacetic acid. We performed the Surveyor nuclease assay using 20 µL of unpurified PCR products incubated with 1 µL of Surveyor nuclease (Transgenomic SURVEYOR mutation detection kit for standard gel electrophoresis), 1 µL of Surveyor Enhancer, and 4 µL of 0.15 M MgCl₂ in a 50-µL reaction. We incubated the mix for 1 hr at 42° and stopped the reaction by adding 4 µL of the stop solution provided in the kit. The reactions were either kept at −20° or used immediately for electrophoresis.