Protocol: CRISPR-based Gene Deletion in Nostoc punctiforme - Overview

Douglas Risser

May 25, 2026

Protocol: CRISPR-based Gene Deletion in Nostoc punctiforme - Overview

This protocol is a draft, published without a DOI.

Douglas Risser¹

¹UCCS

Risser Lab (N. punctiforme)

Douglas Risser

UCCS

Protocol Citation: Douglas Risser 2026. Protocol: CRISPR-based Gene Deletion in Nostoc punctiforme - Overview. protocols.io https://dx.doi.org/

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: May 22, 2026

Last Modified: May 25, 2026

Protocol Integer ID: 317791

Keywords: gene deletion in nostoc punctiforme, based gene deletion, crispr, nostoc punctiforme

Funders Acknowledgements:

NSF

Grant ID: IOS 2420339

Abstract

This protocol provides an overview of the steps for CRISPR-based gene deletion in N. punctiforme

Guidelines

**Protocol Overview**

**I. Vector design**
   a. In silico design of primers and gRNA arrays to construct pCpf1b vectors for deletion of target genes
   b. In silico design of primers to confirm gene deletion

**II. Vector construction**
   a. Preparation of plasmid pCpf1b digested with AvrII and BamHI
   b. PCR amplification of 5’ and 3’ HRTs and gRNA array
   c. HiFi plasmid assembly
   d. Transformation
   e. Plasmid prep
   f. PCR confirmation and sequencing

**III. Conjugal transfer**
   a. Preparation of N. punctiforme cultures for conjugation
   b. Transformation of vectors into E. coli UC585 donor strain
   c. Conjugal transfer to N. punctiforme
   d. PCR confirmation of exconjugants

**IV. Plasmid curing**
   a. Liquid cultures of positive exconjugants
   b. Counterselection on 5% sucrose
   c. Confirmation of neomycin sensitivity
   d. Final PCR verification

Vector design

In silico design of primers and gRNA arrays to construct pCpf1b vectors for deletion of target genes

In silico design of primers to confirm gene deletion

Vector construction

Preparation of plasmid pCpf1b digested with AvrII and BamHI

PCR amplification of 5’ and 3’ HRTs and gRNA array

HiFi plasmid assembly

Transformation

Plasmid prep

PCR confirmation and sequencing

Conjugal transfer

Preparation of N. punctiforme cultures for conjugation

Transformation of vectors into E. coli UC585 donor strain

Conjugal transfer to N. punctiforme

PCR confirmation of exconjugants

Plasmid curing

Liquid cultures of positive exconjugants

Counterselection on 5% sucrose

Confirmation of neomycin sensitivity

Final PCR verification