What are the main steps of using CRISPR in my research?

There are four main steps for using CRISPR: designing, delivering, repairing, and analyzing. 

1. When starting a CRISPR experiment, the first step is to choose the appropriate CRISPR-associated (Cas) enzyme based on the protospacer-adjacent motif (PAM) sequence. The two main Cas enzymes used are Cas9 and Cas12a. Then, design the guide RNA (gRNA) to direct the Cas enzyme to the target site. For Cas9, gRNA can be a single guide RNA (sgRNA) or a 2-part guide RNA containing tracrRNA and crRNA. Cas12a uses single crRNA. Predesigned or custom gRNAs can be also used, with Alt-R modifications enhancing genome editing outcomes and diminishing immune response.
2. In the delivery step, Cas enzyme and gRNA are joined to form a ribonucleoprotein (RNP). The RNP is delivered to cells via lipofection or electroporation. 
3. In the repairing step, once inside the cell, the RNP cuts the genomic DNA, forming a double-strand break (DSB). Cells then go through homology-directed repair (HDR) non-homologous end joining (NHEJ) or pathways. NHEJ creates knockouts for researching gene function, while HDR requires a template and can be used to insert particular mutations. 
4. Mutation identification can be analyzed using various methods, including gel-based techniques for alterations in genomic sequence and next-generation sequencing (NGS) for visualizing unwanted effects. However, for a comprehensive analysis of mutation presence and characterization, next-generation sequencing (NGS) is preferred.